[0001] This application claims priority benefit from U.S. Provisional Patent Application No. 62/385,930 and U.S. Provisional Patent Application No.62/385,933, filed on September 9, 2016, the contents of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 768312000240SEQLIST.txt; date recorded: September 5, 2017, size: 28 KB.

FIELD OF THE INVENTION

[0003] The present invention relates to oncolytic viruses, anti-PD-1 antibodies, and methods of use thereof.

BACKGROUND OF THE INVENTION

[0004] Oncolytic viruses (OVs) are promising agents for cancer treatment, due to their ability to infect, replicate in, and lyse tumor cells and spread through tumor cells in successive rounds of replication (Russell et al., Nat Biotechnol.2012, 30:658-670; Kelly and Russell, Mol Ther.2007, 15:651-659). Their major mode of action is to lyse tumor cells, which may induce antigen- specific T-cell responses to target metastatic diseases, even if the OVs are delivered locally. OV has been tested in both preclinical models and clinical trials, but complete clinical responses have only been rarely observed, highlighting the need for further improvement of OV therapy. Many OVs are associated with limited virus spread through tumors and suboptimal activation of anti-tumor T-cell responses (Heo et al., Nat Med.2013, 19:329-336; Breitbach et al., Nature.2011, 477:99-102; Kim et al., Mol Ther. 2006, 14:361-370; Hwang et al., Mol Ther.2011, 19:1913-1922; Lun et al., Mol Ther. 2010, 18:1927-1936; Heo et al., Mol Ther.2011, 19:1170-1179; Senzer et al., J Clin Oncol.2009, 27:5763-5771; Adair et al., Sci Transl Med.2012, 4:138ra77).

[0005] Increasing evidence has shown that T-cell immunotherapy has the ability to control tumor growth and prolong survival in cancer patients. However, tumor-specific T-cell responses are hard to achieve and sustain, likely due to the limitations of various immune escape mechanisms of tumor cells (Shafer-Weaver et al., Adv Exp Med Biol.2007, 601:357–368; Shafer-Weaver et al., J Immunol.2009, 183:4848–4852). Immune checkpoint molecules are proteins expressed on certain immune cells that need to be activated or inhibited to start an immune response, for example, to attack abnormal cells, such as tumor cells, in the body. The “immune escape” may include several activities by the tumor cells, such as down-regulation of co-stimulatory molecule expression, such as stimulatory immune checkpoint molecules, and up- regulation of inhibitory molecule expression, such as inhibitory immune checkpoint molecules. Blockade of these inhibitory immune checkpoint molecules have shown very promising results in preclinical and clinical tests in cancer treatment. However, there are some unwanted side effects in some cases. For example, blocking these inhibitory immune checkpoint molecules (receptors or ligands) may lead to a disruption in immune homeostasis and self- tolerance, resulting in autoimmune/autoinflammatory side effects (Corsello, S. et al., J Clin Endocrinol Metab (2013) 98(4):1361–7510). The inability for immune checkpoint modulators to actively accumulate at tumor sites may also lead to systemic adverse effects.

[0006] Bispecific engager molecules, such as those comprising a T-cell surface molecule- binding domain and a tumor antigen-binding domain, have provided a way to engage T cells to tumor cells and shown some clinical success, such as killing tumor cells in patients with non- Hodgkin's lymphomas and B-cell precursor acute lymphoblastic leukemia (Bargou et al., Science.2008, 321:974-977; Topp et al., J Clin Oncol.2011, 29:2493-2498; Nagorsen et al., Leuk Lymphoma.2009, 50:886-891). However, bispecific engager molecules, such as bispecific T-cell engagers, have short half-life, which require continuous infusion (Hammond et al., Cancer Res.2007, 67:3927-3935; Lutterbuese et al., Proc Natl Acad Sci USA.2010, 107:12605-12610; Friedrich et al., Mol Cancer Ther.2012, 11:2664–2673; Choi et al., Proc Natl Acad Sci USA.2013, 110:270–275). Moreover, the inability for bispecific T-cell engagers to actively accumulate at tumor sites lead to systemic adverse effects such as within the central nervous system. [0007] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0008] One aspect of the present application provides an oncolytic vaccinia virus (VV) comprising a nucleic acid encoding an immune checkpoint modulator, wherein the nucleic acid is operably linked to a late promoter. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, the late promoter is F17R.

[0009] In some embodiments according to any one of the oncolytic VV described above, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR). In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene.

[0010] In some embodiments according to any one of the oncolytic VV described above, the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule.

[0011] In some embodiments according to any one of the oncolytic VV described above, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the antibody specifically recognizing PD-1 comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising (1) a HVR- L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody specifically recognizing PD-1 comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule, such as PD- L1/PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin. In some embodiments, the Fc fragment is an IgG4 Fc. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator is a ligand that binds to at least two different inhibitory immune checkpoint molecules. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).

[0012] In some embodiments according to any one of the oncolytic VV described above, the oncolytic VV further comprises a second nucleic acid encoding a cytokine, such as GM-CSF.

[0013] Another aspect of the present application provides an oncolytic virus (OV) comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell. [0014] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the OV is selected from the group consisting of vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus, Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, and Newcastle disease virus (NDV). In some embodiments, the OV is an oncolytic VV. In some embodiments, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR). In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene.

[0015] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule.

[0016] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the antibody specifically recognizing PD-1 comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody specifically recognizing PD-1 comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule, such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin. In some embodiments, the Fc fragment is an IgG4 Fc. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).

[0017] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3 (GPC3). In some embodiments, the tumor antigen is EpCAM, FAP, EGFR, or GPC3.

[0018] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the effector cell is selected from the group consisting of T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, and NKT-cell. In some embodiments, the effector cell is a T lymphocyte, such as a cytotoxic T lymphocyte.

[0019] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D. In some embodiments, the cell surface molecule is CD3.

[0020] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the first and/or second antigen-binding domain is a single chain variable fragment (scFv). [0021] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the first antigen-binding domain and the second antigen binding domain are connected by a linker.

[0022] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the first antigen-binding domain is N- terminal to the second antigen-binding domain.

[0023] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the first antigen-binding domain is C- terminal to the second antigen-binding domain.

[0024] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the first nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter. In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the second nucleic acid encoding the bispecific molecule is operably linked to a late promoter. In some embodiments, the late promoter driving the expression of immune checkpoint modulator and/or bispecific molecule is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, the late promoter is F17R.

[0025] In some embodiments according to any one of the OV encoding an immune checkpoint modulator and a bispecific molecule described above, the oncolytic virus further comprises a third nucleic acid encoding a cytokine, such as GM-CSF.

[0026] Further provided is a pharmaceutical composition comprising any one of the oncolytic VV encoding an immune checkpoint modulator or oncolytic virus encoding an immune checkpoint modulator and a bispecific molecule described above, and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a second OV comprising a second nucleic acid encoding a bispecific engager molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific molecules described above), and a pharmaceutical acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a second OV comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described above), and a pharmaceutical acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), a second OV comprising a second nucleic acid encoding a bispecific engager molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific molecules described above), and a third OV comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described above), and a pharmaceutical acceptable carrier.

[0027] Another aspect of the present application provides a method of treating a cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition described above. In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of the bispecific molecules described above), and a second pharmaceutical acceptable carrier. In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described above), and a second pharmaceutical acceptable carrier. In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described above), and a first pharmaceutical acceptable carrier, an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell (such as any one of bispecific molecules described above), and a second pharmaceutical acceptable carrier, and an effective amount of a third pharmaceutical composition comprising a third OV comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described above), and a third pharmaceutical acceptable carrier.

[0028] In some embodiments, the effective amount is about 10 ⁵ to about 10 ¹³ pfu. In some embodiments, the effective amount is about 10 ⁹ pfu. In some embodiments, the pharmaceutical composition is administered systemically, such as intravenously. In some embodiments, the pharmaceutical composition is administered locally, such as intratumorally. In some embodiments, the cancer is a solid tumor, such as colorectal cancer, liver cancer, or breast cancer. In some embodiments, the method of treating cancer described above further comprises administering to the individual an additional cancer therapy, such as surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof. In some embodiments, the individual is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 depicts an exemplary schemes of expression cassette PD1-Ig-VV encoding PD1 (extracellular domain)-IgG4-Fc, expression cassette GPC3-TEA-VV encoding bispecific engager molecule GPC3-CD3 (GPC3-TE), expression cassette FAP-TEA-VV encoding bispecific engager molecule FAP-CD3 (FAP-TE), and expression cassette PD1-Ig-FAP-TEA-VV co- encoding bispecific engager molecule FAP-CD3 (FAP-TE) and PD1-IgG4-Fc, all under control of the late promoter F17R. The YFP-GFP selection marker was removed after confirming the expression of PD1-Ig, GPC3-CD3, FAP-CD3, or FAP-CD3 and PD1-Ig.

[0030] FIG. 2A depicts Western blot of PD1-Ig in the supernatant of human 143 TK- cells infected with VV-GFP or PD1-Ig-VV. FIG 2B depicts successful binding of PD1-Ig on Huh7 tumor cells. Huh7 cells were incubated with supernatant from VV infected 143 TK- cells and binding was assessed by FACS and APC-anti-Fc. An isotype control was used as a negative control.

[0031] FIGS.3A-3C depict the ability of PD1-Ig to enhance GPC3-CD3-dependent Huh7-GFP tumor cell lysis by T cells. FIG. 3A depicts the FACS plot of viable Huh7-GFP-cells. Percentages of CD3+/GFP+ double positive cells are indicated at effector: target ratio of 1:1 (top panels) and 5:1 (bottom panels). FIG. 3B depicts immunofluorescence analysis of viable Huh7- GFP cells. FIG. 3C depicts an increase in cell lysis in Huh7-GFP-cells co-infected with GPC3- CD3-VV and PD1-Ig-VV, as measured by FACS analysis using apoptotic markers Annexin-V and propidium iodide (PI).

[0032] FIG. 4A demonstrates that expression of PD1-Ig in Huh7 cells does not affect T cell phenotype in co-cultured PBMC cells. PBMC were cultured alone or with Huh7 cells infected with GPC3-CD3-VV or co-infected with GPC3-CD3-VV and PD1-Ig-VV. T cells were analyzed by FACS using the CD3 T cell marker and the CD69 T cell activation marker. Percentages of CD3+/CD69+ double positive cells are indicated. FIG. 4B depicts a negative control FACS analysis of T cells using CCR7 and CD45RA markers.

[0033] FIGS. 5A-5C depict the ability of PD1-Ig to augment GPC3-CD3-dependent increase in cytokine production by T cells. PBMC were co-cultured with Huh7 cells infected with GPC3- CD3-VV, co-infected with GPC3-CD3-VV and PD1-Ig-VV, or infected with GPC3-CD3-VV in combination with antibodies against PD-1 or PD-L1. Huh7 cells were infected with VV at a MOI of 1, PBMC were added and 48h post virus infection cell culture was collected for enzyme- linked immunosorbent assay (ELISA). ELISA results show an increase in the production of IFNȖ (FIG. 5A), TNFĮ (FIG. 5B), and IL-2 (FIG. 5C) cytokines by T cells co-cultured with PD1-Ig and GPC3-CD3 expressing Huh7 cells.

[0034] FIGS.6A-6C depict the ability of PD1-Ig to inhibit SK-BR3 tumor growth in vivo. FIG. 6A depicts the scheme whereby 4×10 ⁶ SK-BR3 cells were implanted into the right flank of NSG mice on day 0, followed by i.p. injection of 1×10 ⁸ PFUs of virus or PBS control on day 8 and i.v. implantation of 2×10 ⁷ PBMC on day 11. FIG. 6B depicts tumor volume (mm ³ ) in mice over the course of 21 days. FIG.6C depicts images of tumors in representative mice.

[0035] FIGS. 7A-7B depict the ability of PD1-Ig to inhibit HT-29 tumor growth in vivo. FIG. 7A depicts the scheme whereby 4×10 ⁶ HT-29 cells were implanted into the right flank of NSG mice on day 0, followed by i.p. injection of 1×10 ⁸ PFUs of virus or PBS control on day 8 and i.v. implantation of 2×10 ⁷ PBMC on day 11. FIG. 7B depicts tumor volume (mm ³ ) in mice over the course of 24 days.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention provides an oncolytic vaccinia virus encoding an immune checkpoint modulator (such as immune checkpoint inhibitor) under control of a late promoter, and an oncolytic virus (such as oncolytic VV) encoding an immune checkpoint modulator (such as immune checkpoint inhibitor) and a bispecific engager molecule (hereinafter also referred to as“bispecific molecule”,“engager molecule”, or“engager”) as new strategies to 1) facilitate T- cell activation at tumor sites, 2) effectively lyse tumor cells that are infected or not infected by the oncolytic virus (bystander killing), and/or 3) minimize systemic adverse effects to achieve greater antitumor activity, especially for solid tumors. The present invention is based in part on the finding that oncolytic virus (such as oncolytic VV), immune checkpoint modulator and/or bispecific molecule expressed at the tumor site can provide synergistic effect. Moreover, using a late promoter (such as the late vaccinia viral promoter F17R), which is only activated after viral infection in tumor cells, to drive the expression of immune checkpoint modulator and/or the bispecific molecule can avoid systemic toxicity and allow tumor-site restricted delivery.

[0037] Accordingly, one aspect of the present invention provides an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator, wherein the nucleic acid is operably linked to a late promoter (such as F17R).

[0038] Another aspect of the present invention provides an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as immune checkpoint inhibitor), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen- binding domain specifically recognizing a cell surface molecule on an effector cell.

[0039] Also provided are compositions (such as pharmaceutical compositions), and methods of treating cancer (such as solid cancer) using the oncolytic viruses (such as oncolytic VV).

[0040] Also provided herein are novel anti-PD-1 antibodies and methods of uses thereof.

I. Definitions

[0041] The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y.(2009); Ausubel et al, Short Protocols in Molecular Biology, 3 ^rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001 ); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

[0042] As used herein,“treatment” or“treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. Also encompassed by“treatment” is a reduction of pathological consequence of cancer. The methods of the invention contemplate any one or more of these aspects of treatment.

[0043] The term“prevent,” and similar words such as“prevented,”“preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the recurrence of, a disease or condition, e.g., cancer. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of the symptoms of a disease or condition. As used herein,“prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to recurrence of the disease or condition.

[0044] As used herein,“delaying” the development of cancer means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. A method that“delays” development of cancer is a method that reduces probability of disease development in a given time frame and/or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals. Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT Scan), Magnetic Resonance Imaging (MRI), abdominal ultrasound, clotting tests, arteriography, or biopsy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.

[0045] The term“effective amount” used herein refers to an amount of an agent or a combination of agents, sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0046] As used herein, an“individual” or a“subject” refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0047] The terms“bispecific T-cell engagers” or“BiTEs” are used herein interchangeably to refer to an antibody or fragment thereof that has polyepitopic specificity, with one specificity directed to a T-cell surface molecule.

[0048] The term“multispecific” as used in conjunction with an antibody refers to an antibody having polyepitopic specificity (i.e., is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules).

[0049] The term“bispecific” as used in conjunction with an antibody refers to an antibody capable of specifically binding to two different epitopes on one biological molecule, or capable of specifically binding to epitopes on two different biological molecules. Unless otherwise indicated, the order in which the antigens bound by a bispecific antibody listed is arbitrary. That is, for example, the terms “anti-CD3/EpCAM,” “anti-EpCAM/CD3,” “EpCAM×CD3,” “CD3×EpCAM,”“CD3-EpCAM,” and“EpCAM-CD3” may be used interchangeably to refer to bispecific antibodies that specifically bind to both CD3 and EpCAM.

[0050] As used herein, the term“immune checkpoint inhibitor” refers to a molecule that totally or partially reduces, inhibits or interferes with one or more inhibitory immune checkpoint molecules that may inhibit T-cell activation and function.

[0051] As used herein, the term“activator of a stimulatory immune checkpoint molecule” refers to a molecule that stimulates, activates, or increases the intensity of an immune response mediated by stimulatory immune checkpoint molecules.

[0052] An“isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0053] The term“vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0054] The term“transfected” or“transformed” or“transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or“transformed” or“transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0055] The terms“host cell,”“host cell line,” and“host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include“transformants” and“transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0056] “Adjuvant setting” refers to a clinical setting in which an individual has had a history of cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (e.g., surgery resection), radiotherapy, and chemotherapy. However, because of their history of cancer, these individuals are considered at risk of development of the disease. Treatment or administration in the“adjuvant setting” refers to a subsequent mode of treatment. The degree of risk (e.g., when an individual in the adjuvant setting is considered as“high risk” or “low risk”) depends upon several factors, most usually the extent of disease when first treated.

[0057] “Neoadjuvant setting” refers to a clinical setting in which the method is carried out before the primary/definitive therapy. [0058] It is understood that embodiments of the invention described herein include “consisting” and/or“consisting essentially of” embodiments.

[0059] Reference to“about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to“about X” includes description of“X”.

[0060] As used herein, reference to“not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0061] The term“about X-Y” used herein has the same meaning as“about X to about Y.”

[0062] As used herein and in the appended claims, the singular forms“a,”“or,” and“the” include plural referents unless the context clearly dictates otherwise.

II. Oncolytic viruses expressing immune checkpoint modulators

Oncolytic vaccinia virus expressing immune checkpoint modulator under a late promoter

[0063] The present invention provides an oncolytic vaccinia virus (VV) comprising a nucleic acid encoding an immune checkpoint modulator, wherein the nucleic acid is operably linked to a late promoter. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, the late promoter is F17R. In some embodiments, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR). In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of thymidine kinase (TK) gene and vaccinia virus growth factor (VGF) gene. In some embodiments, the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule (such as activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS). In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor (such as inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73). In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a ligand that binds to an immune checkpoint molecule, such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the oncolytic VV further comprises a second nucleic acid encoding a cytokine (such as GM-CSF).

[0064] Late oncolytic vaccinia virus promoters include but are not limited to F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, the late promoter is F17R.

[0065] Thus, in some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an immune checkpoint modulator, wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule (such as activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS). In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor (such as inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73). In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a ligand that binds to an immune checkpoint molecule, such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0066] The immune checkpoint modulator can be an activator of a stimulatory immune checkpoint molecule, including but are not limited to activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS.

[0067] Thus, in some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an activator of a stimulatory immune checkpoint molecule (such as activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS), wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an activator of a stimulatory immune checkpoint molecule (such as activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS), wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a ligand that binds to an immune checkpoint molecule. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0068] The immune checkpoint modulator can be an immune checkpoint inhibitor, include but are not limited to inhibitors of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73.

[0069] Thus, in some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an immune checkpoint inhibitor, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an immune checkpoint inhibitor, wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a ligand that binds to an immune checkpoint molecule (such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0070] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an antibody specifically recognizing an inhibitory immune checkpoint molecule, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an antibody specifically recognizing an inhibitory immune checkpoint molecule, wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the inhibitory immune checkpoint molecule is PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73. In some embodiments, the inhibitory immune checkpoint molecule is PD-1. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0071] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic vaccinia virus comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the inhibitor of PD-1 is an antibody specifically recognizing PD-1. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0072] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic vaccinia virus comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a F17R late promoter, wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0073] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1 comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a F17R late promoter, wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1 comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0074] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1 comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a F17R late promoter, wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1 comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0075] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1 comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a F17R late promoter, wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1 comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0076] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1 comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a F17R late promoter, wherein the inhibitor of PD-1 is an antibody specifically recognizing PD-1 comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0077] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding a ligand that binds to PD-L1 and/or PD-L2, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding a ligand that binds to PD-L1 and/or PD-L2, wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the ligand that binds to PD-L1 and/or PD-L2 comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0078] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0079] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a late promoter (such as F17R), and wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a F17R late promoter, and wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). [0080] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding a ligand that binds to HHLA2, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding a ligand that binds to HHLA2, wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the ligand that binds to HHLA2 comprises an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0081] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0082] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a late promoter (such as F17R), and wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a F17R late promoter, and wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0083] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding a ligand that binds to CD47 and CXCR4, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding a ligand that binds to CD47 and CXCR4, wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the ligand that binds to CD47 and CXCR4 comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0084] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a F17R late promoter. In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF).

[0085] In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a late promoter (such as F17R), and wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, there is provided an oncolytic VV comprising a nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), wherein the nucleic acid is operably linked to a F17R late promoter, and wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). Oncolytic virus expressing immune checkpoint modulator and bispecific engager molecule

[0086] The present invention also provides an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus, Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, and Newcastle disease virus (NDV). In some embodiments, the oncolytic virus is an oncolytic VV. In some embodiments, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR). In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic virus comprises double deletion of TK gene and VGF gene. In some embodiments, the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule (such as activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS). In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a ligand that binds to an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a ligand of PD-L1/PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3 (GPC3). In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is selected from the group consisting of T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, and NKT-cell. In some embodiments, the effector cell is a T lymphocyte (such as cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D. In some embodiments, the cell surface molecule is CD3. In some embodiments, the first and/or second antigen-binding domain is a single chain variable fragment (scFv). In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first antigen-binding domain is C-terminal to the second antigen-binding domain. In some embodiments, the first nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter. In some embodiments, the second nucleic acid encoding the bispecific molecule is operably linked to a late promoter. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, the late promoter is F17R. In some embodiments, the oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0087] The oncolytic virus expressing an immune checkpoint modulator and a bispecific engager molecule described herein can: 1) facilitate T-cell activation at tumor sites, 2) effectively lyse tumor cells that are infected or not infected by the oncolytic virus (bystander killing); 3) minimize systemic autoimmune/autoinflammatory side effects due to non-tumor-restricted blockade of immune checkpoint molecules, by delivering and sustaining immune checkpoint modulators selectively within the tumor; 4) minimize systemic adverse events by delivering and sustaining bispecific engager molecules selectively within the tumor; and/or 5) enhance tumor lytic activity mediated by bispecific engager molecule in the presence of T cells.

[0088] In some embodiments, there is provided an oncolytic VV comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the oncolytic VV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0089] The immune checkpoint modulator can be an activator of a stimulatory immune checkpoint molecule, include but are not limited to activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS.

[0090] Thus, in some embodiments, there is provided an OV comprising a first nucleic acid encoding an activator of a stimulatory immune checkpoint molecule (such as activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator an antibody specifically recognizing a stimulatory immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a ligand that binds to a stimulatory immune checkpoint molecule. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0091] The immune checkpoint modulator can be an immune checkpoint inhibitor, include but are not limited to inhibitors of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73.

[0092] Thus, in some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint inhibitor (such as inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0093] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an inhibitor of PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0094] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0095] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0096] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR- L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0097] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0098] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding a ligand that binds to PD-L1 and/or PD-L2, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the ligand that binds to PD-L1 and/or PD-L2 comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0099] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0100] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0101] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding a ligand that binds to HHLA2, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the ligand that binds to HHLA2 comprises an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0102] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding a ligand that binds to CD47 and CXCR4, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the ligand that binds to CD47 and CXCR4 comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is a T lymphocyte (such as a cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is CD3. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0103] Tumor antigens can be a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, TAA or TSA is expressed on a cell of a solid tumor. Tumor antigens include, but are not limited to, EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3.

[0104] Effector cells include, but are not limited to T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, NKT-cell, or the like. In some embodiments, the effector cell is a T lymphocyte. In some embodiments, the effector cell is a cytotoxic T lymphocyte.

[0105] Cell surface molecules on an effector cell include, but are not limited to CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, NKG2D, or the like. In some embodiments, the cell surface molecule is CD3.

[0106] Thus, in some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). [0107] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0108] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0109] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0110] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0111] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint inhibitor (such as inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint inhibitor is an antibody specifically recognizing an inhibitory immune checkpoint molecule. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to an inhibitory immune checkpoint molecule (such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0112] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint inhibitor (such as inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint inhibitor is an antibody specifically recognizing an inhibitory immune checkpoint molecule. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to an inhibitory immune checkpoint molecule (such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0113] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint inhibitor (such as inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint inhibitor is an antibody specifically recognizing an inhibitory immune checkpoint molecule. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to an inhibitory immune checkpoint molecule (such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0114] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint inhibitor (such as inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3 and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint inhibitor is an antibody specifically recognizing an inhibitory immune checkpoint molecule. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to an inhibitory immune checkpoint molecule (such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0115] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint inhibitor (such as inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint inhibitor is an antibody specifically recognizing an inhibitory immune checkpoint molecule. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to an inhibitory immune checkpoint molecule (such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0116] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an inhibitor of PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0117] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an inhibitor of PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0118] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an inhibitor of PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0119] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an inhibitor of PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3 and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). [0120] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an inhibitor of PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0121] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0122] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0123] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti- PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0124] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3 and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti- PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing GPC3 and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). [0125] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the anti- PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).

[0126] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0127] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0128] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti- PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0129] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3 and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti- PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing GPC3 and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0130] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the anti- PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).

[0131] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM, and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N- terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0132] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP, and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N- terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0133] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR, and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N- terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0134] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3, and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N- terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0135] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).

[0136] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM, and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). [0137] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP, and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0138] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR, and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0139] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3, and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0140] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N- terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0141] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (e.g., EpCAM, FAP, EGFR, or GPC3), and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). [0142] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (e.g., EpCAM, FAP, EGFR, or GPC3), and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing EGFR, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0143] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (e.g., EpCAM, FAP, EGFR, or GPC3), and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing GPC3, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second antigen- binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). [0144] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (e.g., EpCAM, FAP, EGFR, or GPC3), and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EpCAM, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing EGFR, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing GPC3, and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second antigen- binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes, wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, both first and second antigen-binding domains are scFvs. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).

[0145] The bispecific molecule described herein can be of any format. In some embodiments, the first antigen-binding domain is a scFv. In some embodiments, the second antigen-binding domain is a scFv. In some embodiments, both the first and second antigen-binding domains are scFvs. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first antigen-binding domain is C-terminal to the second antigen-binding domain. [0146] Thus, in some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the first scFv is N-terminal to the second scFv. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the first scFv and the second scFv are connected by a linker. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0147] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an inhibitor of PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an inhibitor of PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the first scFv is N- terminal to the second scFv. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, the first scFv and the second scFv are connected by a linker. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0148] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti- PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0149] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0150] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0151] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0152] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3 and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0153] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0154] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR- L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0155] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR- L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0156] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR- L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0157] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3 and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR- L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an antibody specifically recognizing PD-1, and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the anti-PD-1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0158] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the first scFv is N-terminal to the second scFv. In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0159] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM, and a second scFv specifically recognizing CD3 on T lymphocytes. In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N- terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0160] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP, and a second scFv specifically recognizing CD3 on T lymphocytes. In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0161] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR, and a second scFv specifically recognizing CD3 on T lymphocytes. In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0162] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3, and a second scFv specifically recognizing CD3 on T lymphocytes. In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3, and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0163] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (e.g., EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing CD3 on T lymphocytes. In some embodiments, the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM- CSF).

[0164] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (e.g., EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N- terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0165] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (e.g., EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing CD3 on T lymphocytes. In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N-terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0166] In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (e.g., EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EpCAM, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing FAP, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing EGFR, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing GPC3, and a second scFv specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, there is provided an oncolytic virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc), and a second nucleic acid encoding a bispecific molecule comprising a first scFv specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second scFv specifically recognizing CD3 on T lymphocytes, wherein the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29, and the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the OV is an oncolytic VV. In some embodiments, the OV is a WR strain VV. In some embodiments, the OV comprises double deletion of TK and VGF genes. In some embodiments, the first scFv is N- terminal to the second scFv. In some embodiments, the first and/or second nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).

[0167] The nucleic acids encoding the bispecific molecule, the immune checkpoint modulator, and/or the cytokine described herein can be operably linked to a promoter. In some embodiments, at least two of the nucleic acids encoding the bispecific molecule, the immune checkpoint modulator, and the cytokine are operably linked to the same promoter. In some embodiments, all of the nucleic acids encoding the bispecific molecule, the immune checkpoint modulator, and the cytokine are operably linked to the same promoter. In some embodiments, all of the nucleic acids encoding the bispecific molecule, the immune checkpoint modulator, and the cytokine are operably linked to different promoters. In some embodiments, the promoter is a late promoter. In some embodiments, the promoter is a vaccinia virus promoter. In some embodiments, the promoter is a late VV promoter. In some embodiments, the promoter is F17R.

Oncolytic virus

[0168] Oncolytic viruses are capable of selective replication in dividing cells (e.g. cancer cell) while leaving non dividing cells (e.g. normal cells) unharmed. As the infected dividing cells are destroyed by lysis, they release new infectious virus particles to infect the surrounding dividing cells. Cancer cells are ideal hosts for many viruses because they have the antiviral interferon pathway inactivated or have mutated tumor suppressor genes that enable viral replication to proceed unhindered (Chernajovsky et al., 2006, British Med. J.332: 170-2).

[0169] Exemplary oncolytic virus include without limitation vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus (AdV), herpes simplex virus (HSV, such as HSV1 and HSV2), reovirus, myxoma virus (MYXV), poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, Newcastle disease virus (NDV), or the like (see, e.g. , Kirn et al., Nat. Med. 7:781 (2001); Coffey et al., Science 282:1332 (1998); Lorence et al., Cancer Res. 54:6017 (1994); and Peng et al., Blood 98:2002 (2001)). In some embodiments, the oncolytic virus described herein is an oncolytic vaccinia virus (VV). [0170] Oncolytic viruses may utilize DNA or RNA as their genetic material. Oncolytic DNA viruses may have capsid symmetry that is icosahedral or complex. Icosahedral oncolytic DNA viruses may be naked or comprise an envelope. Families of oncolytic DNA viruses include the Adenoviridae (for example, Adenovirus, having a genome size of 36-38kb), Herpesviridae (for example, HSV1, having a genome size of 120-200 kb) and Poxviridae (for example, Vaccinia virus and myxoma virus, having a genome size of 130-280 kb). Oncolytic RNA viruses include those having icosahedral or helical capsid symmetry. Icosahedral oncolytic viruses are naked without envelope and include Reoviridae (for example, Reovirus, having a genome of 22-27 kb) and Picornaviridae (for example, Poliovirus, having a genome size of 7.2-8.4 kb). Helical oncolytic RNA viruses are enveloped and include Rhabdoviridae (for example, VSV, having genome size of 13-16 kb) and Paramyxoviridae (for example MV and NDV, having genome sizes of 16-20 kb).

[0171] In some embodiments, the oncolytic virus is a vaccinia virus (VV). In some embodiments, the VV can be Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, or Western Reserve (WR) strain. In some embodiments, the VV is a WR strain.

[0172] In some embodiments, the oncolytic virus (such as oncolytic VV) of the present invention is modified by altering for one or more viral gene(s). Said modification(s) preferably lead(s) to the synthesis of a defective protein unable to ensure the activity of the protein produced under normal conditions by the unmodified gene (or lack of synthesis). Modifications encompass deletion, mutation and/or substitution of one or more nucleotide(s) (contiguous or not) within the viral gene or its regulatory elements. Modification(s) can be made in a number of ways known to those skilled in the art using conventional recombinant techniques. Exemplary modifications are disclosed in the literature with a specific preference for those altering viral genes involved in DNA metabolism, host virulence, IFN pathway (see e.g. Guse et al., 2011, Expert Opinion Biol. Ther.11(5): 595-608) and the like.

[0173] In some embodiments, the oncolytic virus (such as oncolytic VV) comprises an inactivating mutation in a thymidine kinase (TK) gene to produce a negative TK phenotype. In some embodiments, the TK gene of the VV is deleted. The TK enzyme is involved in the synthesis of deoxyribonucleotides. TK is needed for viral replication in normal cells as these cells have generally low concentration of nucleotides whereas it is dispensable in dividing cells which contain high nucleotide concentration. Therefore TK deletion limits virus replication significantly in resting cells allowing efficient virus replication to occur only in actively dividing cells (e.g. cancer cells).

[0174] Alternatively or in combination, other strategies may also be pursued to further increase the virus tumor-specificity. A representative example of suitable modification includes disruption of the VGF-encoding gene from the viral genome. VGF (for VV growth factor) is a secreted protein which is expressed early after cell infection and its function seems important for virus spread in normal cells. Replication of VGF deleted vaccinia viruses is highly attenuated in resting (non-cancer) cells. In some embodiments, the VV of the present invention does not express functional vaccinia growth factor (VGF). In some embodiments, the oncolytic virus of the present invention is a vaccinia virus defective for both TK and VGF activities. The effects of TK and VGF deletions have been shown to be synergistic.

[0175] Thus, in some embodiments, there is provided an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic VV comprises double deletion of TK gene and VGF gene.

[0176] In some embodiments, there is provided an oncolytic virus (such as oncolytic VV) comprising a nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), further comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). In some embodiments, the oncolytic virus is an oncolytic VV. In some embodiments, the oncolytic virus is a WR strain. In some embodiments, the oncolytic virus comprises double deletion of TK gene and VGF gene. [0177] It is envisaged that the present invention also relates to an oncolytic viral vector comprising the nucleic acid molecule described in the present disclosure. As used herein, the term“viral vector” is used according to its art-recognized meaning. It refers to a nucleic acid vector construct that includes at least one element of viral origin and can be packaged into a viral vector particle. The viral vector particles can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Oncolytic viral vectors include, but are not limited to, vaccinia virus (VV) vectors, Seneca Valley virus (SVV) vectors, adenovirus (AdV) vectors, herpes simplex virus vectors (e.g. HSV1 vector), reovirus vectors, myxoma virus (MYXV) vectors, poliovirus vectors, vesicular stomatitis virus (VSV) vectors, measles virus (MV) vectors, lentiviral vectors, retrovirus vectors, morbillivirus vectors, influenza virus vectors, Sinbis virus vectors, and Newcastle disease virus (NDV) vectors.

[0178] In some embodiments, the oncolytic viral vector is an oncolytic VV vector. The present invention therefor relates to an oncolytic VV vector comprising a nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), wherein the nucleic acid is operably linked to a late promoter (such as F17R). There is also provided an oncolytic virus vector (such as oncolytic VV vector) comprising a nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), further comprising a nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes).

[0179] The present application also provides an immune checkpoint modulator (such as any of the immune checkpoint modulators described herein). These immune checkpoint modulators can be incorporated into oncolytic viruses, such as oncolytic VV, or can be provided in isolated forms. Immune checkpoint modulators

[0180] Immune checkpoints are molecules in the immune system that either turn up (stimulatory molecules) or turn down a signal (inhibitory molecules). Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Stimulatory checkpoint molecules include, but are not limited to, CD27, CD40, OX40, GITR and CD137, which belong to tumor necrosis factor (TNF) receptor superfamily, as well as CD28 and ICOS, which belong to the B7-CD28 superfamily. Inhibitory checkpoint molecules include, but are not limited to, program death 1 (PD-1), Cytotoxic T- Lymphocyte-Associated protein 4 (CTLA-4), Lymphocyte Activation Gene-3 (LAG-3), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3, HAVCR2), V-domain Ig suppressor of T cell activation (VISTA, B7-H5), B7-H3, B7-H4 (VTCN1), HHLA2 (B7-H7), B and T Lymphocyte Attenuator (BTLA), Indoleamine 2,3-dioxygenase (IDO), Killer-cell Immunoglobulin-like Receptor (KIR), adenosine A2A receptor (A2AR), T cell immunoreceptor with Ig and ITIM domains (TIGIT), 2B4 (CD244) and ligands thereof. Numerous checkpoint proteins have been studied extensively, such as CTLA-4 and its ligands CD80 (B7-1) and CD86, and PD-1 with its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) (See, for example, Pardoll, Nature Reviews Cancer 12: 252-264 (2012)).

[0181] Immune checkpoint modulators can be immune checkpoint inhibitors (inhibitors of inhibitory immune checkpoint molecules) or activators of stimulatory immune checkpoint molecules. Immune checkpoint inhibitors (inhibitors of inhibitory immune checkpoint molecules) are of particular interest in the present invention, such as inhibitors of PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7-DC, CD273), LAG-3, TIM-3 (HAVCR2), BTLA, CTLA-4, TIGIT, VISTA (B7-H5), B7-H4 (VTCN1), CD160 (BY55), HHLA2 (B7-H7), CXCR4, 2B4 (CD244), CD73, B7-1 (CD80), B7-H3 (CD276), KIR, or IDO.

[0182] In some embodiments, the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule. In some embodiments, the activator of a stimulatory immune checkpoint molecule is a natural or engineered ligand of a stimulatory immune checkpoint molecule, including, for example, ligands of OX40 (e.g., OX40L), ligands of CD28 (e.g., CD80, CD86), ligands of ICOS (e.g., B7RP1), ligands of 4-1BB (e.g., 4-1BBL, Ultra 4- 1BBL), ligands of CD27 (e.g., CD70), ligands of CD40 (e.g., CD40L), and ligands of TCR (e.g., MHC class I or class II molecules, IMCgp100). In some embodiments, the activator of a stimulatory immune checkpoint molecule is a secreted protein. In some embodiments, the activator of a stimulatory immune checkpoint molecule is an antibody (such as an agonist antibody), such as anti-CD28, anti-OX40, anti-ICOS, anti-GITR, anti-4-1BB, anti-CD27, anti- CD40, anti-CD3, and anti-HVEM.

[0183] In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets T cells. In some embodiments, the immune checkpoint inhibitor targets tumor cells. For example, in some cases, tumor cells can turn off activated T cells, when they attach to specific T-cell receptors. However, immune checkpoint inhibitors may prevent tumor cells from attaching to T cells so that T cells stay activated (see, for example, Howard West, JAMA Oncol. 1(1):115 (2015)). In some embodiments, the immune checkpoint inhibitor is a natural or engineered ligand of an inhibitory immune checkpoint molecule, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM-3 (e.g., Galectin-9), ligands of A2A Receptor (e.g., adenosine, Regadenoson), ligands of LAG-3 (e.g., MHC class I or MHC class II molecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g., MHC class I or MHC class II molecules), ligands of PD-1 (e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, Indoximod, NLG919), ligands of HHLA2 (e.g., TMIGD2), ligands of CXCR4 (e.g., CXCL12), and ligands of CD47 (e.g., SIRP-Į receptor). In some embodiments, the immune checkpoint inhibitor is secreted. In some embodiments, the immune checkpoint inhibitor is an antibody (such as antagonist antibody) that targets an inhibitory immune checkpoint protein, including but not limited to, anti-CTLA-4, anti- TIM-3, anti-LAG-3, anti-KIR, anti-PD-1, anti-PD-L1, anti-CD73, anti-B7-H3, anti-CD47, anti- BTLA, anti-VISTA, anti-A2AR, anti-B7-1, anti-B7-H4, anti-CD52, anti-IL-10, anti-IL-35, and anti-TGF-ȕ. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an inhibitory checkpoint molecule selected from the group consisting of PD-1, PD-L1, LAG-3, TIM-3, HHLA2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, and VISTA. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing PD-1. In some embodiments, the immune checkpoint modulator is a ligand that binds to an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator inhibitor is a ligand that binds to PD-L1 and/or PD-L2. In some embodiments, the immune checkpoint modulator is an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin. In some embodiments, the Fc fragment is an IgG4 Fc. In some embodiments, the immune checkpoint modulator is a ligand that binds to HHLA2. In some embodiments, the immune checkpoint modulator is an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator is a ligand that binds to at least two different inhibitory immune checkpoint molecules (e.g. bispecific), such as a ligand that binds to both CD47 and CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc.

PD-1

[0184] PD-1 is a part of the B7/CD28 family of co-stimulatory molecules that regulate T-cell activation and tolerance, and thus antagonistic anti-PD-1 antibodies can be useful for overcoming tolerance. PD-1 has been defined as a receptor for B7-4. B7-4 can inhibit immune cell activation upon binding to an inhibitory receptor on an immune cell. Engagement of the PD- 1/PD-L1 pathway results in inhibition of T-cell effector function, cytokine secretion and proliferation. (Turnis et al., OncoImmunology 1(7):1172-1174, 2012). High levels of PD-1 are associated with exhausted or chronically stimulated T cells. Moreover, increased PD-1 expression correlates with reduced survival in cancer patients. Agents for down modulating PD- 1, B7-4, and the interaction between B7-4 and PD-1 inhibitory signal in an immune cell can result in enhancement of the immune response.

[0185] PD-L1 (Programmed cell death-ligand 1) is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1 serves as a ligand for PD-1 to play a major role in suppressing the immune system during particular events such as pregnancy, tissue allographs, autoimmune disease and other disease states such as hepatitis and cancer. The formation of PD-1 receptor/PD-L1 ligand complex transmits an inhibitory signal which reduces the proliferation of CD8+ T cells at the lymph nodes.

[0186] PD-L2 (Programmed cell death 1 ligand 2) is also known as B7-DC. PD-L2 serves as a ligand for PD-1. Under certain circumstances, PD-L2 and its inhibitor can be used as a substitute for PD-L1 and its inhibitor respectively. TMIGD2

[0187] HHLA2 is widely expressed in many human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, esophagus and hematological malignancies of leukemia and lymphoma. HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and is an attractive target for human cancer therapy. TMIGD2 has been identified as a receptor for HHLA2. Blocking of HHLA2/TMIGD2 could be an effective strategy for cancer immunotherapy.

CD47

[0188] CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPĮ). Interference with the CD47-SIRPĮ interaction could enhance anti-tumor immune responses.

CXCR4

[0189] The chemokine CXCL12 and its receptor CXCR4 are expressed widely in human cancers, including ovarian cancer, in which they are associated with disease progression at the levels of tumor cell proliferation, invasion, and angiogenesis. CXCL12 produced by tumor tissue and surrounding stroma stimulates VEGF-mediated angiogenesis and the recruitment of endothelial progenitor cells from the bone marrow. CXCL12 has also been shown to recruit suppressive CD11b+Gr1+ myeloid cells and pDCs at tumor sites, and induce intratumoral T regulatory cells (Tregs) localization, which impede immune mechanisms of tumor destruction. Therefore, modulation of the CXCL12/CXCR4 axis could impact multiple aspects of tumor pathogenesis including immune dysregulation. Several CXCR4 antagonists have demonstrated antitumor efficacy in preclinical models and have been evaluated in early clinical trials.

[0190] In some embodiments, the oncolytic virus (such as oncolytic VV) of the present may comprise a nucleic acid encoding any antibodies or antigen-binding fragments of an immune checkpoint molecule described herein. For example, the oncolytic virus (such as oncolytic VV) of the present may comprise a nucleic acid encoding a scFv version of the anti-PD-1 antibody described above. In some embodiments, the oncolytic virus (such as oncolytic VV) of the present may comprise a nucleic acid encoding a fusion protein comprising any antibody fragment or any other functional variants or derivatives of a full-length antibody described herein. For example, the oncolytic virus (such as oncolytic VV) of the present may comprise a nucleic acid encoding antigen binding domains of the anti-PD-1 antibody described above fused to an IgG4 fragment.

[0191] The immune checkpoint modulators contemplated herein are proteins or peptides. In some embodiments, the immune checkpoint modulator comprises a single polypeptide chain. In some embodiments, the immune checkpoint modulator comprises more than one (such as any of 2, 3, 4, or more) polypeptide chains. The polypeptide chain(s) of the immune checkpoint modulator may be of any length, such as at least about any of 10, 20, 50, 100, 200, 300, 500, or more amino acids long. In the cases of multi-chain immune checkpoint modulators, the nucleic acid sequences encoding the polypeptide chains may be operably linked to the same promoter or to different promoters.

[0192] In some embodiments, the immune checkpoint modulator is a secreted protein. In some embodiments, the immune checkpoint modulator is an antibody. Native antibodies, such as monoclonal antibodies, are immunoglobulin molecules that are immunologically reactive with a particular antigen. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is an antagonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of V _H , V _L , V _NAR , V _H H, Fab, Fab’, F(ab’) ₂ , Fv, minibody, scFv, sc(Fv) ₂ , tribody, tetrabody, scFv- scFv (such as BiTE ^® ), minibody, scFv-Fc, triabody, and other antigen-binding subsequences of the full length antibody or engineered combinations thereof. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a multivalent antibody, such as a divalent antibody or a tetravalent antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a single domain antibody (sdAb). In some embodiments, the antibody is a heavy chain-only antibody, such as a camelid antibody or a derivative thereof. In some embodiments, the antibody is a single-chain antibody. In some embodiments, the antibody is a scFv. In some embodiments, the antibody is a fusion protein comprising an antibody fragment (such as an Fc-containing fusion protein, e.g. PD-1 extracellular domain-Fc fusion protein) or any other functional variants or derivatives of a full-length antibody. [0193] In some embodiments, the immune checkpoint modulator is an antibody comprising a heavy chain and a light chain. In some embodiments, the heavy chain comprises a V _H domain. In some embodiments, the heavy chain further comprises one or more constant domains, such as C _H 1, C _H 2, C _H 3, or any combination thereof. In some embodiments, the light chain comprises a V _L domain. In some embodiments, the light chain further comprises a constant domain, such as C _L . In some embodiments, the heavy chain and the light chain are connected to each other via a plurality of disulfide bonds. In some embodiments, the antibody comprises an Fc, such as an Fc fragment of the human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody does not comprise an Fc fragment. In some embodiments, the immune checkpoint modulator is a Fab. In some embodiments, the immune checkpoint modulator is a full length anti-PD-1 antibody.

[0194] The oncolytic virus (such as oncolytic VV) may express any number (such as any of 1, 2, 3, 4, 5, 6, or more) of immune checkpoint modulators. In some embodiments, the oncolytic virus comprises a nucleic acid encoding a single immune checkpoint modulator. In some embodiments, the oncolytic virus comprises one or more nucleic acids encoding at least two immune checkpoint modulators. In some embodiments, the nucleic acids encoding the at least two immune checkpoint modulators are operably linked to the same promoter. In some embodiments, the nucleic acids encoding the at least two immune checkpoint modulators are operably linked to different promoters. In some embodiments, the nucleic acids encoding the immune checkpoint modulator(s) and the bispecific molecule of the present invention are operably linked to the same promoter. In some embodiments, the nucleic acids encoding the immune checkpoint modulator(s) and the bispecific molecule of the present invention are operably linked to different promoters.

[0195] The heavy chain polypeptide and the light chain polypeptide of multi-chain immunomodulatory antibodies are co-expressed in the oncolytic virus (such as oncolytic VV) of the present invention, either by a single nucleic acid, or by two nucleic acids. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed at equimolar ratio. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed at a ratio of about any of 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 3:2, 4:3, 5:4, 1:1, 4:5, 3:4, 2:3, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed at a ratio of any of about 1:10 to about 1:5, about 1:5 to about 1:3, about 1:4 to about 1:2, about 1:2 to about 1:1, about 1:1 to about 2:1, about 2:1 to about 4:1, about 3:1 to about 5:1, about 5:1 to about 10:1, about 1:2 to about 2:1, about 1:3 to about 3:1, about 1:5 to about 5:1, or about 1:10 to about 10:1. The optimal expression ratio between the heavy chain polypeptide and the light chain polypeptide may facilitate the antibody folding and assembly process. See, for example, Schlatter S et al., Biotechnol Prog.21(1): 122-33 (2005).

[0196] The various expression ratio between the heavy chain polypeptide and the light chain polypeptide of a multi-chain immunomodulatory antibody may be achieved by manipulating the copy numbers of the heterologous nucleic acids and/or the nucleic acids encoding the heavy chain and the light chain, and/or the induction sequence and/or the strength of the promoters linked to the nucleic acids encoding the heavy chain and the light chain. In some embodiments, the nucleic acid encoding the heavy chain and the nucleic acid encoding the light chain are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the heavy chain and the nucleic acid encoding the light chain are operably linked to different promoters. In some embodiments, the promoter for the nucleic acid encoding the heavy chain and the promoter for the nucleic acid encoding the light chain can be simultaneously induced. In some embodiments, the promoter for the nucleic acid encoding the heavy chain and the promoter for the nucleic acid encoding the light chain can be sequentially induced. In some embodiments, the promoter for the nucleic acid encoding the heavy chain is induced prior to the induction of the promoter for the nucleic acid encoding the light chain. In some embodiments, the promoter for the nucleic acid encoding the heavy chain is induced after the induction of the promoter for the nucleic acid encoding the light chain. In some embodiments, the promoter for the nucleic acid encoding the heavy chain and the promoter for the nucleic acid encoding the light chain have a strength ratio of about any of 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 3:2, 4:3, 5:4, 1:1, 4:5, 3:4, 2:3, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, or 1:10. In some embodiments, the promoter for the nucleic acid encoding the heavy chain and the promoter for the nucleic acid encoding the light chain have a strength ratio of any of about 1:10 to about 1:5, about 1:5 to about 1:3, about 1:4 to about 1:2, about 1:2 to about 1:1, about 1:1 to about 2:1, about 2:1 to about 4:1, about 3:1 to about 5:1, about 5:1 to about 10:1, about 1:2 to about 2:1, about 1:3 to about 3:1, about 1:5 to about 5:1, or about 1:10 to about 10:1.

[0197] In some embodiments, the immune checkpoint modulator is a fusion protein comprising an antibody fragment or any other functional variants or derivatives of a full-length antibody. In some embodiments, the immune checkpoint modulator is an Fc-containing fusion protein. In some embodiments, the immune checkpoint modulator comprises antigen binding domains (such as fragments comprising the CDRs) of an antibody described herein fused to an Fc fragment. For example, the oncolytic virus of the present invention can comprise a nucleic acid encoding the antigen-binding domain of an anti-PD-1 antibody fused to an IgG4 fragment. In some embodiments, the immune checkpoint modulator is an extracellular domain of a ligand of the inhibitory immune checkpoint molecule described herein (such as the extracellular domain of PD-1) fused to Fc fragment. In some embodiments, the Fc fragment can be an Fc fragment of the human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc fragment is an IgG4 Fc.

[0198] In some embodiments, the immune checkpoint modulator is PD-1 extracellular domain- Fc fusion protein. In some embodiments, the Fc fragment is an IgG4 Fc. In some embodiments, the PD-1 extracellular domain comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the PD-1 extracellular domain is encoded by a nucleic acid sequence comprising SEQ ID NO: 26.

[0199] In some embodiments, the immune checkpoint modulator is TMIGD2 extracellular domain-Fc fusion protein. In some embodiments, the Fc fragment is an IgG4 Fc. In some embodiments, the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the TMIGD2 extracellular domain-Fc fusion protein further comprises a signal peptide comprising amino acid sequence of SEQ ID NO: 28.

[0200] In some embodiments, the immune checkpoint modulator specifically binds two different immune checkpoint molecules, such as CD47 and CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin. In some embodiments, the Fc fragment is an IgG4 Fc. In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker comprising amino acid sequence of SEQ ID NO: 31. In some embodiments, the SIRPĮ-CXCL12-Fc fusion protein further comprises a signal peptide comprising amino acid sequence of SEQ ID NO: 32. In some embodiments, the SIRPĮ-CXCL12-Fc fusion protein has the configuration of (N’ to C’) signal peptide-SIRPĮ extracellular domain-linker-CXCL12-Fc fragment.

Cytokines

[0201] The term“cytokine” or“cytokines” as used herein refers to the general class of biological molecules, which affect cells of the immune system. The definition is meant to include, but is not limited to, those biological molecules that act locally or may circulate in the blood, and which, when used in the present invention serve to regulate or modulate an individual’s immune response to cancer. Exemplary cytokines for use in practicing the invention include, but are not limited to, interferons (such as IFN-Į, IFN-ȕ, IFN-Ȗ), all interleukins (e.g., IL-1 to IL-29, in particular, IL-1, IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, IL-23, IL-24, and IL-27), tumor necrosis factors (e.g., TNF-Į and TNF-ȕ), erythropoietin (EPO), MIP3a, ICAM, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells and fibroblasts that functions as a cytokine. GM-CSF induces activation, proliferation, and differentiation of a variety of immunologically active cell populations, thereby facilitating the development of both humoral and cellular-mediated immunity (Warren and Weiner, 2000). In some embodiments, the cytokine is GM-CSF.

[0202] In some embodiments, there is provided an oncolytic vaccinia virus comprising a first nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), wherein the first nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter (such as F17R), further comprising a second nucleic acid encoding a cytokine (such as GM- CSF). In some embodiments, the nucleic acid encoding the immune checkpoint modulator and the cytokine are operably linked to the same late promoter (such as F17R). In some embodiments, the nucleic acids encoding the immune checkpoint modulator and the cytokine are operably linked to different promoters. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor (such as an antibody that specifically recognizing an immune checkpoint molecule). In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule (such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4). In some embodiments, the immune checkpoint modulator is a PD-1 extracellular domain-Fc fusion protein. In some embodiments, the Fc fragment is an IgG4 Fc. In some embodiments, the PD-1 extracellular domain comprises amino acid sequence of SEQ ID NO: 25. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31.

[0203] In some embodiments, there is provided an oncolytic vaccinia virus comprising a first nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the first nucleic acid encoding the anti-PD-1 antibody or antigen-binding fragment thereof is operably linked to a late promoter (such as F17R), further comprising a second nucleic acid encoding GM-CSF.

[0204] In some embodiments, there is provided an oncolytic vaccinia virus comprising a first nucleic acid encoding a PD-1 extracellular domain-Fc fusion protein, wherein the first nucleic acid encoding the PD-1 extracellular domain-Fc fusion protein is operably linked to a late promoter (such as F17R), further comprising a second nucleic acid encoding GM-CSF. In some embodiments, the PD-1 extracellular domain comprises amino acid sequence of SEQ ID NO: 25.

[0205] In some embodiments, there is provided an oncolytic vaccinia virus comprising a first nucleic acid encoding a TMIGD2 extracellular domain-Fc fusion protein, wherein the first nucleic acid encoding the TMIGD2 extracellular domain-Fc fusion protein is operably linked to a late promoter (such as F17R), further comprising a second nucleic acid encoding GM-CSF. In some embodiments, the TMIGD2 extracellular domain comprises amino acid sequence of SEQ ID NO: 27.

[0206] In some embodiments, there is provided an oncolytic vaccinia virus comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein, wherein the first nucleic acid encoding the extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein is operably linked to a late promoter (such as F17R), further comprising a second nucleic acid encoding GM-CSF. In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31.

[0207] In some embodiments, there is provided an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), further comprising a third heterologous nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, at least two of the nucleic acids encoding the immune checkpoint modulator, the bispecific engager molecule, and the cytokine are operably linked to the same promoter. In some embodiments, all of the nucleic acids encoding the immune checkpoint modulator, the bispecific engager molecule, and the cytokine are operably linked to the same promoter. In some embodiments, all of the nucleic acids encoding the immune checkpoint modulator, the bispecific engager molecule, and the cytokine are operably linked to different promoters. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor (such as an antibody that specifically recognizing an immune checkpoint molecule). In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule (such as PD-L1/PD-L2, HHLA-2, CD47, or CXCR4). In some embodiments, the immune checkpoint modulator is a PD-1 extracellular domain-Fc fusion protein. In some embodiments, the PD-1 extracellular domain comprises amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fc fragment is an IgG4 Fc. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the TMIGD2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31.

[0208] In some embodiments, there is provided an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof, and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), further comprising a third nucleic acid encoding GM-CSF.

[0209] In some embodiments, there is provided an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding a PD-1 extracellular domain-Fc fusion protein, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), further comprising a third nucleic acid encoding GM-CSF. In some embodiments, the PD-1 extracellular domain comprises amino acid sequence of SEQ ID NO: 25. [0210] In some embodiments, there is provided an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding a TMIGD2 extracellular domain-Fc fusion protein, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), further comprising a third nucleic acid encoding GM-CSF. In some embodiments, the TMIGD2 extracellular domain comprises amino acid sequence of SEQ ID NO: 27.

[0211] In some embodiments, there is provided an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), further comprising a third nucleic acid encoding GM-CSF. In some embodiments, the SIRPĮ extracellular domain comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPĮ extracellular domain by a linker, such as IgG1 hinge, or a linker comprising amino acid sequence of SEQ ID NO: 31.

Regulatory sequence

[0212] It is evident to the person skilled in the art that regulatory sequences may be added to the OV nucleic acid molecules comprised in the disclosure. Such regulatory sequences (control elements) are known to the artisan and may include a promoter, additional elements for proper initiation, regulation and/or termination of transcription (e.g. polyA transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), stability (e.g. introns and non-coding 5’ and 3’ sequences), translation (e.g. an initiator Met, tripartite leader sequences, IRES ribosome binding sites, signal peptides, etc.), and insertion site for introducing an insert into the viral vector. In some embodiments, the regulatory sequences are promoters, transcriptional enhancers and/or sequences that allow for proper expression of the bispecific molecule, the immune checkpoint modulator, and the cytokine of the disclosure may be employed.

[0213] The term“regulatory sequence” refers to DNA sequences that are necessary to affect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoters, ribosomal binding sites, and terminators. In eukaryotes generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term“control sequence” is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components.

[0214] The term“operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence“operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. In case the control sequence is a promoter, it is obvious for a skilled person that double-stranded nucleic acid is preferably used.

[0215] As used herein, a“promoter”, a promoter region or a promoter element or regulatory region or regulatory element refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked. The promoter region includes specific sequences that are involved in RNA polymerase recognition, binding and transcription initiation. In addition, the promoter includes sequences that modulate recognition, binding and transcription initiation activity of RNA polymerase (i.e., binding of one or more transcription factors). These sequences can be cis acting or can be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, can be constitutive or regulated. Regulated promoters can be inducible or environmentally responsive (e.g. respond to cues such as pH, anaerobic conditions, osmoticum, temperature, light, or cell density). Many such promoter sequences are known in the art. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,928; 5,759,828; 5,888,783; 5,919,670, and, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). [0216] It is envisaged that the present invention also relates to an oncolytic viral vector (such as oncolytic VV vector) comprising the nucleic acid molecules described in the present disclosure. As used herein, the term“viral vector” is used according to its art-recognized meaning. It refers to a nucleic acid vector construct that includes at least one element of viral origin and can be packaged into a viral vector particle. The viral vector particles can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. In some embodiment, the oncolytic viral vector is a VV vector. The VV may be the Wyeth or Western Reserve (WR) strain. The VV may have a deletion in its genome or a mutation in one or more genes. The thymidine kinase gene of the vaccinia virus may have been deleted. The vaccinia virus may have a mutation in a gene encoding vaccinia virus growth factor. In some embodiments, the oncolytic viral vector is a lentiviral vector. Lentiviral vectors are commercially available, including from Clontech (Mountain View, Calif.) or GeneCopoeia (Rockville, Md.), for example.

[0217] “Expression vector” is a construct that can be used to transform a selected host and provides for expression of a coding sequence in the selected host. Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors. Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells they comprise normally promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Examples of regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.

[0218] Beside elements that are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the recited nucleic acid sequence and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous nucleic acid sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pEF-DHFR and pEF-ADA, (Raum et al., Cancer Immunol Immunother (2001) 50(3), 141-150) or pSPORT1 (GIBCO BRL).

[0219] It will be appreciated by those skilled in the art that the choice of the regulatory sequences can depend on such factors as the nucleic acid molecule itself, the virus into which it is inserted, the host cell or subject, the level of expression desired, etc. The promoter is of special importance. In the context of the invention, it can be constitutive directing expression of the nucleic acid molecule in many types of host cells or specific to certain host cells (e.g. tumor- specific regulatory sequences) or regulated in response to specific events or exogenous factors (e.g. by temperature, nutrient additive, hormone, etc.) or according to the phase of a viral cycle (e.g. late or early). One may also use promoters that are repressed during the production step in response to specific events or exogenous factors, in order to optimize virus production and circumvent potential toxicity of the expressed polypeptide(s).

[0220] In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming of transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and as desired, the collection and purification of the polypeptide of the disclosure may follow.

[0221] Promoters can be native or heterologous (with respect to the oncolytic virus described herein). Any suitable promoters, including synthetic and naturally-occurring and modified promoters, can be used. As used herein, a native promoter is a promoter that is endogenous to the virus and is unmodified with respect to its nucleotide sequence and its position in the viral genome as compared to a wild-type virus. A synthetic promoter is a heterologous promoter that has a nucleotide sequence that is not found in nature. A synthetic promoter can be a nucleic acid molecule that has a synthetic sequence or a sequence derived from a native promoter or portion thereof. A synthetic promoter can also be a hybrid promoter composed of different elements derived from different native promoters. See, e.g. US9005602 for exemplary vaccinia virus synthetic promoters.

[0222] Viral promoters can include, but are not limited to, VV promoter, poxvirus promoter, adenovirus late promoter, Cowpox ATI promoter, or T7 promoter. The promoter may be a vaccinia virus promoter, a synthetic promoter, a promoter that directs transcription during at least the early phase of infection, a promoter that directs transcription during at least the intermediate phase of infection, a promoter that directs transcription during early/late phase of infection, or a promoter that directs transcription during at least the late phase of infection.

[0223] Promoters may be roughly categorized as constitutive promoters or regulated promoters, such as inducible promoters.

[0224] Promoters suitable for constitutive expression in mammalian cells include but are not limited to the cytomegalovirus (CMV) immediate early promoter (US 5,168,062), the RSV promoter, the adenovirus major late promoter, the phosphoglycerate kinase (PGK) promoter (Adra et al., 1987, Gene 60: 65-74), the thymidine kinase (TK) promoter of herpes simplex virus (HSV)-l and the T7 polymerase promoter (WO98/10088). Vaccinia virus promoters are particularly adapted for expression in oncolytic poxviruses. Representative examples include without limitation the vaccinia 7.5K, H5R, 11K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15(1): 18-28), TK, p28, pll, pB2R, pA35R and K1L promoters, as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23: 1094-7; Hammond et al, 1997, J. Virol Methods 66: 135-8; and Kumar and Boyle, 1990, Virology 179: 151-8) as well as early/late chimeric promoters. Promoters suitable for oncolytic measles viruses include without limitation any promoter directing expression of measles transcription units (Brandler and Tangy, 2008, CIMID 31: 271).

[0225] Inducible promoters belong to the category of regulated promoters. The inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the host cell, or the physiological state of the host cell, an inducer (i.e., an inducing agent), or a combination thereof.

[0226] Appropriate promoters for expression can be tested in vitro (e.g. in a suitable cultured cell line) or in vivo (e.g. in a suitable animal model or in the subject). When the encoded immune checkpoint modulator(s) comprise(s) an antibody and especially a mAb, examples of suitable promoters for expressing the heavy component of said immune checkpoint modulator comprise CMV, SV and vaccinia virus pH5R, F17R and pllK7.5 promoters; examples of suitable promoters for expressing the light component of said immune checkpoint modulator comprise PGK, beta-actin and vaccinia virus p7.5K, F17R and pA35R promoters.

[0227] Promoters can be replaced by stronger or weaker promoters, where replacement results in a change in the attenuation of the virus. As used herein, replacement of a promoter with a stronger promoter refers to removing a promoter from a genome and replacing it with a promoter that effects an increased the level of transcription initiation relative to the promoter that is replaced. Typically, a stronger promoter has an improved ability to bind polymerase complexes relative to the promoter that is replaced. As a result, an open reading frame that is operably linked to the stronger promoter has a higher level of gene expression. Similarly, replacement of a promoter with a weaker promoter refers to removing a promoter from a genome and replacing it with a promoter that decreases the level of transcription initiation relative to the promoter that is replaced. Typically, a weaker promoter has a lessened ability to bind polymerase complexes relative to the promoter that is replaced. As a result, an open reading frame that is operably linked to the weaker promoter has a lower level of gene expression. The viruses can exhibit differences in characteristics, such as attenuation, as a result of using a stronger promoter versus a weaker promoter. For example, in vaccinia virus, synthetic early/late and late promoters are relatively strong promoters, whereas vaccinia synthetic early, P7.5k early/late, P7.5k early, and P28 late promoters are relatively weaker promoters (see e.g., Chakrabarti et al. (1997) BioTechniques 23 (6) 1094-1097).

[0228] In some embodiments, the promoter is a vaccinia virus promoter. Exemplary vaccinia viral promoters for use in the present invention can include, but are not limited to, P _7.5k , P _11k , P _SE , P _SEL , P _SL , H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b or K1 promoters.

[0229] In some embodiments, the promoter is a vaccinia virus native promoter. As used herein, a native promoter is a promoter that is endogenous to the virus and is unmodified with respect to its nucleotide sequence and its position in the viral genome as compared to a wild-type virus. In some embodiments, the promoter is a vaccinia virus synthetic promoter (see, e.g. US9005602). A synthetic promoter is a heterologous promoter that has a nucleotide sequence that is not found in nature. A synthetic promoter can be a nucleic acid molecule that has a synthetic sequence or a sequence derived from a native promoter or portion thereof. A synthetic promoter can also be a hybrid promoter composed of different elements derived from different native promoters.

[0230] Exemplary vaccinia early, intermediate and late stage promoters include, for example, vaccinia P _7.5k early/late promoter, vaccinia P _EL early/late promoter, vaccinia P ₁₃ early promoter, vaccinia P _11k late promoter and vaccinia promoters listed elsewhere herein. Exemplary synthetic promoters include, for example, P _SE synthetic early promoter, P _SEL synthetic early/late promoter, P _SL synthetic late promoter, vaccinia synthetic promoters listed elsewhere herein (Patel et al., Proc. Natl. Acad. Sci. USA 85: 9431-9435 (1988); Davison and Moss, J Mol Biol 210: 749- 769 (1989); Davison et al., Nucleic Acids Res.18: 4285-4286 (1990); Chakrabarti et al., BioTechniques 23: 1094-1097 (1997)). Combinations of different promoters can be used to express different gene products in the same virus or two different viruses.

[0231] In some embodiments, the promoter directs transcription during at least the late phase of infection (such as F17R promoter). In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter.

[0232] In some embodiments, the promoter directs transcription during at least the late phase of infection (such as F17R promoter) is employed. The late vaccinia viral promoter F17R is only activated after VV infection in tumor cells, thus tumor selective expression of transgene from VV will be further enhanced by the use of F17R promoter. The late expression of the immune checkpoint modulator, the bispecific engager molecule, and/or cytokine (such as GM-CSF) of the present invention will also allow for sufficient viral replication before T-cell activation and mediated tumor lysis.

[0233] Thus in some embodiments of the present invention, there is provided an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), wherein the nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter. In some embodiments, the late promoter is F17R. In some embodiments, the oncolytic vaccinia virus expressing the immune checkpoint modulator further comprises a second nucleic acid encoding a cytokine. In some embodiments, the nucleic acid encoding the cytokine is also operably linked to a promoter. In some embodiments, the nucleic acid encoding the cytokine is linked to a late promoter. In some embodiments, the nucleic acid encoding the cytokine is linked to F17R. In some embodiments, the cytokine is GM-CSF.

[0234] In some embodiments, there is provided an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), wherein the nucleic acid encoding the immune checkpoint modulator and/or the nucleic acid encoding the bispecific molecule is operably linked to a promoter. In some embodiments, the promoter is an oncolytic vaccinia virus promoter. In some embodiments, the promoter is a late promoter. In some embodiments, the promoter is F17R promoter. In some embodiments, the nucleic acid encoding the immune checkpoint modulator and the nucleic acid encoding the bispecific molecule are operably linked under one promoter. In some embodiments, the nucleic acid encoding the immune checkpoint modulator and the nucleic acid encoding the bispecific molecule are operably linked to two same promoters. In some embodiments, the nucleic acid encoding the immune checkpoint modulator and the nucleic acid encoding the bispecific molecule are operably linked to different promoters. In some embodiments, the nucleic acid encoding the immune checkpoint modulator and the nucleic acid encoding the bispecific molecule are directed to transcribe during the same or similar phase of viral infection. In some embodiments, the nucleic acid encoding the immune checkpoint modulator and the nucleic acid encoding the bispecific molecule are directed to transcribe during different phases of viral infection. In some embodiments, the nucleic acid encoding the immune checkpoint modulator and the nucleic acid encoding the bispecific molecule are operably linked to promoters of same or similar strength. In some embodiments, the nucleic acid encoding the immune checkpoint modulator and the nucleic acid encoding the bispecific molecule are operably linked to promoters of different strength. In some embodiments, the nucleic acid encoding the bispecific molecule is operably linked to a stronger promoter than that operably linked to the nucleic acid encoding the immune checkpoint modulator. In some embodiments, the nucleic acid encoding the bispecific molecule is operably linked to a weaker promoter than that operably linked to the nucleic acid encoding the immune checkpoint modulator.

[0235] Additional regulatory elements may include transcriptional as well as translational enhancers. Advantageously, the above-described oncolytic viral vectors of the disclosure comprise a selectable and/or scorable marker. Selectable marker genes useful for the selection of transformed cells are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life-Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485). Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2- (difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).

[0236] Useful scorable markers are also known to those skilled in the art and are commercially available. Advantageously, said marker is a gene encoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47), YFP, or ȕ-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). Scorable markers are particularly useful for simple and rapid screening of cells, tissues and organisms containing a recited vector. [0237] When appropriate, it may be advantageous to include additional regulatory elements to facilitate expression, trafficking and biological activity of at least one of gene inserted into the viral genome of the oncolytic virus of the invention (i.e. the bispecific engager molecule, the immune checkpoint modulator, and/or the cytokine). For example, a signal peptide (or leader sequence) may be included for facilitating secretion from the infected cell. The signal peptide is typically inserted at the N-terminus of the protein immediately after the Met initiator. The choice of signal peptides is wide and is accessible to persons skilled in the art. One may also envisage addition of a trans-membrane domain to facilitate anchorage of the encoded protein(s) in a suitable membrane (e.g. the plasmic membrane) of the infected cells. The trans-membrane domain is typically inserted at the C-terminus of the protein just before or at close proximity of the STOP codon. A vast variety of trans-membrane domains are available in the art (see e.g. WO99/03885).

[0238] In some embodiments, the immune checkpoint modulator described herein further comprises a signal peptide fused at N-terminal of the immune checkpoint modulator. In some embodiments, the immune checkpoint modulator comprises a signal peptide fused at N-terminal of the VH domain, such as SEQ ID NO: 21. In some embodiments, the immune checkpoint modulator comprises a signal peptide fused at N-terminal of the VL domain, such as SEQ ID NO: 23. In some embodiments, the signal peptide can be encoded by nucleic acids of SEQ ID NO: 22 or SEQ ID NO: 24. In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 32.

[0239] As an additional example, a peptide tag (typically a short peptide sequence able to be recognized by available antisera or compounds) may be also be added for following expression, trafficking or purification of the encoded gene product. A vast variety of tag peptides can be used in the context of the invention including, without limitation, PK tag, FLAG octapeptide, MYC tag, HIS tag (usually a stretch of 4 to 10 histidine residues) and e-tag (US 6,686,152). The tag peptide(s) may be independently positioned at the N-terminus of the protein or alternatively at its C-terminus or alternatively internally or at any of these positions when several tags are employed. Tag peptides can be detected by immunodetection assays using anti-tag antibodies. As another example, the glycosylation can be altered so as to increase biological activity of the encoded gene product (e.g. to increase). Such modifications can be accomplished, for example, by mutating one or more residues within the site(s) of glycosylation. Altered glycosylation patterns may increase the ADCC ability of antibodies and/or their affinity for their target.

[0240] The described nucleic acid molecule or vector that is introduced in the host may either integrate into the genome of the host or it may be maintained extrachromosomally.

[0241] The host can be any eukaryotic cell or prokaryotic cell. It is particularly envisaged that the recited host may be a mammalian cell. Host cells include, but are not limited to, CV-1, BS-C- 1, HuTK-143B, BHK-21, CEF, CHO cells, COS cells, myeloma cell lines like SP2/0 or NS/0 cells.

Bispecific engager molecules

[0242] In some embodiments, the oncolytic virus (such as oncolytic VV) expressing the immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein) of the present invention further comprises a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes).

[0243] The present invention discloses an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), further comprising a second nucleic acid encoding an engager molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing one or more tumor antigens (such as EpCAM, FAP EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte). In some embodiments, the engager molecule is a bispecific molecule. In some embodiments, the cell surface molecule is a T lymphocyte surface marker (such as CD3), thus the engager molecule is a T-cell engager (TE). In some embodiments, the present invention discloses an oncolytic virus (such as oncolytic VV) expressing an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), further expressing a bispecific T-cell engager (BiTE).

[0244] Once the engager molecule's second antigen-binding domain has bound to effector cells, the second antigen-binding domain can activate the effector cells. In some embodiments, when the second antigen-binding domain of the engager molecule binds to the cell surface molecule on an immune cell, and the first antigen-binding domain binds to the tumor cell antigen, the immune cell kills the tumor cell.

Tumor antigen

[0245] In some embodiments, the antigen specifically recognized by the first antigen-binding domain of the engager molecule is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiment, the TAA or TSA is expressed on a cancer cell. In some embodiments, the TAA or TSA is expressed on a blood cancer cell. In some embodiments, the TAA or TSA is expressed on a cell of a solid tumor. Certain forms of solid tumor cancer include, by way of non-limiting example, a glioblastoma, a non-small cell lung cancer, a lung cancer other than a non-small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, liver cancer, colorectal cancer, stomach cancer, a cancer of the spleen, skin cancer (such as melanoma), a brain cancer other than a glioblastoma, a kidney cancer, a thyroid cancer, head and neck tumors, bladder cancer, esophageal cancer, or the like. In some embodiments, the TAA or TSA is one or more of, e.g., an scFv on the engager is specific for one or more of EphA2, HER2, GD2, GPC3, 5T4, 8H9, Į _v ȕ ₆ integrin, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, fetal AchR, Folate Receptor a, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, TEM8, VEGFR2, carcinoembryonic antigen, HMW-MAA, VEGF receptors, and other exemplary antigens are antigens that are present with in the extracellular matrix of tumors, such as oncofetal variants of fibronectin, tenascin, or necrotic regions of tumors.

[0246] In some embodiments, the antigen specifically recognized by the first antigen-binding domain of the engager molecule is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and GPC3. In some embodiments, the tumor antigen is EpCAM, FAP, EGFR, or GPC3.

[0247] In some embodiments, the antigen specifically recognized by the first antigen-binding domain of the engager molecule is Epithelial cell adhesion molecule (EpCAM, CD326), also known as 17-1A, ESA, AUA1, EGP40, etc., which is a 40 kDa transmembrane glycoprotein composed of 314 amino acid. EpCAM is specifically expressed in various types of epithelial cells, and major types of human malignancies. For example, EpCAM is highly expressed in colon cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, breast cancer and ovarian cancer.

[0248] In some embodiments, the antigen specifically recognized by the first antigen-binding domain of the engager molecule is fibroblast activation protein (FAP). Fibroblasts are connective tissue cells which secrete an extracellular matrix rich in collagen and other macromolecules. Fibroblasts in the tumor-stroma (i.e., tumor supporting tissue) synthesize FAP, a type II transmembrane protein that functions as a serine protease. FAP is selectively overexpressed in over 90% of stromal fibroblasts associated with colon, breast and lung carcinomas. FAP is also expressed in some tumor cells, such as human malignant gliomas cell line U87 and murine lewis lung cancer cell line LL2 (Kraman et al., Science 330: 827-830 (2010)). Overexpression of FAP reportedly leads to promotion of tumor growth and increases in metastatic potential, whereas treatment with anti-FAP antibodies inhibits tumor growth.

[0249] In some embodiments, the antigen specifically recognized by the first antigen-binding domain of the engager molecule is Epidermal growth factor receptor (EGFR). EGFR is a member of the ErbB family of closely related receptors including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). Activation of EGFR leads to receptor tyrosine kinase activation and a series of downstream signaling events that mediate cellular proliferation, motility, adhesion, invasion, and resistance to chemotherapy as well as inhibition of apoptosis, processes that are crucial to the continual proliferation and survival of cancer cells. Expression of the EGFR is associated with poor prognosis in a number of tumor types including stomach, colon, urinary bladder, breast, prostate, endometrium, kidney and brain (e.g., glioma).

[0250] EphA2 is referred to as EPH receptor A2 (ephrin type-A receptor 2; EPHA2; ARCC2; CTPA; CTPP1; or ECK), which is a protein that in humans is encoded by the EPHA2 gene in the ephrin receptor subfamily of the protein-tyro sine kinase family. Receptors in this subfamily generally comprise a single kinase domain and an extracellular region comprising a Cys-rich domain and 2 fibronectin type III repeats; embodiments of the antibodies of the disclosure may target any of these domains. The ephrin receptors are divided into two groups as a result of the similarity of their respective extracellular domain sequences and also their affinities for binding ephrin-A and ephrin-B ligands, and EphA2 encodes a protein that binds ephrin-A ligands. An exemplary human EphA2 nucleic sequence is in GenBank® Accession No. NM_004431, and an exemplary human EphA2 polypeptide sequence is in GenBank® Accession No. NP_004422, both of which sequences are incorporated herein in their entirety.

[0251] HER2 is referred to as human Epidermal Growth Factor Receptor 2 (Neu, ErbB-2, CD340, or pi 85), which is a protein that in humans is encoded by the ERBB2 gene in the epidermal growth factor receptor (EFR/ErbB) family. HER2 contains an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with a multitude of signaling molecules.

[0252] GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans. GD2 is present and concentrated on cell surfaces, with the two hydrocarbon chains of the ceramide moiety embedded in the plasma membrane and the oligosaccharides located on the extracellular surface, where they present points of recognition for extracellular molecules or surfaces of neighboring cells.

[0253] In some embodiments, the antigen specifically recognized by the first antigen-binding domain of the engager molecule is Glypican-3 (GPC3). Glypican-3 (GPC3) is an oncofetal antigen re-expressed in a high frequency of neoplastic hepatocytes. The GPC3 gene encodes a 70-kDa precursor core protein, which can be cleaved by furin to generate a 40-kDa amino (N) terminal protein and a 30-kDa membrane-bound carboxyl (C) terminal protein. The C-terminus is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. [0254] Vascular endothelial growth factor receptor 2 (VEGFR2, KDR3) is a VEGF receptor, which is one of the most potent and specific positive regulators of angiogenesis. VEGFR2 is highly expressed in tumor associated endothelial cells and contributes to tumor growth, invasion and metastasis (Dias et al., J Clin Invest.106(4):511-521, 2000; Santos et al., Blood 103(10):3883-3889, 2004; St. Croix et al., Science 289:1197-1202, 2000). In addition, VEGFR2 is also expressed on the surface of several tumor cells including: B cell lymphoma and leukemia, multiple myeloma, urothelial bladder cancer, breast cancer, and lung cancer, among others (El-Obeid et al., Leuk Res.28(2): 133-137, 2004; Kumar et al., Leukemia 17(10):2025- 2031, 2003; Gakiopoulou-Givalou et al., Histopathology 43(3):272-279, 2003; Kranz et al., Int J Cancer 84(3):293-298, 1999; Decaussin et al., J Pathol.188(4):369-377, 1999). The relatively high level of expression on tumor cells relative to normal vascular endothelial cells suggests that VEGFR2 is a suitable target of tumor therapy.

[0255] Thus, in some embodiments, the bispecific molecule comprises a first antigen-binding domain (such as scFv) specifically recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte).

[0256] In some embodiments, the bispecific molecule comprises a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte).

[0257] In some embodiments, the bispecific molecule comprises a first antigen-binding domain (such as scFv) specifically recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte).

[0258] In some embodiments, the bispecific molecule comprises a first antigen-binding domain (such as scFv) specifically recognizing GPC3 and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on a T lymphocyte). Effector cell surface molecules

[0259] Exemplary effector cell includes without limitation a T lymphocyte, a B lymphocyte, a natural killer (NK) cell, a dendritic cell (DC), a macrophage, a monocyte, a neutrophil, an NKT- cell, or the like. In some embodiments, the effector cell is a T lymphocyte. In some embodiments, the effector cell is a cytotoxic T lymphocyte. In some embodiments, the effector cell is allogenic. In some embodiments, the effector cell is autologous.

[0260] A cell surface molecule on an effector cell of the present invention is a molecule found on the external cell wall or plasma membrane of a specific cell type or a limited number of cell types. Examples of cell surface molecules include, but are not limited to, membrane proteins such as receptors, transporters, ion channels, proton pumps, and G protein-coupled receptors; extracellular matrix molecules such as adhesion molecules (e.g., integrins, cadherins, selectins, or NCAMS); see, e.g., U.S. Pat. No. 7,556,928, which is incorporated herein by reference in its entirety. Cell surface molecules on an effector cell include but not limited to CD3, CD4, CD5, CD8, CD16, CD27, CD28, CD40, CD64, CD89, CD134, CD137, CD278, NKp46, NKp30, NKG2D, and an invariant TCR.

[0261] The cell surface molecule-binding domain of an engager molecule can provide activation to immune cells. The skilled artisan recognizes that immune cells have different cell surface molecules. For example CD3 is a cell surface molecule on T-cells, whereas CD16, NKG2D, or NKp30 are cell surface molecules on NK cells, and CD3 or an invariant TCR are the cell surface molecules on NKT-cells. Engager molecules that activate T-cells may therefore have a different cell surface molecule-binding domain than engager molecules that activate NK cells. In some embodiments, e.g., wherein the immune cell is a T-cell, the activation molecule is one or more of CD3, e.g., CD3Ȗ, CD3į or CD3İ; or CD27, CD28, CD40, CD134, CD137, and CD278. In other some embodiments, e.g., wherein the immune cell is a NK cell, the cell surface molecule is CD16, NKG2D, or NKp30, or wherein the immune cell is a NKT-cell, the cell surface molecule is CD3 or an invariant TCR.

[0262] CD3 comprises three different polypeptide chains (İ, į and Ȗ chains), is an antigen expressed by T cells. The three CD3 polypeptide chains associate with the T-cell receptor (TCR) and the ȗ-chain to form the TCR complex, which has the function of activating signaling cascades in T cells. Currently, many therapeutic strategies target the TCR signal transduction to treat diseases using anti-human CD3 monoclonal antibodies. The CD3 specific antibody OKT3 is the first monoclonal antibody approved for human therapeutic use, and is clinically used as an immunomodulator for the treatment of allogenic transplant rejections.

[0263] In some embodiments, the second antigen-binding domain specifically binds to CD3 on a T lymphocyte. In some embodiments, the VH and VL regions of the human CD3 specific domain are derived from an CD3 specific antibody such as X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, WT31, F101.01, or Blinatumomab (Blincyto®, CD19-CD3 bispecific antibody). These CD3-specific antibodies are well known in the art and, inter alia, described in Tunnacliffe et al., Int Immunol.1(5):546- 50 (1989). In some embodiments, VH and VL regions are derived from antibodies/antibody derivatives and the like that are capable of specifically recognizing the human CD3-İ chain or human CD3-ȗ chain. In some embodiments, the VH and VL regions of the human CD3 specific domain are derived from Blinatumomab (Blincyto®, CD19-CD3 bispecific antibody). In some embodiments, the second antigen-binding domain specifically binds to an epitope within the human CD3-İ chain or human CD3-ȗ chain.

[0264] The skilled artisan will recognize that the TCR complex is an octomeric complex of variable TCR Į and ȕ chains with three dimeric signaling modules CD3į/İ, CD3Ȗ/İ and CD3ȗ/ȗ or ȗ/^. Although in some cases the engager molecule described herein targets CD3İ with a scFv, targeting other CD3 molecules, especially CD3ȗ, or the TCR Į and ȕ chains, with a specific scFv is also encompassed in the disclosure. In some embodiments, targeting molecules that are not part of the TCR complex (for example, CD27, CD28, CD40, CD134, CD137, and CD278) is encompassed in the disclosure.

[0265] Thus, in some embodiments, the bispecific molecule comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on a T lymphocyte.

[0266] In some embodiments, the bispecific molecule comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing CD16 on an NK cell.

[0267] In some embodiments, the bispecific molecule comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing NKG2D on an NK cell.

[0268] In some embodiments, the bispecific molecule comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing NKp30 on an NK cell.

Engager molecule format

[0269] The engager molecule described herein can be of any format known in the art. Such engager molecules generally comprise a tumor antigen-binding domain and an effector cell surface molecule-binding domain. The engager molecule's antigen-binding domain may be designed so as to bind to one or more tumor antigens present on target cells, while engager molecule's effector cell surface molecule-binding domain may be designed so as to bind to one or more cell surface molecules present on effector cells, for example, T lymphocytes. Once the engager molecule's effector cell surface molecule-binding domain has bound effector cells, the effector cell surface molecule-binding domain can activate the effector cells. In some embodiments, when the effector cell surface molecule-binding domain of the engager binds to the cell surface molecule on the effector cell, and the tumor antigen-binding domain binds to the tumor antigen on tumor cells, the effector cell kills the tumor cells.

[0270] In some embodiments, the engager molecule is bipartite (e.g., comprising one tumor antigen-binding domain and one effector cell surface molecule-binding domain that may optionally be joined by a linker), or may be tripartite or multipartite (e.g., comprising one or more tumor antigen-binding domains and/or one or more effector cell surface molecule-binding domains, or other domains, including one or more co-stimulatory domains and/or one or more dimerization, trimerization or multimerization domains). [0271] In some embodiments, the engager is bispecific (e.g., comprising one tumor antigen- binding domain and one effector cell surface molecule-binding domain that may optionally be joined by a linker, wherein the tumor antigen and cell surface molecule are different). Engagers may also be multispecific (e.g., comprising one tumor antigen-binding domain and two effector cell surface molecule-binding domains that may optionally be joined by a linker, wherein the tumor antigen and two cell surface molecules are all different).

[0272] The engager molecule can be in any format known in the art (see, e.g., Weidle et al., Cancer Genomics Proteomics, 10(1):1-18, 2013; Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015; Stamova et al., Antibodies, 1(2):172-198, 2012). The engager may be a format class of “IgG-derived molecules” comprising Fc regions. For example, the engager molecule can be in the format of, but are not limited to, Common LC (light chain), DAF (dual acting Fab), which comprises evolved Fvs with dual specificity), CrossMab, IgG-dsscFv2 (disulfide-stabilized scFv2), DVD (dual variable domain), IgG-dsFv (disulfide-stabilized Fv), processed IgG-dsFv, IgG-scFab (single chain Fab), scFab-dsscFv, or Fv2-Fc. Knobs-into-holes technologies can be used for heterodimerization of different H-chains in, for example, common LC, CrossMab, IgG-dsF, IgG-scFab or Fv2-Fc. The engager may also be an“Fc-less bispecific” format class, which usually comprises individual scFvs of Fabs of different specificities fused together via linkers. For example, the engager can be in the format of, but are not limited to, Fab- scFv2, Fab-scFv, scFv-scFv (such as BiTE ^® ), diabody, scBsDb (single-chain bispecific diabody), DART (dual-affinity retargeting molecule), TandAb (tetravalent tandem antibody), scBsTaFv (single-chain bispecific tandem variable domain), DNL-F(ab) ₃ (dock-and-lock trivalent Fab), scFv-HSA-scFv (scFv-human serum albumin-scFv), or bssdAb (bispecific single-domain antibody). Bi- or trivalent Fab-Fv or Fab-Fv2 formats are generated by fusion of VH-CH1 and/or L chains to scFvs. scFv-scFv molecules (such as BiTE ^® ) are generated by fusions of scFvs of different specificities. The linker peptide length can be modulated so that VH and VL correctly pair, such as in Diabodies, DARTs, and TandAbs. These molecules can be further stabilized by interchain disulfide bonds (e.g. in DART, or between VH and VL of antibodies comprising scFvs). The engager molecule may also be a format class of an antibody mimetics, which are small engineered proteins comprising antigen-binding domains reminiscent of antibodies (Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and comprise a single polypeptide. Exemplary engagers in the format class of antibody mimetics can be a Designed ankyrin repeat protein (DARPin; comprising 3-5 fully synthetic ankyrin repeats flanked by N- and C-terminal Cap domains), an avidity multimer (avimer; a high-affinity protein comprising multiple A domains, each domain with low affinity for a target), or an Anticalin (based on the scaffold of lipocalins, with four accessible loops, the sequence of each can be randomized). The engager molecule to be employed in accordance with the disclosure can be chemically modified derivative of any of the aforementioned engager formats, or it may comprise ligands, peptides, or combinations thereof. The engager molecule to be employed in accordance with the disclosure can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) (e.g. posttranslational and chemical modifications, such as glycosylation and phosphorylation) known in the art either alone or in combination. Chemical/biochemical or molecular biological methods for such modifications are known in the art and described inter alia in laboratory manuals (see Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition 1989 and 3rd edition 2001; Gerhardt et al.; Methods for General and Molecular Bacteriology; ASM Press, 1994; Lefkovits; Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press, 1997; Golemis; Protein-Protein Interactions: A Molecular Cloning Manual; Cold Spring Harbor Laboratory Press, 2002).

[0273] In some embodiments, the bispecific engager molecule of the present invention is a bispecific single chain Fv (scFv). A scFv in general contains a VH and VL domain connected by a linker peptide. The secretable engager is composed of a signal peptide (to allow for secretion) from cells, followed by 2 scFvs connected by linker peptides (Lx, Ly, Lz). Linkers may be of a length and sequence sufficient to ensure that each of the first and second domains can, independently from one another, retain their differential binding specificities. Bispecific single chain molecules are known in the art and are described in WO 99/54440; Mack, J. Immunol. (1997), 158, 3965-3970; Mack, PNAS, (1995), 92, 7021-7025; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197; Loffler, Blood, (2000), 95, 6, 2098-2103; and Bruhl, J. Immunol., (2001), 166, 2420-2426.

[0274] In some embodiments, an exemplary molecular format of the disclosure provides an oncolytic virus (such as oncolytic VV) comprising a nucleic acid encoding a polypeptide comprising a signal peptide followed by two scFvs, wherein the first scFv specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and the second scFv specifically recognizes a cell surface molecule on an effector cell (such as CD3 on T lymphocytes). Each scFv comprises one V _H and one V _L region. Bispecific scFv may be tandem bi-scFv or diabody. Bispecific scFvs can be arranged in different formats:

Thus, bispecific scFvs with the above

possible arrangements are particular embodiments of the bispecific single chain engager molecule. Linkers Lx, Ly, and Lz can be the same or different.

[0275] The linkers can be peptide linkers of any length. In some embodiments, the peptide linker between VH and VL of an antigen-binding domain (such as scFv) is from 1 amino acids to 20 amino acids long, from 2 amino acids to 19 amino acids long, from 3 amino acids to 18 amino acids long, from 4 amino acids to 17 amino acids long, from 5 amino acids to 17 amino acids long, from 6 amino acids to 17 amino acids long, from 7 amino acids to 18 amino acids long, from 8 amino acids to 17 amino acids long, from 9 amino acids to 17 amino acids long, from 10 amino acids to 17 amino acids long, from 11 amino acids to 16 amino acids long, from 12 amino acids to 17 amino acids long, from 13 amino acids to 16 amino acids long, from 14 amino acids to 16 amino acids long, or from 14 amino acids to 15 amino acids long. In some embodiments, the peptide linker between VH and VL of an antigen-binding domain (such as scFv) is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In some embodiments, the peptide linker between VH and VL of an antigen-binding domain (such as scFv) is 14 or 15 amino acids long. In some embodiments, the peptide linker between the first and second antigen-binding domains (such as scFv) is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In some embodiments, the peptide linker between the first and second antigen-binding domains (such as scFv) is 5 amino acids long.

[0276] An essential technical feature of such peptide linker is that said peptide linker does not comprise any polymerization activity. The characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in Dall’Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21- 30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). A particularly preferred amino acid in context of the“peptide linker” is Gly. Furthermore, peptide linkers that also do not promote any secondary structures are preferred. The linkage of the domains to each other can be provided by, e.g., genetic engineering. Methods for preparing fused and operatively linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well-known in the art (e.g. WO 99/54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y. 1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001).

[0277] The peptide linker can be a stable linker, which is not cleavable by protease, especially by Matrix metalloproteinases (MMPs).

[0278] The linker can also be a flexible linker. Exemplary flexible linkers include glycine polymers (G) _n , glycine-serine polymers (including, for example, (GS) _n , (GSGGS) _n and (GGGS) _n , where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). The ordinarily skilled artisan will recognize that design of a bispecific antibody molecule can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired bispecific antibody molecule structure.

[0279] In some embodiments, the VH and VL domains of the first antigen-binding domain (such as scFv) are linked together by a linker of sufficient length to enable the domains to fold in such a way as to permit binding to the tumor antigen (such as EpCAM, FAP, EGFR, or GPC3). Further to this embodiment, such a linker may comprise, for example, the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 33), or GGGGSGGGGSGGSA (SEQ ID NO: 34). In some embodiments, the VH domain and VL domains of the second antigen-binding domain (such as scFv) are linked together by a linker of sufficient length to enable the domains to fold in such a way as to permit binding to cell surface molecule on an effector cell, such as CD3 on T lymphocytes. Further to this embodiment, such a linker may comprise, for example, the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 33). In some embodiments, the first and second antigen-binding domains (such as scFvs) are linked together by a linker of sufficient length to enable the domains to fold in such a way as to permit binding both to the tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and to the cell surface molecule on effector cells (such as CD3 on T lymphocytes). Further to this embodiment, such a linker may comprise, for example, the amino acid sequence of GGGGS (SEQ ID NO: 35).

[0280] In some embodiments, the engager molecule additionally comprises one or more other domains, e.g., one or more of a cytokine, a costimulatory domain, a domain that inhibits negative regulatory molecules of T-cell activation, or a combination thereof. In some embodiments, the cytokine is IL-15, IL-2, and/or IL-7. In some embodiments, the co-stimulatory domain is CD27, CD80, CD83, CD86, CD134, or CD137. In some embodiments, the domain that inhibits negative regulatory molecules of T-cell activation is PD- 1, PD-L1, CTLA4, or B7-H4.

[0281] Thus, in some embodiments, the engager molecule described herein comprises a first antigen-binding domain (such as scFv) recognizing EpCAM and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the first antigen-binding domain is an scFv. In some embodiments, the second antigen-binding domain is an scFv. In some embodiments, both the first and second antigen-binding domains are scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is at the N-terminus of the bispecific molecule. In some embodiments, the first antigen-binding domain is at the C-terminus of the bispecific molecule.

[0282] In some embodiments, the engager molecule comprises a first antigen-binding domain (such as scFv) recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the first antigen-binding domain is an scFv. In some embodiments, the second antigen-binding domain is an scFv. In some embodiments, both the first and second antigen-binding domains are scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is at the N-terminus of the bispecific molecule. In some embodiments, the first antigen-binding domain is at the C-terminus of the bispecific molecule.

[0283] In some embodiments, the engager molecule comprises a first antigen-binding domain (such as scFv) recognizing EGFR and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the first antigen-binding domain is an scFv. In some embodiments, the second antigen-binding domain is an scFv. In some embodiments, both the first and second antigen-binding domains are scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is at the N-terminus of the bispecific molecule. In some embodiments, the first antigen-binding domain is at the C-terminus of the bispecific molecule.

[0284] In some embodiments, the engager molecule comprises a first antigen-binding domain (such as scFv) recognizing GPC3 and a second antigen-binding domain (such as scFv) specifically recognizing CD3 on T lymphocytes. In some embodiments, the first antigen-binding domain is an scFv. In some embodiments, the second antigen-binding domain is an scFv. In some embodiments, both the first and second antigen-binding domains are scFv. In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is at the N-terminus of the bispecific molecule. In some embodiments, the first antigen-binding domain is at the C-terminus of the bispecific molecule.

[0285] Further provided by the present application are pharmaceutical compositions comprising an effective amount of any one of the oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein) described herein, wherein the nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter (such as F17R), and optionally a pharmaceutically acceptable carrier. The present application also provides pharmaceutical compositions comprising an effective amount of any one of the oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and a second nucleic acid encoding a bispecific molecule described herein, and optionally a pharmaceutically acceptable carrier.

[0286] In some embodiments, the present application also provides a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described herein, e.g. GM-CSF), and optionally a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and a third OV (e.g. oncolytic VV) comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described herein, e.g. GM- CSF) and optionally a pharmaceutically acceptable carrier. In some embodiments, the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus, Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, and Newcastle disease virus (NDV). In some embodiments, the OV is an oncolytic VV. In some embodiments, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia, Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR). In some embodiments, the oncolytic VV is a WR strain. In some embodiments, the oncolytic virus comprises double deletion of TK gene and VGF gene. In some embodiments, the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule (such as activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS). In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a ligand that binds to an immune checkpoint molecule. In some embodiments, the immune checkpoint modulator is a ligand of PD-L1/PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3 (GPC3). In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell is selected from the group consisting of T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, and NKT-cell. In some embodiments, the effector cell is a T lymphocyte (such as cytotoxic T lymphocyte). In some embodiments, the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D. In some embodiments, the cell surface molecule is CD3. In some embodiments, the first and/or second antigen-binding domain is a single chain variable fragment (scFv). In some embodiments, the first and second antigen-binding domains are connected by a linker. In some embodiments, the first antigen-binding domain is N-terminal to the second antigen-binding domain. In some embodiments, the first antigen-binding domain is C-terminal to the second antigen-binding domain. In some embodiments, the first nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter. In some embodiments, the second nucleic acid encoding the bispecific molecule is operably linked to a late promoter. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter. In some embodiments, the late promoter is F17R.

[0287] “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

[0288] Pharmaceutical compositions comprising such carriers can be formulated by well- known conventional methods. The solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples include sterile water, physiological saline (e.g. sodium chloride), Ringer's solution, glucose, trehalose or saccharose solutions, Hank's solution, and other aqueous physiologically balanced salt solutions (see, for example, the most current edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins).

[0289] The pharmaceutical compositions of the disclosure may be administered locally (such as intratumorally) or systematically. Administration will generally be parenteral, e.g., intravenous; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti- oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present disclosure might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin. Various virus formulation are available in the art either in frozen, liquid form or lyophilized form (e.g. WO98/02522, WO01/66137, WO03/053463, WO2007/056847 and WO2008/114021, etc). Solid (e.g. dry powdered or lyophilized) compositions can be obtained by a process involving vacuum drying and freeze- drying (see e.g. WO2014/053571). It is envisaged that the pharmaceutical composition of the disclosure might comprise, in addition to the immune checkpoint modulator (and/or bispecific engager molecule, cytokine) or nucleic acid molecules or vectors encoding the same (as described in this disclosure), further biologically active agents, depending on the intended use of the pharmaceutical composition.

[0290] In some embodiments, the pharmaceutical composition described herein is suitably buffered for human use. Suitable buffers include without limitation phosphate buffer (e.g. PBS), bicarbonate buffer and/or Tris buffer capable of maintaining a physiological or slightly basic pH (e.g. from approximately pH 7 to approximately pH 9). In some embodiments, the pharmaceutical composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.

[0291] In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved. Therapeutic uses of oncolytic virus

[0292] One aspect of the present application relates to methods of treating cancer comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator (such as an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), wherein the nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter (such as F17R), and optionally a pharmaceutical acceptable carrier. In some embodiments, the individual is a cancer patients or patent susceptible to cancer or suspected of having cancer. The disclosure includes nucleic acid sequence that encodes an immune checkpoint modulator, vector(s) (such as oncolytic viral vectors) that encodes an immune checkpoint modulator, as contemplated herein and/or produced by a process as contemplated herein.

[0293] Another aspect of the present application relates to methods of treating cancer comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, the individual is a cancer patients or patent susceptible to cancer or suspected of having cancer. The disclosure includes nucleic acid sequence that encodes an immune checkpoint modulator, vector(s) (such as oncolytic viral vectors) that encodes an immune checkpoint modulator, as contemplated herein and/or produced by a process as contemplated herein.

[0294] The present invention contemplates, in part, viruses, protein constructs (such as bispecific molecule or immune checkpoint modulator), nucleic acid molecules and/or vectors (such as oncolytic viral vector) that can be administered either alone or in any combination with another therapy, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In some embodiments, prior to administration of the viruses or protein constructs, they may be combined with suitable pharmaceutical carriers and excipients that are well known in the art. The compositions prepared according to the disclosure can be used for the treatment or delaying of onset or worsening of cancer.

[0295] In some embodiments, there is provided a method of treating cancer in an individual (such as a human), comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator (such as an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), wherein the nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter (such as F17R), and optionally a pharmaceutical acceptable carrier. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is extracellular domain of PD-1 fused to Fc fragment of an immunoglobulin, such as IgG4 Fc. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) prolonging survival; (8) prolonging time to cancer progression; (9) preventing, inhibiting, or reducing the likelihood of the recurrence of a cancer; (10) inducing redistribution of peripheral T cells; and (11) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node). In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (uninfected by the oncolytic VV) death rate of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the tumor size. In some embodiments, the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis. In some embodiments, the method of prolonging survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging time to cancer progression mediated by the pharmaceutical composition described herein can prolongs the time to cancer progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0296] In some embodiments, there is provided a method of treating cancer in an individual (such as a human), comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as an inhibitor of PD-1, PD- L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is extracellular domain of PD-1 fused to Fc fragment of an immunoglobulin, such as IgG4 Fc. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting proliferation of cancer cells; (3) inducing redistribution of peripheral T cells; (4) inducing immune response in a tumor; (5) reducing tumor size; (6) alleviating one or more symptoms in an individual having cancer; (7) inhibiting tumor metastasis; (8) prolonging survival; (9) prolonging time to cancer progression; (10) preventing, inhibiting, or reducing the likelihood of the recurrence of a cancer; (11) inducing stromal destruction or killing tumor stromal cells in a tumor; (12) promoting oncolytic virus spread through tumors; (13) facilitating T cell infiltration in tumors, and (14) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node). In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (uninfected by the oncolytic VV) death rate of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the tumor size. In some embodiments, the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis. In some embodiments, the method of prolonging survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging time to cancer progression mediated by the pharmaceutical composition described herein can prolongs the time to cancer progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0297] In some embodiments, there is also provided a method of treating cancer in an individual (such as human), comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. immune checkpoint inhibitor, e.g. anti-PD-1 antibody, extracellular domain of PD-1 fused to IgG4 Fc, extracellular domain of TMIGD2 fused to IgG4 Fc, or extracellular domain of SIRPĮ and a CXCL12 fragment fused to IgG4 Fc), and a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutically acceptable carrier. In some embodiments, there is also provided a method of treating cancer in an individual (such as human), comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g., immune checkpoint inhibitor, e.g. anti-PD-1 antibody, extracellular domain of PD-1 fused to IgG4 Fc, extracellular domain of TMIGD2 fused to IgG4 Fc, or extracellular domain of SIRPĮ and a CXCL12 fragment fused to IgG4 Fc), and a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described herein, e.g. GM-CSF), and optionally a pharmaceutically acceptable carrier. In some embodiments, there is also provided a method of treating cancer in an individual (such as human), comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g., immune checkpoint inhibitor, e.g. anti-PD-1 antibody, extracellular domain of PD-1 fused to IgG4 Fc, extracellular domain of TMIGD2 fused to IgG4 Fc, or extracellular domain of SIRPĮ and a CXCL12 fragment fused to IgG4 Fc), a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and a third OV (e.g. oncolytic VV) comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described herein, e.g. GM-CSF), and optionally a pharmaceutically acceptable carrier. In some embodiments, there is also provided a method of treating cancer in an individual (such as human), comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g., immune checkpoint inhibitor, e.g. anti-PD-1 antibody, extracellular domain of PD-1 fused to IgG4 Fc, extracellular domain of TMIGD2 fused to IgG4 Fc, or extracellular domain of SIRPĮ and a CXCL12 fragment fused to IgG4 Fc), and optionally a pharmaceutically acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a second pharmaceutically acceptable carrier. In some embodiments, there is provided a method of treating cancer in an individual (such as human), comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g., immune checkpoint inhibitor, e.g. anti-PD-1 antibody, extracellular domain of PD-1 fused to IgG4 Fc, extracellular domain of TMIGD2 fused to IgG4 Fc, or extracellular domain of SIRPĮ and a CXCL12 fragment fused to IgG4 Fc), and optionally a first pharmaceutically acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a cytokine (such as any one of the cytokines described herein, e.g. GM-CSF), and optionally a second pharmaceutically acceptable carrier. In some embodiments, there is provided a method of treating cancer in an individual (such as human), comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g., immune checkpoint inhibitor, e.g. anti-PD-1 antibody, extracellular domain of PD-1 fused to IgG4 Fc, extracellular domain of TMIGD2 fused to IgG4 Fc, or extracellular domain of SIRPĮ and a CXCL12 fragment fused to IgG4 Fc), and optionally first pharmaceutically acceptable carrier, an effective amount of a second pharmaceutical composition comprising a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally pharmaceutically acceptable carrier, and an effective amount of a third pharmaceutical composition comprising a third OV (e.g. oncolytic VV) comprising a third nucleic acid encoding a cytokine (such as any one of the cytokines described herein, e.g. GM-CSF), and optionally a third pharmaceutically acceptable carrier. In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting proliferation of cancer cells; (3) inducing redistribution of peripheral T cells; (4) inducing immune response in a tumor; (5) reducing tumor size; (6) alleviating one or more symptoms in an individual having cancer; (7) inhibiting tumor metastasis; (8) prolonging survival; (9) prolonging time to cancer progression; (10) preventing, inhibiting, or reducing the likelihood of the recurrence of a cancer; (11) inducing stromal destruction or killing tumor stromal cells in a tumor (e.g., when a FAP-CD3 T-cell engager is expressed); (12) promoting oncolytic virus spread through tumors (e.g., when a FAP-CD3 T-cell engager is expressed); (13) facilitating T cell infiltration in tumors (e.g., when a FAP-CD3 T-cell engager is expressed), and (14) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node). In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve a bystander tumor cell (uninfected by the OV) death rate of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the tumor size. In some embodiments, the method of inhibiting tumor metastasis mediated by the pharmaceutical composition described herein can inhibit at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis. In some embodiments, the method of prolonging survival of an individual (such as a human) mediated by the pharmaceutical composition described herein can prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging time to cancer progression mediated by the pharmaceutical composition described herein can prolongs the time to cancer progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0298] The efficacy of oncolytic vaccinia virus encoding an immune checkpoint modulator, and the efficacy of oncolytic virus (such as oncolytic VV) expressing an immune checkpoint inhibitor and a bispecific engager molecule described herein may be limited by tumor stroma as physical barrier for virus spread and T cells. Preclinical studies have shown that co-targeting tumor cells and tumor stroma significantly enhanced antitumor activity of immunotherapies. In some embodiments, the oncolytic virus (such as oncolytic VV) of the present invention comprises a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (e.g. scFv) specifically recognizing the tumor antigen FAP, and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g. CD3 on T lymphocytes). In some embodiments, the oncolytic virus co-expressing immune checkpoint modulator and bispecific molecule is an oncolytic VV. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a PD-1 extracellular domain-Fc fusion protein. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).

[0299] Accordingly in some embodiments, there is provided a method of inducing stromal destruction or killing tumor stroma cells in a tumor in an individual (such as a human), comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen- binding domain (such as scFv) specifically recognizing tumor antigen FAP and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, there is provided a method of inducing stromal destruction or killing tumor stroma cells in a tumor in an individual (such as a human), comprising administering to the individual a first pharmaceutical composition comprising a first OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. immune checkpoint inhibitor, e.g., anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and optionally a first pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising an OV encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a second pharmaceutically acceptable carrier (and optionally a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of cytokines described herein), and optionally a third pharmaceutically acceptable carrier). In some embodiments, there is provided a method of inducing stromal destruction or killing tumor stroma cells in a tumor in an individual (such as a human), comprising administering to the individual a pharmaceutical composition comprising a first OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. immune checkpoint inhibitor, e.g., anti- PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and a second OV encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and/or a third OV encoding a cytokine (such as any one of cytokines described herein, e.g. GM-CSF), and optionally a pharmaceutical acceptable carrier. In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the method further induces bystander stromal destruction or kills bystander tumor stroma cells (not infected by the oncolytic virus described herein) in the presence of T cells. In some embodiments, the method further comprises administering to the individual an effective amount of another pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a nucleic acid encoding a bispecific molecule (such as any one of bispecific molecules described herein) comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen that is not FAP (e.g. EpCAM, EGFR, or GPC3) and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g. CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, the oncolytic virus in the another pharmaceutical composition is a vaccinia virus. In some embodiments, the FAP-T cell engager armed oncolytic virus (FAP-TEA- OV) enhances the antitumor activity of the non-FAP-TEA-OV, such as EpCAM-TEA-OV (also hereinafter referred to as EpCAM-CD3-OV), EGFR-TEA-OV (also hereinafter referred to as EGFR-CD3-OV), or GPC3-TEA-OV (also hereinafter referred to as GPC3-CD3-OV). In some embodiments, the immune checkpoint modulator expressed by the oncolytic virus co-expressing FAP-TE (also hereinafter referred to as FAP-CD3) further enhances the anti-tumor effect of FAP-TE and/or non-FAP-TE. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a PD-1 extracellular domain-IgG4 Fc. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).

[0300] In some embodiments, there is also provided a method of promoting oncolytic virus spread through tumors in an individual (such as a human), comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing tumor antigen FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, there is provided a method of promoting oncolytic virus spread through tumors in an individual (such as a human), comprising administering to the individual a first pharmaceutical composition comprising a first OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein,e.g. immune checkpoint inhibitor, e.g., anti-PD- 1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and optionally a first pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising an OV encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a second pharmaceutically acceptable carrier (and optionally a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of cytokines described herein), and optionally a third pharmaceutically acceptable carrier). In some embodiments, there is provided a method of promoting oncolytic virus spread through tumors in an individual (such as a human), comprising administering to the individual a pharmaceutical composition comprising a first OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. immune checkpoint inhibitor, e.g., anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and a second OV encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing FAP and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and/or a third OV encoding a cytokine (such as any one of cytokines described herein, e.g. GM-CSF), and optionally a pharmaceutical acceptable carrier. In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the method further comprises administering to the individual an effective amount of another pharmaceutical composition comprising an oncolytic virus comprising a nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (e.g. scFv) specifically recognizing a tumor antigen that is not FAP (e.g. EpCAM, EGFR, or GPC3) and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g. CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, the oncolytic virus in the another pharmaceutical composition is a vaccinia virus. In some embodiments, the FAP-T cell engager armed OV (FAP-TEA-OV) enhances the spread of the non-FAP-TEA-OV, such as EpCAM-TEA-OV, EGFR-TEA-OV, or GPC3-TEA-OV. In some embodiments, the immune checkpoint modulator expressed by the oncolytic virus co-expressing FAP-TE further enhances the effect of FAP-TE and/or non-FAP-TE. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a PD-1 extracellular domain-IgG4 Fc. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).

[0301] In some embodiments, there is provided a method of increasing T cell tumor infiltration in an individual (such as a human), comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, there is provided a method of increasing T cell tumor infiltration in an individual (such as a human), comprising administering to the individual a first pharmaceutical composition comprising a first OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. immune checkpoint inhibitor, e.g., anti- PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and optionally a first pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising an OV encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen- binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a second pharmaceutically acceptable carrier (and optionally a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of cytokines described herein), and optionally a third pharmaceutically acceptable carrier). In some embodiments, there is provided a method of increasing T cell tumor infiltration in an individual (such as a human), comprising administering to the individual a pharmaceutical composition comprising a first OV encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. immune checkpoint inhibitor, e.g., anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), and a second OV encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and/or a third OV encoding a cytokine (such as any one of cytokines described herein, e.g. GM-CSF), and optionally a pharmaceutical acceptable carrier. In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the method induces T cell immune response in bystander tumor cells (not infected by the oncolytic virus described herein) in the presence of T cells. In some embodiments, the method further comprises administering to the individual an effective amount of another pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (e.g. scFv) specifically recognizing the tumor antigen FAP and a second antigen-binding domain (e.g. scFv) specifically recognizing a cell surface molecule on an effector cell (e.g. CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, the oncolytic virus in the another pharmaceutical composition is a vaccinia virus. In some embodiments, the FAP-TEA- VV (also hereinafter referred to as FAP-CD3-VV) of the another pharmaceutical composition enhances the infiltration of T cells to tumor cells infected with oncolytic virus described herein and/or to bystander tumor cells not infected with oncolytic virus described herein. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is a PD-1 extracellular domain-IgG4 Fc. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc).

[0302] The methods described herein are suitable for treating a variety of cancers, including both solid cancer and liquid cancer. The methods are applicable to cancers of all stages, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (such as T-cell therapy), bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio- frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting (i.e., the method may be carried out before the primary/definitive therapy). In some embodiments, the method is used to treat an individual who has previously been treated. In some embodiments, the cancer has been refractory to prior therapy. In some embodiments, the method is used to treat an individual who has not previously been treated.

[0303] Examples of cancers that may be treated by the methods of the invention include, but are not limited to, adenocortical carcinoma, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas/carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. The administration of the pharmaceutical composition(s) described herein is useful for all stages and types of cancer, including for minimal residual disease, early solid tumor, advanced solid tumor and/or metastatic solid tumor.

[0304] In some embodiments, the method is suitable for treating cancers with aberrant PD-1 or PD-L1/PD-L2 expression, activity and/or signaling include, by way of non-limiting example, hematological cancer and/or solid tumors. Some cancers whose growth may be inhibited using the antibodies of the invention include cancers typically responsive to immunotherapy. Non- limiting examples of other cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer). Additionally, the invention includes refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the invention. Examples of other cancers that may be treated using the antibodies of the invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoidcancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. The present invention is also useful for treatment of metastatic cancers, especially metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol.17:133-144).

[0305] Thus, in some embodiments, there is provided a method of treating immunotherapy- responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant PD- 1 or PD-L1/PD-L2 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator (such as an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), wherein the nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter (such as F17R), and optionally a pharmaceutical acceptable carrier. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is PD-1 extracellular domain-Fc fusion protein. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node); (8) prolonging survival; and/or (9) prolonging time to cancer progression. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer, colon cancer, or liver cancer.

[0306] In some embodiments, there is provided a method of treating immunotherapy- responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant PD- 1 or PD-L1/PD-L2 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, there is provided a method of treating immunotherapy- responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant PD- 1 or PD-L1/PD-L2 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a third OV encoding a cytokine (such as any one of the cytokines described herein, e.g., GM-CSF). In some embodiments, there is provided a method of treating immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant PD-1 or PD-L1/PD-L2 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and optionally a first pharmaceutically acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a second pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering to the individual an effective amount of a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of the cyhtokines described herein, e.g., GM-CSF), and optionally a third pharmaceutically acceptable carrier. In some embodiments, the immune checkpoint modulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint modulator is PD-1 extracellular domain-Fc fusion protein. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node); (8) prolonging survival; (9) prolonging time to cancer progression; (10) increasing T cell tumor infiltration; and/or (11) enhancing any of the mentioned effect. For example, the co-expression of PD-1 extracellular domain-Fc fusion protein may enhance tumor cell killing by activated T cells in the presence of the co-expressed bispecific engager molecule, increase cytokine production of the T cell, or releasing or diminishing the tumor escape effect due to inhibitory immune checkpoint molecules expressed by tumor cells. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer, colon cancer, or liver cancer.

[0307] In some embodiments, the method is suitable for treating cancers with aberrant HHLA2 and/or TMIGD2 expression, activity and/or signaling, including but not limited to cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, esophagus and hematological malignancies of leukemia and lymphoma.

[0308] Thus in some embodiments, there is provided a method of treating immunotherapy- responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant HHLA2 and/or TMIGD2 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD- 1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), wherein the nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter (such as F17R), and optionally a pharmaceutical acceptable carrier. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node); (8) prolonging survival; and/or (9) prolonging time to cancer progression. In some embodiments, the immunotherapy- responsive solid tumor is breast cancer, colon cancer, or liver cancer.

[0309] In some embodiments, there is provided a method of treating immunotherapy- responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant HHLA2 and/or TMIGD2 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, there is provided a method of treating immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant HHLA2 and/or TMIGD2 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a third OV encoding a cytokine (such as any one of the cytokines described herein, e.g., GM-CSF). In some embodiments, there is provided a method of treating immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant HHLA2 and/or TMIGD2 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and optionally a first pharmaceutically acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a second pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering to the individual an effective amount of a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of cytokines described herein, e.g., GM-CSF), and optionally a third pharmaceutically acceptable carrier. In some embodiments, the immune checkpoint modulator comprises TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node); (8) prolonging survival; (9) prolonging time to cancer progression; (10) increasing T cell tumor infiltration; and/or (11) enhancing any of the mentioned effect. For example, the co-expression of TMIGD2 extracellular domain-Fc fusion protein may enhance tumor cell killing by activated T cells in the presence of the co-expressed bispecific engager molecule, increase cytokine production of the T cell, or releasing or diminishing the tumor escape effect due to inhibitory immune checkpoint molecules expressed by tumor cells. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer, colon cancer, or liver cancer. [0310] In some embodiments, the method is suitable for treating cancers with aberrant CD47/SIRPĮ expression, activity and/or signaling, and/or aberrant CXCL12/CXCR4 expression, activity and/or signaling, including but not limited to ovarian cancer, melanoma, prostate cancer, ovarian cancer, multiple myeloma, breast cancer, lung cancer, liver cancer.

[0311] Thus in some embodiments, there is provided a method of treating immunotherapy- responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant CD47/SIRPĮ and/or CXCL12/CXCR4 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), wherein the nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter (such as F17R), and optionally a pharmaceutical acceptable carrier. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node); (8) prolonging survival; and/or (9) prolonging time to cancer progression. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer or liver cancer.

[0312] In some embodiments, there is provided a method of treating immunotherapy- responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant CD47/SIRPĮ and/or CXCL12/CXCR4 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutical acceptable carrier. In some embodiments, there is provided a method of treating immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant CD47/SIRPĮ and/or CXCL12/CXCR4 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a third OV encoding a cytokine (such as any one of the cytokines described herein, e.g., GM-CSF). In some embodiments, there is provided a method of treating immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancers with aberrant CD47/SIRPĮ and/or CXCL12/CXCR4 expression, activity and/or signaling) in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV (e.g. oncolytic VV) comprising a first nucleic acid encoding an immune checkpoint modulator (such as any one of the immune checkpoint modulators described herein, e.g. an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73), and optionally a first pharmaceutically acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any one of the bispecific molecules described herein) comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes), and optionally a second pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering to the individual an effective amount of a third pharmaceutical composition comprising a third OV encoding a cytokine (such as any one of the cytokiness described herein, e.g., GM-CSF), and optionally a third pharmaceutically acceptable carrier. In some embodiments, the immune checkpoint modulator comprises an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting proliferation of cancer cells; (3) inducing immune response in a tumor; (4) reducing tumor size; (5) alleviating one or more symptoms in an individual having cancer; (6) inhibiting tumor metastasis; (7) reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node); (8) prolonging survival; (9) prolonging time to cancer progression; (10) increasing T cell tumor infiltration; and/or (11) enhancing any of the mentioned effect. For example, the co-expression of extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein may enhance tumor cell killing by activated T cells in the presence of the co-expressed bispecific engager molecule, increase cytokine production of the T cell, or releasing or diminishing the tumor escape effect due to inhibitory immune checkpoint molecules expressed by tumor cells. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer or liver cancer.

[0313] In some embodiments, the cancer is EpCAM-, FAP-, EGFR-, or GPC3-positive, for example. In some embodiments, the cancer is positive for, e.g., displays on its cell surface, any of the tumor associated antigens or tumor specific antigens listed herein.

[0314] In some embodiments, the method described herein is suitable for treating cancers that overexpress EpCAM on the surface of the cancer cells, such as EpCAM-positive solid cancers. For example, the cancer cells in the individual (such as a human) may express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of EpCAM compared to normal cells. EpCAM-positive solid cancer can be a carcinoma or adenocarcinoma. EpCAM-positive solid cancer include, but are not limited to, small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer (such as melanoma), renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

[0315] In some embodiments, the method described herein is suitable for treating cancers that overexpress FAP on tumor cells or tumor stromal fibroblasts, such as FAP-positive solid cancers. For example, the tumor stromal fibroblasts in the individual (such as a human) express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of FAP compared to normal fibroblasts. FAP-positive solid cancer can be a carcinoma or adenocarcinoma. In some embodiments, the FAP-positive solid cancer is selected from the group consisting of colorectal cancer, breast cancer, brain cancer, lung cancer, and skin cancer (such as melanoma).

[0316] In some embodiments, the method described herein is suitable for treating cancers that overexpress EGFR on the surface of the cancer cells, such as EGFR-positive solid cancers. For example, the cancer cells in the individual (such as a human) express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of EGFR compared to normal cells. EGFR-positive solid cancer can be carcinoma or adenocarcinoma. EGFR-mediated cancers include, but are not limited to, glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retina cancer, skin cancer, liver cancer, genital- urinary cancer, and bladder cancer.

[0317] In some embodiments, the method described herein is suitable for treating cancers that overexpress GPC3 on the surface of the cancer cells, such as GPC3-positive solid cancers. For example, the cancer cells in the individual (such as a human) may express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of GPC3 compared to normal cells. GPC3-positive solid cancer can be, e.g., lung squamous cell carcinoma, or hepatocellular carcinoma (HCC).

[0318] In some embodiments, the method described herein is suitable for treating a colorectal cancer, such as adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, leiomysarcoma, melanoma, or squamous cell carcinoma. In some embodiments, the method described herein is suitable for treating a liver cancer, such as liver cell carcinoma, fibrolamellar variants of hepatocellular carcinoma, or mixed hepatocellular cholangiocarcinoma. In some embodiments, the method described herein is suitable for treating a breast cancer, such as early stage breast cancer, non-metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in an adjuvant setting, or breast cancer in a neoadjuvant setting. In some embodiments, the breast cancer is fibroadenoma, or intraductal papilloma. In some embodiments, the breast cancer is HER2 positive or HER2 negative. In some embodiments, the breast cancer is a triple negative breast cancer.

[0319] The number of viruses (such as VV) employed in an effective amount of the pharmaceutical composition described herein will depend upon a number of circumstances, such as the purpose for the introduction, the particular type and stage of cancer being treated, the protocol to be used, for example, the number and route of administrations, the stability of the viruses, the activity of the encoded bispecific molecules and immune checkpoint modulator, and the like. The dosage regimen of the pharmaceutical composition described herein will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. In some embodiments, that effective amount of the pharmaceutical composition described herein is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the pharmaceutical composition is administered to the individual.

[0320] In some embodiments, the oncolytic virus (such as oncolytic VV) in the effective amount of the pharmaceutical composition described herein is from about 10 ⁵ to about 10 ¹³ pfu, including for example any of about 10 ⁵ to about 10 ¹² pfu, about 10 ⁶ to about 10 ¹³ pfu, about 10 ⁶ to about 10 ¹² pfu, about 10 ⁷ to about 10 ¹³ pfu, about 10 ⁷ to about 10 ¹² pfu, about 10 ⁷ to about 10 ¹¹ pfu, about 10 ⁷ to about 10 ¹⁰ pfu, about 10 ⁷ to about 10 ⁹ pfu, about 10 ⁸ to about 10 ¹³ pfu, about 10 ⁸ to about 10 ¹² pfu, about 10 ⁸ to about 10 ¹¹ pfu, about 10 ⁸ to about 10 ¹⁰ pfu, about 10 ⁸ to about 10 ⁹ pfu, about 10 ⁹ to about 10 ¹³ pfu, about 10 ⁹ to about 10 ¹² pfu, about 10 ⁹ to about 10 ¹¹ pfu, about 10 ⁹ to about 10 ¹⁰ pfu, about 10 ¹⁰ to about 10 ¹³ pfu, about 10 ¹¹ to about 10 ¹³ pfu, or about 10 ¹² to about 10 ¹³ pfu. In some embodiments, the OV (such as oncolytic VV) in the effective amount of the pharmaceutical composition described herein is about 10 ¹³ pfu, about 10 ¹² pfu, about 10 ¹¹ pfu, about 10 ¹⁰ pfu, about 10 ⁹ pfu, about 10 ⁸ pfu, about 10 ⁷ pfu, about 10 ⁶ pfu, or about 10 ⁵ pfu. In some embodiments, the OV (such as oncolytic VV) in the effective amount of the pharmaceutical composition described herein is about 10 ⁵ to about 10 ¹³ pfu. In some embodiments, the OV (such as oncolytic VV) in the effective amount of the pharmaceutical composition described herein is about 10 ⁷ to about 10 ⁹ pfu. In some embodiments, the OV (such as oncolytic VV) in the effective amount of the pharmaceutical composition described herein is about 10 ⁹ pfu.

[0321] In some embodiments, the pharmaceutical composition is administered for a single time (e.g. bolus injection). In some embodiments, the pharmaceutical composition is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times). If multiple administrations, they may be performed by the same or different routes and may take place at the same site or at alternative sites. The pharmaceutical composition may be administered twice per week, 3 times per week, 4 times per week, 5 times per week, daily, daily without break, once per week, weekly without break, once per 2 weeks, once per 3 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, or once per year. The interval between administrations can be about any one of 24h to 48h, 2 days to 3 days, 3 days to 5 days, 5 days to 1 week, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 month to 3 months, 3 months to 6 months, or 6 months to a year. Intervals can also be irregular (e.g. following tumor progression). In some embodiments, there is no break in the dosing schedule. In some embodiments, the pharmaceutical composition comprising the oncolytic virus described herein may be administered once or several time (e.g. 2, 3, 4, 5, 6, 7 or 8 times, etc.) at a dose within the range of from about 10 ⁵ pfu to about l0 ¹³ pfu (such as from about 10 ⁷ pfu to about l0 ⁹ pfu, or about l0 ⁹ pfu). The time interval between each administration can vary from approximately 1 day to approximately 8 weeks, from approximately 2 days to approximately 6 weeks, from approximately 3 days to approximately 4 weeks, from approximately 1 week to approximately 3 weeks, or every two weeks. In some embodiments, the pharmaceutical composition comprising the oncolytic virus (such as oncolytic VV) described herein is administered 2 to 5 times (e.g. 3 times) intravenously or intratumorally in the range of about 10 ⁵ pfu to about l0 ¹³ pfu (such as from about 10 ⁷ pfu to about l0 ⁹ pfu, or about l0 ⁹ pfu) at approximately 1 or 2 weeks interval. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0322] The pharmaceutical composition described herein may be suitable for a variety of modes of administration, including for example systemic or localized administration. In some embodiments, the pharmaceutical composition is administered parenterally, transdermally (into the dermis), intraluminally, intra-arterially (into an artery), intramuscularly (into muscle), intrathecally or intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously (under the skin). In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition described herein is administered to the individual via infusion or injection. In some embodiments, the pharmaceutical composition is directly injected to tumor sites (intratumorally - - into tumor or at its close proximity). Pharmaceutical compositions comprising oncolytic viral vectors encoding the bispecific molecule described herein and a pharmaceutically acceptable carrier are also encompassed in the present invention. Oncolytic viral vectors may be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Administrations may use conventional syringes and needles (e.g. Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or improving delivery of the active agent(s) in the subject.

[0323] In some embodiments, the individual to be treated is a mammal. Examples of mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cattle and the like. In some embodiments, the individual is a human.

IV. Antibodies

[0324] Various aspects of the present application make use of antibodies. The present application provides novel antibodies, antibody fragments, or antigen-binding fragments thereof. These antibodies can be used either independently, or can be incorporated into any of the OV (such as oncolytic VV), or oncolytic virus vector (such as oncolytic VV vector) described herein. [0325] The term“antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0326] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen-binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (V _H ) followed by three constant domains (C _H ) for each of the Į and Ȗ chains and four C _H domains for ^ and İ isotypes. Each L chain has at the N-terminus, a variable domain (V _L ) followed by a constant domain at its other end. The V _L is aligned with the V _H and the C _L is aligned with the first constant domain of the heavy chain (C _H 1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a V _H and V _L together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C _H ), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated Į, į, İ, Ȗ and ^, respectively. The Ȗ and Į classes are further divided into subclasses on the basis of relatively minor differences in the C _H sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

[0327] A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0328] The term“recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

[0329] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR (hereinafter defined) of the recipient are replaced by residues from an HVR of a non- human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, framework (“FR”) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos.6,982,321 and 7,087,409.

[0330] The term“chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies can comprise a murine variable region and a human constant region.“Chimeric antibodies” can also be those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as“class-switched antibodies”. Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See e.g. Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81 (1984) 6851- 6855; US Patent Nos.5,202,238 and 5,204,244.

[0331] The term“monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post- translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 ^nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human- like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol.7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol.14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0332] The terms“full-length antibody,”“intact antibody” or“whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically full-length 4-chain antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0333] An“antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fabƍ, F(abƍ) ₂ and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual“Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V _H ), and the first constant domain of one heavy chain (C _H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(abƍ) ₂ fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fabƍ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C _H 1 domain including one or more cysteines from the antibody hinge region. Fabƍ-SH is the designation herein for Fabƍ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(abƍ) ₂ antibody fragments originally were produced as pairs of Fabƍ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0334] The term“constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the C _H 1, C _H 2 and C _H 3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0335] The“light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“^”) and lambda (“^”), based on the amino acid sequences of their constant domains. [0336] The term“diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med.9:129-134 (2003).

[0337] The“Fc” fragment comprises the carboxy-terminal portions of both H chains held together by di-sulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0338] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0339] “Single-chain Fv” also abbreviated as“sFv” or“scFv” are antibody fragments that comprise the V _H and V _L antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the V _H and V _L domains which enables the scFv to form the desired structure for antigen binding. scFvs are known in the art, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

[0340] The“variable region” or“variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as“V _H ” and“V _L ”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.

[0341] The term“variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.

[0342] The term“hypervariable region,”“HVR,” or“HV,” when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, 4-chain antibodies comprise six HVRs; three in the V _H (H1, H2, H3), and three in the V _L (L1, L2, L3). In native 4-chain antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol.3:733-736 (1996).

[0343] A number of HVR delineations are in use and are encompassed herein. The Kabat “Complementarity Determining Regions” (or“CDRs”) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below in Table 1.

Table 1. HVR delineations.

[0344] HVRs may comprise“extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56 or 50- 56 (L2) and 89-97 or 89-96 (L3) in the V _L and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94- 102, or 95-102 (H3) in the V _H . The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0345] The expression“variable-domain residue-numbering as in Kabat” or“amino-acid- position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence.

[0346] “Framework” or“FR” residues are those variable-domain residues other than the HVR residues as herein defined. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0347] Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. The“EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.

[0348] As used herein, the term“binds”,“specifically binds to”,“specifically recognizes” or “is specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to, or specifically recognizes, or specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

[0349] “Percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0350] In some embodiments, there is provided an antibody, antibody fragment or antigen- binding domain that specifically binds to PD-1 (hereinafter also referred to as“anti-PD-1 antibody”, “anti-PD-1 antibody fragment”, “PD-1 binding fragment”, or “PD-1 binding domain”). Two exemplary anti-PD-1 antibodies or antigen-binding fragments thereof are 1H7e3 and 4F11C3.

[0351] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as a full length antibody or scFv) comprises a heavy chain variable region (VH) comprising one, two or three HVRs from SEQ ID NO: 13, and/or a light chain variable region (VL) comprising one, two or three HVRs from SEQ ID NO: 14. In some embodiments, the anti- PD-1 antibody or antigen-binding fragment thereof (such as a full length antibody of scFv) comprises a heavy chain variable region (VH) comprising three HVRs from SEQ ID NO: 13, and/or a light chain variable region (VL) comprising three HVRs from SEQ ID NO: 14.

[0352] In some embodiments, the anti-PD-1 antibody comprises an antigen-binding domain (such as an scFv) comprising a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0353] In some embodiments, the anti-PD-1 antibody comprises an antigen-binding domain (such as an scFv) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as an scFv) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14.

[0354] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as a full length antibody or scFv) comprises a heavy chain variable region (VH) comprising one, two or three HVRs from SEQ ID NO: 15, and/or a light chain variable region (VL) comprising one, two or three HVRs from SEQ ID NO: 16. In some embodiments, the anti- PD-1 antibody or antigen-binding fragment thereof (such as a full length antibody of scFv) comprises a heavy chain variable region (VH) comprising three HVRs from SEQ ID NO: 15, and/or a light chain variable region (VL) comprising three HVRs from SEQ ID NO: 16.

[0355] In some embodiments, the anti-PD-1 antibody comprises an antigen-binding domain (such as an scFv) comprising a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and/or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0356] In some embodiments, the anti-PD-1 antibody comprises an antigen-binding domain (such as an scFv) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as an scFv) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.

[0357] In some embodiments, the anti-PD-1 antibody is a full-length antibody. In some embodiments, the full-length anti-PD-1 antibody comprises an Fc sequence from an immunoglobulin, such as IgA, IgD, IgE, IgG, and IgM. In some embodiments, the full-length anti-PD-1 antibody comprises an Fc sequence of IgG, such as any of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the full-length anti-PD-1 antibody comprises an Fc sequence of a human immunoglobulin. In some embodiments, the full-length anti-PD-1 antibody comprises an Fc sequence that has been altered or otherwise changed so that it has enhanced antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) effector function.

[0358] Also provided is an isolated antibody or an antigen-binding fragment thereof which competes with any of the anti-PD-1 antibodies described herein for binding with PD-1. In some embodiments, there is provided an isolated antibody or an antigen-binding fragment thereof which binds to the same epitope as any of the anti-PD-1 antibodies described herein. Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0359] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a monoclonal antibody has the same specificity as a monoclonal antibody of the invention (e.g., the anti-PD-1 antibody having a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 14, or the anti-PD-1 antibody having a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 16) by ascertaining whether the former prevents the latter from binding to PD-1. If the monoclonal antibody being tested competes with the monoclonal antibody of the invention, as shown by a decrease in binding by the monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same, or a closely related, epitope.

[0360] An alternative method for determining whether a monoclonal antibody has the specificity of monoclonal antibody of the invention is to pre-incubate the monoclonal antibody of the invention with soluble PD-1 protein (with which it is normally reactive), and then add the monoclonal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind PD-1. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention.

[0361] In some embodiments, the anti-PD-1 antibody is a monoclonal antibody, such as a monovalent antibody. In some embodiments, the anti-PD-1 antibody is a full length antibody. In some embodiments, the anti-PD-1 antigen-binding fragment is in the form of a Fab, Fab’, a F(ab’) ₂ , single-chain Fv (scFv), an Fv fragment, a diabody, or a linear antibody.

[0362] In some embodiments, the anti-PD-1 antibody is a multispecific antibody that binds to PD-1, but also binds one or more other targets and optionally inhibits their function. Multispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for two or more different antigens (e.g., bispecific antibodies have binding specificities for at least two antigens). In some embodiments, the anti-PD-1 antibody is a bispecific molecule, wherein the bispecific molecule further comprises a second antigen-binding fragment specifically recognizing another inhibitory immune checkpoint molecule described herein (e.g. PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73). In some embodiments, the anti-PD-1 antibody is a bispecific molecule, wherein the bispecific molecule further comprises a second antigen- binding fragment specifically recognizing and activating an stimulatory immune checkpoint molecule described herein (e.g. OX40L, CD80, CD86, B7RP1, 4-1BBL, Ultra 4-1BBL, CD70, CD40L, or MHC class I or class II molecules, IMCgp100).

[0363] In some embodiments, the multi-specific anti-PD-1 molecule is, for example, a diabody (Db), a single-chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a di-diabody, a tandem scFv, a tandem di-scFv (e.g., a bispecific T cell engager), a tandem tri-scFv, a tri(a)body, a bispecific Fab ₂ , a di-miniantibody, a tetrabody, an scFv-Fc-scFv fusion, a dual-affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, an IgG-scFab, an scFab-ds-scFv, an Fv2-Fc, an IgG-scFv fusion, a dock and lock (DNL) antibody, a knob-into-hole (KiH) antibody (bispecific IgG prepared by the KiH technology), a DuoBody (bispecific IgG prepared by the Duobody technology), a heteromultimeric antibody, or a heteroconjugate antibody. In some embodiments, the multi- specific anti-PD-1 molecule is a tandem scFv (e.g., a tandem di-scFv, such as a bispecific T-cell engager).

[0364] Further provided are fusion proteins, conjugates, or isolated cells comprising any of the anti-PD-1 antibodies or antigen-binding fragments thereof described above.

[0365] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent (e.g., cytotoxic agent, a radioisotope and a chemotherapeutic agent) or a label for detecting PD-1 in patient samples or in vivo by imaging (e.g., radioisotope, fluorescent dye and enzyme). In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is conjugated to a toxin.

[0366] The anti-PD-1 antibodies or antigen-binding fragments described herein can be used in a variety of therapeutic and diagnostic methods. Further provided are methods of treating cancer in an individual, comprising administering an effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof described above or pharmaceutical compositions thereof to the individual. For example, the anti-PD-1 antibodies (or antigen-binding fragments thereof) can be used alone or in combination with other agents in treating a disease characterized by abnormal PD-L1 or PD-1 expression, or cancer responsive to immunotherapy, including, but not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, liver cancer and lung cancer (e.g. non-small cell lung cancer). The anti-PD-1 antibodies or antigen-binding fragments of present invention may also be useful for treatment of metastatic cancers, especially metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol.17:133-144). The antibodies provided herein can also be used for detecting PD-1 protein in patients or patient samples.

[0367] Further provided are isolated nucleic acid encoding the anti-PD-1 antibodies or antigen- binding fragments thereof, OV (such as oncolytic VV) comprising the nucleic acid encoding the anti-PD-1 antibodies or antigen-binding fragments thereof, isolated cells expressing the anti-PD- 1 antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising any of the anti-PD-1 antibodies or antigen-binding fragments thereof, OV (such as oncolytic VV) encoding thereof, host cells expressing thereof, methods of treating a cancer in an individual using such pharmaceutical compositions. In some embodiments, the pharmaceutical composition is administered to the individual to be treated intravenously. In some embodiments, the pharmaceutical composition is administered to the individual to be treated intratumorally. In some embodiments, the individual to be treated is a human.

Monoclonal antibodies

[0368] Screening of monoclonal antibodies of the invention, can be also carried out, e.g., by measuring PD-1-mediated signaling, and determining whether the test monoclonal antibody is able to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with PD-1- mediated signaling. These assays can include competitive binding assays. Additionally, these assays can measure a biologic readout.

[0369] Various procedures known in the art may be used for the production of monoclonal antibodies directed against PD-1, or against derivatives, fragments, analogs homologs or orthologs thereof. See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference. Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the HVRs, arise from human genes. Such antibodies are termed“human antibodies” or“fully human antibodies” herein. Human monoclonal antibodies are prepared, for example, using the procedures described in the Examples provided below. Human monoclonal antibodies can be also prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0370] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No.8 (April 17, 2000), pp.25-28).

[0371] The PD-1 antibodies of the invention are monoclonal antibodies. Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with PD-1-mediated cell signaling are generated, e.g., by immunizing an animal with membrane bound and/or soluble PD-1, such as, for example, human PD-1or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding PD-1 such that PD-1 is expressed and associated with the surface of the transfected cells. Alternatively, the antibodies are obtained by screening a library that contains antibody or antigen-binding domain sequences for binding to PD-1. This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e.,“phage displayed library”). Hybridomas resulting from myeloma/B cell fusions are then screened for reactivity to PD-1.

[0372] Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0373] The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

[0374] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)).

[0375] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity for the target antigen.

[0376] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco’s Modified Eagle’s Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0377] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0378] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibodies). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as Chinese hamster ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, simian COS cells, PER.C6®, NS0 cells, SP2/0, YB2/0, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody, or can be substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody.

Human Antibodies and Humanization of Antibodies

[0379] Monoclonal antibodies of the invention include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. [0380] An anti-PD-1 antibody can be generated using any procedures known in the art. For example, anti-PD-1 antibodies can be identified using a modified RIMMS (Repetitive Immunization Multiple Sites) immunization strategy in mice and subsequent hybridoma generation. In other, alternative methods, an anti-PD-1 antibody is developed, for example, using phage-display methods using antibodies containing only human sequences. Such approaches are well-known in the art, e.g., in WO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated by reference. In this approach, a combinatorial library of phage carrying random pairs of light and heavy chains are screened using natural or recombinant source of PD-1 or fragments thereof. In another approach, an anti-PD-1 antibody can be produced by a process wherein at least one step of the process includes immunizing a transgenic, non-human animal with human PD-1 protein. In this approach, some of the endogenous heavy and/or kappa light chain loci of this xenogenic non-human animal have been disabled and are incapable of the rearrangement required to generate genes encoding immunoglobulins in response to an antigen. In addition, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into the animal. Thus, in response to an administered antigen, the human loci rearrange to provide genes encoding human variable regions immunospecific for the antigen. Upon immunization, therefore, the xenomouse produces B-cells that secrete fully human immunoglobulins.

[0381] A variety of techniques are well-known in the art for producing xenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181 and No. 6,150,584, which is hereby incorporated by reference in its entirety. This general strategy was demonstrated in connection with generation of the first XenoMouse™ strains as published in 1994. See Green et al. Nature Genetics 7:13-21 (1994), which is hereby incorporated by reference in its entirety. See also, U.S. Patent Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181; and 5,939,598 and Japanese Patent Nos.3068180 B2, 3068506 B2, and 3068507 B2 and European Patent No., EP 0463 151 B1 and International Patent Applications No. WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related family members.

[0382] In an alternative approach, others have utilized a“minilocus” approach in which an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more D _H genes, one or more J _H genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. See e.g., U.S. Patent Nos. 5,545,806; 5,545,807; 5,591,669; 5,612,205;5,625,825; 5,625,126; 5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429; 5,789,215; 5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and 6,255,458; and European Patent No. 0 546 073 B1; and International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and related family members.

[0383] Generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced, has also been demonstrated. See European Patent Application Nos.773288 and 843961.

[0384] Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and an immune variable region, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in chronic or multi-dose utilizations of the antibody. Thus, it would be desirable to provide fully human antibodies against PD-1 in order to vitiate or otherwise mitigate concerns and/or effects of HAMA or HACA response.

[0385] The production of antibodies with reduced immunogenicity is also accomplished via humanization, chimerization and display techniques using appropriate libraries. It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See e.g., Winter and Harris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). The antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (see WO 92102190 and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,792;, 5,714,350; and 5,777,085). Also, the use of Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol.139:3521 (1987)). mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA. The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant regions genes may be found in Kabat et al. (1991) Sequences of Proteins of immunological Interest, N.I.H. publication no.91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effecter functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.

[0386] Antibody fragments, such as Fv, F(ab’) ₂ and Fab may be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F(ab’) ₂ fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.

[0387] Consensus sequences of H, L, and J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

[0388] Expression vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e.g., SV-40 early promoter (Okayama et al. Mol. Cell. Bio.3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murine leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also, as will be appreciated, native Ig promoters and the like may be used.

[0389] Further, human antibodies or antibodies from other species can be generated through display type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art. Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes and Plückthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol.17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel et al. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A (1992), and U.S. Patent No.5,733,743. If display technologies are utilized to produce antibodies that are not human, such antibodies can be humanized as described above.

[0390] Using these techniques, antibodies can be generated to PD-1 expressing cells, soluble forms of PD-1, epitopes or peptides thereof, and expression libraries thereto (see e.g., U.S. Patent No. 5,703,057) which can thereafter be screened as described above for the activities described herein.

[0391] The anti-PD-1 antibodies of the invention can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

[0392] These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93/64701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic.

[0393] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87:1149 (1990)), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995)) and Adeno-associated Virus Vectors (see Kaplitt, M. G., et al., Nat. Genet.8:148 (1994)).

[0394] Pox viral vectors introduce genes into the cellular cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. Vaccinia virus vectors are capable to multiply in a large number of different types of cells. The particular vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaPO ₄ precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0395] The vector can be employed to target essentially any desired target cell. For example, stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell (see Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.

[0396] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of PD-1 in a sample. The anti-PD-1 antibody can also be used to try to bind to and disrupt PD-1-mediated signaling.

[0397] Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No.4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab’) ₂ fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F _(ab’)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F _v fragments.

[0398] The invention also includes F _v , Fab, Fab’ and F(ab’) ₂ anti-PD-1 fragments, single chain anti-PD-1 antibodies, single domain antibodies (e.g., nanobodies or VHHs), multispecific (such as bispecific) anti-PD-1 antibodies, and heteroconjugate anti-PD-1 antibodies.

[0399] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0400] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0401] According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0402] Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab’) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’-TNB derivatives is then reconverted to the Fab’-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab’-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0403] Additionally, Fab’ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab’) ₂ molecule. Each Fab’ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

[0404] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V _H ) connected to a light-chain variable domain (V _L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V _H and V _L domains of one fragment are forced to pair with the complementary V _L and V _H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. See, Gruber et al., J. Immunol.152:5368 (1994).

[0405] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al., J. Immunol.147:60 (1991).

[0406] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcȖR), such as FcȖRI (CD64), FcȖRII (CD32) and FcȖRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.

[0407] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980), and for treatment of HIV infection (see WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.4,676,980.

[0408] It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating diseases and disorders associated with aberrant PD-1 signaling. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities (see Stevenson et al., Anti- Cancer Drug Design, 3: 219-230 (1989)).

[0409] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), a radioactive isotope (i.e., a radioconjugate), or a label for detecting the target antigen (such as PD-1) in patient samples or in vivo by imaging (e.g., radioisotope, fluorescent dye and enzyme).

[0410] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

[0411] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94/11026). [0412] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies of the invention (see, for example,“Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0413] Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0414] Preferred linkers are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N- hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dith io)- toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC. [0415] The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

[0416] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556.

[0417] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

Use of antibodies against PD-1

[0418] It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P.“Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W.“Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN“Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

[0419] In some embodiments, anti-PD-1 antibodies of the invention may be used as therapeutic agents. Such agents will generally be employed to diagnose, prognose, monitor, treat, alleviate, and/or prevent a disease or pathology associated with aberrant PD-1 or PD-L1/PD-L2 expression, activity and/or signaling in a subject. A therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a disease or disorder associated with aberrant PD-1 or PD-L1/PD-L2 expression, activity and/or signaling, e.g., a cancer or other neoplastic disorder, using standard methods. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Administration of the antibody may abrogate or inhibit or interfere with the expression, activity and/or signaling function of the target (e.g., PD-1). Administration of the antibody may abrogate or inhibit or interfere with the binding of the target (e.g., PD-1) with an endogenous ligand to which it naturally binds. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with PD-1 expression, activity and/or signaling.

[0420] Diseases or disorders related to aberrant PD-1 or PD-L1/PD-L2 expression, activity and/or signaling include, by way of non-limiting example, hematological cancer and/or solid tumors. Cancers whose growth may be inhibited using the antibodies of the invention include cancers typically responsive to immunotherapy. Examples of cancers for treatment include, but are not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, liver cancer and lung cancer (e.g. non-small cell lung cancer). The present invention is also useful for treatment of metastatic cancers, especially metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol.17:133-144).

[0421] Optionally, antibodies to PD-1 can be combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines (He et al (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. The anti-PD-1 antibodies of the present invention can also be combined with bispecific engager molecules described herein, such as a bispecific molecule comprising a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3), and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 on T lymphocytes).

[0422] Symptoms associated with cancers and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain, often localized to the inflamed area, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue and abdominal symptoms such as, for example, abdominal pain, diarrhea or constipation.

[0423] A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of non-limiting example, from about 0.1 mg/kg body weight to about 100 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week or biweekly.

[0424] Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular inflammatory-related disorder. Alleviation of one or more symptoms of the inflammatory-related disorder indicates that the antibody confers a clinical benefit.

[0425] In another embodiment, antibodies directed against PD-1 may be used in methods known within the art relating to the localization and/or quantitation of PD-1 (e.g., for use in measuring the level of PD-1 within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific to PD-1, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as“Therapeutics”).

[0426] In another embodiment, an antibody specific for PD-1 can be used to isolate a PD-1 polypeptide, by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Antibodies directed against the PD-1 protein (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ^-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

[0427] In some embodiments, an anti-PD-1 antibody according to the invention can be used as an agent for detecting the presence of PD-1 (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. Antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., scFv) is used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995;“Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti- analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0428] In some embodiments, the individual is a mammal. Examples of mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cattle and the like. In some embodiments, the individual is a human.

[0429] There is also provided a pharmaceutical composition comprising an effective amount of the anti-PD-1 antibodies descried herein, host cells expressing the anti-PD-1 antibodies descried herein, or oncolytic virus (such as oncolytic VV) encoding the anti-PD-1 antibodies descried herein, and optionally a pharmaceutically acceptable carrier.

[0430] There is also provided a method of treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition comprising the anti-PD-1 antibodies descried herein, host cells expressing the anti-PD-1 antibodies descried herein, or oncolytic virus (such as oncolytic VV) encoding the anti-PD-1 antibodies descried herein, and optionally a pharmaceutically acceptable carrier. The treatment effects may include, but are not limited to, killing cancer cells, inhibiting proliferation of cancer cells, inducing redistribution of peripheral T cells, inducing immune response in a tumor, reducing tumor size, inhibiting tumor metastasis, reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node), prolonging survival of an individual, prolonging time to cancer progression, etc. In some embodiments, the pharmaceutical composition is administered to the individual intravenously or intratumorally. In some embodiments, the individual is a human.

V. Methods of preparation

[0431] The anti-PD-1 antibodies (or antigen-binding fragments thereof), PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein described herein may be prepared by any of the known protein expression and purification methods in the art.

[0432] In some embodiments, the present application provides isolated nucleic acids encoding one or more of the polypeptide chains of any one of the anti-PD-1 antibodies (or antigen-binding fragments thereof) described herein, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein.

[0433] In some embodiments, the isolated nucleic acid comprises a first nucleic acid sequence of SEQ ID NO: 17, and a second nucleic acid sequence of SEQ ID NO: 18. The isolated nucleic acids may be DNA or RNA. In some embodiments, the isolated nucleic acid described herein is operably linked to a promoter. In some embodiments, the promoter is a late promoter. In some embodiments, the promoter is a VV promoter. In some embodiments, the promoter is a VV late promoter. In some embodiments, the promoter is F17R.

[0434] In some embodiments, the isolated nucleic acid comprises a first nucleic acid sequence of SEQ ID NO: 19, and a second nucleic acid sequence of SEQ ID NO: 20. The isolated nucleic acids may be DNA or RNA. In some embodiments, the isolated nucleic acid described herein is operably linked to a promoter. In some embodiments, the promoter is a late promoter. In some embodiments, the promoter is a VV promoter. In some embodiments, the promoter is a VV late promoter. In some embodiments, the promoter is F17R.

[0435] In some embodiments, the isolated nucleic acid is inserted into a vector, such as an expression vector, a viral vector (such as oncolytic VV vector) , or a cloning vector. For expression of the nucleic acids, the vector may be introduced into a host cell to allow expression of the nucleic acids within the host cell. The expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, the promoter sequences may be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter. EF1a promoter, CMV promoter, SV40 promoter, and vaccinia virus promoter (such as F17R). Enhancer sequences may be selected to enhance the transcription of the nucleic acids. Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside of the host cell. In some embodiments, the isolated nucleic acids further comprise a nucleic acid sequence encoding a signal peptide.

[0436] In some embodiments, the oncolytic virus encoding the immune checkpoint modulators (and/or bispecific engager molecules, cytokines, such as those described herein) is a vaccinia virus. Vaccinia virus is appealing for cancer gene therapy due to several characteristics. It has natural tropism towards cancer cells and the selectivity can be significantly enhanced by deleting some of the viral genes. In some embodiments, the oncolytic virus (such as oncolytic VV) encoding the immune checkpoint modulators (and/or bispecific engager molecules, cytokines, such as those described herein) comprises double deletion of thymidine kinase (TK) gene and vaccinia virus growth factor (VGF) gene (vvDD strain). TK and VGF genes are needed for virus to replicate in normal but not in cancer cells. The TK or VGF deletion may be engineered in the TK or VGF region conferring activity, respectively. For example, this can by generated by recombination of a pSEM-1 shuttle plasmid containing the immune checkpoint modulator (and/or bispecific engager molecules, cytokines, such as those described herein) into the TK gene of the VSC20 strain (VGF deleted strain) of Western Reserve vaccinia virus (WR VV). The VSC20 train can be constructed by inserting lacZ gene under the control of the p11 promoter into two copies of the viral VGF genes, thus inactivating VGF. The shuttle vector pSEM-1 can be constructed to have the immune checkpoint modulator (and/or bispecific engager molecules, cytokines, such as those described herein) expressed under the transcriptional control of a promoter, such as the F17R late promoter which allows for sufficient viral replication before T-cell activation. In some embodiments, the VV can further express a marker, such as DsRed2, YFP, GFP, or YFP-GFP, to allow for virus selection. In some embodiments, the infectivity monitoring marker can be expressed under the transcriptional control of the same promoter that drives the expression of the immune checkpoint modulator (and/or bispecific engager molecule described herein). In some embodiments, the virus selection marker can be expressed under the transcriptional control of a different promoter, such as Pse/I promoter, or P7.5 promoter. In some embodiments, the virus selection marker can be flanked by loxP sites in the same orientation. For constructing the recombinant virus encoding the immune checkpoint modulator (and/or bispecific engager molecules, cytokines, such as those described herein), the shuttle vectors pSEM-1 containing the immune checkpoint modulator (and/or bispecific engager molecules, cytokines, such as those described herein) can be transfected into human 143 TK- cells. Cells can then be infected with virus VSC20 at a multiplicity of infection (MOI) of 0.1. After 3-5 (e.g., five) rounds of plaque selection and amplification with confirmation of the expression of immune checkpoint modulator (and/or bispecific engager molecules, cytokines, such as those described herein), one of the clones can be selected for amplification and purification. In some embodiments, the selection marker, e.g., YFP-GFP cassette can be removed from the recombinant viruses. For example, in some embodiments, viruses can be passaged on a U2OS cell line expressing a cytoplasmic form of Cre recombinase (U2OS-Cre). After 3-5 (e.g., five) rounds of plaque selection and amplification to confirm the expression of immune checkpoint modulator (and/or bispecific engager molecules, cytokines, such as those described herein), one of the selectable marker-negative (e.g., YFP-GFP-negative) clones can be selected for amplification and purification.

[0437] One of skill in the art will recognize that any suitable method can be used for generating inactivating mutations in a gene of interest, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering technique known to a person of skill in the art. Mutation can involve modification of a nucleotide sequence, a single gene, or blocks of genes. A mutation may involve a single nucleotide (such as a point mutation, which involves the removal, addition or substitution of a single nucleotide base within a DNA sequence) or it may involve the insertion or deletion of large numbers of nucleotides. Mutations can arise spontaneously as a result of events such as errors in the fidelity of DNA replication, or induced following exposure to chemical or physical mutagens. A mutation can also be site- directed through the use of particular targeting methods that are well known to persons of skill in the art.

[0438] The obtained virus of the present invention can be replicated by conventional methods for viral replication, e.g. infecting host cells such as 293 cells with the virus.

[0439] It is envisaged for further purposes that oncolytic viral nucleic acid molecules may contain, for example, thioester bonds and/or nucleotide analogues. The modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. The nucleic acid molecules may be transcribed by an appropriate oncolytic vector comprising a chimeric gene that allows for the transcription of the nucleic acid molecule in the cell. In this respect, it is also to be understood that such polynucleotides can be used for“gene targeting” or“gene therapeutic” approaches. The nucleic acid molecules may also be labeled. Methods for the detection of nucleic acids are well known in the art, e.g., Southern and Northern blotting, PCR or primer extension. This embodiment may be useful for screening methods for verifying successful introduction of the nucleic acid molecules described above, for example during gene therapy approaches.

[0440] In some embodiments, there is provided an isolated host cell comprising an isolated nucleic acid encoding the anti-PD-1 antibody (or antigen-binding fragment thereof) described above, isolated nucleic acid encoding the PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein described above. The host cells comprising the isolated nucleic acid described herein may be useful in expression or cloning of the anti-PD-1 antibody (or antigen- binding fragment thereof), PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein described herein. Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. The expression of antibodies and antigen-binding fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. BioTechnology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of antibodies or antigen-binding fragments thereof, see recent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560. Higher eukaryotic cells, in particular, those derived from multicellular organisms can be used for expression of glycosylated polypeptides. Suitable higher eukaryotic cells include, without limitation, invertebrate cells and insect cells, and vertebrate cells.

[0441] The vector can be introduced to the host cell using any suitable methods known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art. In some embodiments, the host cells comprise a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In some embodiments, the host cells comprise a single vector comprising isolated nucleic acids encoding a first polypeptide and a second polypeptide.

[0442] In some embodiments, the present application provides methods of expressing any of the anti-PD-1 antibodies (or antigen-binding fragments thereof), PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein and/or the bispecific engager molecule described herein , comprising culturing the isolated host cell containing the vector and recovering the anti-PD-1 antibodies (or antigen-binding fragment thereof), PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein, and/or the bispecific engager molecule from the cell culture. The isolated host cells are cultured under conditions that allow expression of the isolated nucleic acids inserted in the vectors. Suitable conditions for expression of polynucleotides may include, without limitation, suitable medium, suitable density of host cells in the culture medium, presence of necessary nutrients, presence of supplemental factors, suitable temperatures and humidity, and absence of microorganism contaminants. A person with ordinary skill in the art can select the suitable conditions as appropriate for the purpose of the expression.

[0443] In some embodiments, the polypeptides expressed in the host cell can form a dimer and thus produce an anti-PD-1 antibody (or antigen-binding fragment thereof) and/or the bispecific engager molecule described herein . In some embodiments, the polypeptide expressed in the host cell can form a polypeptide complex which is a homodimer. In some embodiments, wherein the host cells express a first polynucleotide and a second polynucleotide, the first polynucleotide and the second polynucleotide can form a polypeptide complex which is a heterodimer.

[0444] In some embodiments, the polypeptide complex (such as the bispecific engager molecule or the anti-PD-1 antibody or antigen-binding fragment thereof) may be formed inside the host cell. For example, the dimer may be formed inside the host cell with the aid of relevant enzymes and/or cofactors. In some embodiments, the polypeptide complex may be secreted out of the cell. In some embodiments, a first polypeptide and a second polypeptide may be secreted out of the host cell and form a dimer outside of the host cell.

[0445] In some embodiments, a first polypeptide and a second polypeptide may be separately expressed and allowed to dimerize to form the bispecific engager molecule or the anti-PD-1 antibody (or antigen-binding fragment thereof) under suitable conditions. For example, the first polypeptide and the second polypeptide may be combined in a suitable buffer and allow the first protein monomer and the second protein monomer to dimerize through appropriate interactions such as hydrophobic interactions. In some embodiments, the first polypeptide and the second polypeptide may be combined in a suitable buffer containing an enzyme and/or a cofactor which can promote the dimerization of the first polypeptide and the second polypeptide. In some embodiments, the first polypeptide and the second polypeptide may be combined in a suitable vehicle and allow them to react with each other in the presence of a suitable reagent and/or catalyst.

[0446] The expressed polypeptide(s) and/or the polypeptide complex can be collected using any suitable methods. The polypeptide(s) and/or the polypeptide complex can be expressed intracellularly, in the periplasmic space or be secreted outside of the cell into the medium. If the polypeptide and/or the polypeptide complex are expressed intracellularly, the host cells containing the polypeptide and/or the polypeptide complex may be lysed and polypeptide and/or the polypeptide complex may be isolated from the lysate by removing the unwanted debris by centrifugation or ultrafiltration. If the polypeptide and/or the polypeptide complex is secreted into periplasmic space of E. coli, the cell paste may be thawed in the presence of agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)). If the polypeptide and/or the polypeptide complex is secreted into the medium, the supernatant of the cell culture may be collected and concentrated using a commercially available protein concentration filter, for example, an Amincon or Millipore Pellicon ultrafiltration unit. A protease inhibitor and/or a antibiotics may be included in the collection and concentration steps to inhibit protein degradation and/or growth of contaminated microorganisms.

[0447] The expressed polypeptide(s) and/or the polypeptide complex can be further purified by a suitable method, such as without limitation, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (see, for review, Bonner, P. L., Protein purification, published by Taylor & Francis. 2007; Janson, J. C., et al, Protein purification: principles, high resolution methods and applications, published by Wiley-VCH, 1998).

[0448] In some embodiments, the polypeptides and/or polypeptide dimer complexes can be purified by affinity chromatography. In some embodiments, protein A chromatography or protein A/G (fusion protein of protein A and protein G) chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising a component derived from antibody CH2 domain and/or CH3 domain (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)); Zettlit, K. A., Antibody Engineering, Part V, 531-535, 2010). In some embodiments, protein G chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising IgG Ȗ3 heavy chain (Guss et al., EMBO J. 5:1567 1575 (1986)). In some embodiments, protein L chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising ^ light chain (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, B. H. K. at al, J. Biol. Chem., 267, 2234- 2239 (1992)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.

VI. Articles of manufacture and kits

[0449] Any of the compositions described herein may be comprised in a kit (e.g., oncolytic vaccinia virus expressing immune checkpoint modulator (e.g., anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), oncolytic virus expressing an immune checkpoint modulator and a bispecific engager molecule, the anti-PD-1 antibody compositions, host cells expressing the anti-PD-1 antibodies, or oncolytic virus encoding the anti-PD-1 antibodies). In a non-limiting example, one or more viruses and/or the reagents to generate or manipulate the virus may be comprised in a kit. The kit components are provided in suitable container means.

[0450] Some components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. Various combinations of components may also be comprised in a vial. The kits also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0451] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.

[0452] The components of the kit may also be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.

[0453] In some embodiments, viruses for use in therapy are provided in a kit, and in some cases the viruses are essentially the sole component of the kit. The kit may comprise reagents and materials to modify the desired virus. In specific embodiments, the reagents and materials include expression constructs, primers for amplifying desired sequences, restriction enzymes, one or more DNAs for inclusion in the virus, nucleotides, suitable buffers or buffer reagents, salt, and so forth, and in some cases the reagents include vectors and/or DNA that encodes an engager molecule as described herein and/or regulatory elements therefor.

[0454] In some embodiments, there are one or more apparatuses in the kit suitable for extracting one or more samples from an individual. The apparatus may be a syringe, scalpel, and so forth.

[0455] In some embodiments, the kit, in addition to the virus embodiments, also includes a second cancer therapy, such as chemotherapy, hormone therapy, and/or immunotherapy, for example. The kit(s) may be tailored to a particular cancer for an individual and comprise respective second cancer therapies for the individual. [0456] The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.

[0457] The present application further provides articles of manufacture comprising the compositions (such as pharmaceutical compositions) described herein in suitable packaging. Suitable packaging for compositions (such as oncolytic vaccinia virus expressing immune checkpoint modulator (e.g., anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or extracellular domain of SIRPĮ and a CXCL12 fragment-Fc fusion protein), oncolytic virus expressing an immune checkpoint modulator and a bispecific engager molecule, the anti-PD-1 antibody compositions, host cells expressing the anti-PD-1 antibodies, or oncolytic virus encoding the anti-PD-1 antibodies) described herein are known in the art, and include, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed.

EXEMPLARY EMBODIMENTS

[0458] Embodiment 1. An oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint modulator, wherein the nucleic acid is operably linked to a late promoter.

[0459] Embodiment 2. The oncolytic vaccinia virus of embodiment 1, wherein the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter.

[0460] Embodiment 3. The oncolytic vaccinia virus of embodiment 2, wherein the late promoter is F17R.

[0461] Embodiment 4. The oncolytic vaccinia virus of any one of embodiments 1-3, wherein the oncolytic vaccinia virus is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR). [0462] Embodiment 5. The oncolytic vaccinia virus of embodiment 4, wherein the oncolytic vaccinia virus is a WR strain.

[0463] Embodiment 6. The oncolytic vaccinia virus of embodiment 5, wherein the oncolytic vaccinia virus comprises double deletion of thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene.

[0464] Embodiment 7. The oncolytic vaccinia virus of any one of embodiments 1-6, wherein the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule.

[0465] Embodiment 8. The oncolytic vaccinia virus of any one of embodiments 1-6, wherein the immune checkpoint modulator is an immune checkpoint inhibitor.

[0466] Embodiment 9. The oncolytic vaccinia virus of embodiment 8, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73.

[0467] Embodiment 10. The oncolytic vaccinia virus of embodiment 9, wherein the immune checkpoint inhibitor is an inhibitor of PD-1.

[0468] Embodiment 11. The oncolytic vaccinia virus of any one of embodiments 1-10, wherein the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule.

[0469] Embodiment 12. The oncolytic vaccinia virus of embodiment 11, wherein the immune checkpoint modulator is an anti-PD-1 antibody comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising (1) a HVR- L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0470] Embodiment 13. The oncolytic vaccinia virus of embodiment 11, wherein the immune checkpoint modulator is an anti-PD-1 antibody comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (VL) comprising (1) a HVR- L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0471] Embodiment 14. The oncolytic vaccinia virus of any one of embodiments 1-9, wherein the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule.

[0472] Embodiment 15. The oncolytic vaccinia virus of embodiment 14, wherein the immune checkpoint molecule is PD-L1, PD-L2, HHLA-2, CD47, or CXCR4.

[0473] Embodiment 16. The oncolytic vaccinia virus of embodiment 15, wherein the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin, a TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin, or an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin.

[0474] Embodiment 17. The oncolytic vaccinia virus of embodiment 16, wherein the Fc fragment is an IgG4 Fc.

[0475] Embodiment 18. The oncolytic vaccinia virus of any one of embodiments 1-17, further comprising a second nucleic acid encoding a cytokine.

[0476] Embodiment 19. The oncolytic vaccinia virus of embodiment 18, wherein the cytokine is GM-CSF.

[0477] Embodiment 20. An oncolytic virus comprising a first nucleic acid encoding an immune checkpoint modulator, and a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell.

[0478] Embodiment 21. The oncolytic virus of embodiment 20, wherein the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Seneca Valley virus (SVV), adenovirus, Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sinbis virus, and Newcastle disease virus (NDV). [0479] Embodiment 22. The oncolytic virus of embodiment 21, wherein the oncolytic virus is an oncolytic vaccinia virus.

[0480] Embodiment 23. The oncolytic virus of embodiment 22, wherein the oncolytic vaccinia virus is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia. Ankara (MVA), Lister, King, IHD, Evans, USSR, and Western Reserve (WR).

[0481] Embodiment 24. The oncolytic virus of embodiment 23, wherein the oncolytic vaccinia virus is a WR strain.

[0482] Embodiment 25. The oncolytic virus of embodiment 24, wherein the oncolytic vaccinia virus comprises double deletion of thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene.

[0483] Embodiment 26. The oncolytic virus of any one of embodiments 20-25, wherein the immune checkpoint modulator is an activator of a stimulatory immune checkpoint molecule.

[0484] Embodiment 27. The oncolytic virus of any one of embodiments 20-25, wherein the immune checkpoint modulator is an immune checkpoint inhibitor.

[0485] Embodiment 28. The oncolytic virus of embodiment 27, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73.

[0486] Embodiment 29. The oncolytic virus of embodiment 28, wherein the immune checkpoint inhibitor is an inhibitor of PD-1.

[0487] Embodiment 30. The oncolytic virus of any one of embodiments 20-29, wherein the immune checkpoint modulator is an antibody specifically recognizing an immune checkpoint molecule.

[0488] Embodiment 31. The oncolytic virus of embodiment 30, wherein the immune checkpoint modulator is an anti-PD-1 antibody comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising (1) a HVR- L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0489] Embodiment 32. The oncolytic virus of embodiment 30, wherein the immune checkpoint modulator is an anti-PD-1 antibody comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (VL) comprising (1) a HVR- L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0490] Embodiment 33. The oncolytic virus of any one of embodiments 20-28, wherein the immune checkpoint modulator is a ligand that binds to the immune checkpoint molecule.

[0491] Embodiment 34. The oncolytic virus of embodiment 33, wherein the immune checkpoint molecule is PD-L1, PD-L2, HHLA-2, CD47, or CXCR4.

[0492] Embodiment 35. The oncolytic virus of embodiment 34, wherein the immune checkpoint modulator comprises an extracellular domain of PD-1 fused to an Fc fragment of an immunoglobulin, a TMIGD2 extracellular domain fused to an Fc fragment of an immunoglobulin, or an extracellular domain of SIRPĮ and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin.

[0493] Embodiment 36. The oncolytic virus of embodiment 35, wherein the Fc fragment is an IgG4 Fc.

[0494] Embodiment 37. The oncolytic virus of any one of embodiments 20-36, wherein the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3 (GPC3).

[0495] Embodiment 38. The oncolytic virus of embodiment 37, wherein the tumor antigen is EpCAM.

[0496] Embodiment 39. The oncolytic virus of embodiment 37, wherein the tumor antigen is FAP. [0497] Embodiment 40. The oncolytic virus of embodiment 37, wherein the tumor antigen is EGFR.

[0498] Embodiment 41. The oncolytic virus of embodiment 37, wherein the tumor antigen is GPC3.

[0499] Embodiment 42. The oncolytic virus of any one of embodiments 20-41, wherein the effector cell is selected from the group consisting of T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, and NKT-cell.

[0500] Embodiment 43. The oncolytic virus of embodiment 42, wherein the effector cells is a T lymphocyte.

[0501] Embodiment 44. The oncolytic virus of embodiment 43, wherein the T lymphocyte is a cytotoxic T lymphocyte.

[0502] Embodiment 45. The oncolytic virus of any one of embodiments 20-44, wherein the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D.

[0503] Embodiment 46. The oncolytic virus of embodiment 45, wherein the cell surface molecule is CD3.

[0504] Embodiment 47. The oncolytic virus of any one of embodiments 20-46, wherein the first antigen-binding domain is a single chain variable fragment (scFv).

[0505] Embodiment 48. The oncolytic virus of any one of embodiments 20-47, wherein the second antigen-binding domain is a scFv.

[0506] Embodiment 49. The oncolytic virus of any one of embodiments 20-48, wherein the first antigen-binding domain and the second antigen binding domain are connected by a linker.

[0507] Embodiment 50. The oncolytic virus of any one of embodiments 20-49, wherein the first antigen-binding domain is N-terminal to the second antigen-binding domain.

[0508] Embodiment 51. The oncolytic virus of any one of embodiments 20-49, wherein the first antigen-binding domain is C-terminal to the second antigen-binding domain. [0509] Embodiment 52. The oncolytic virus of any one of embodiments 20-51, wherein the first nucleic acid encoding the immune checkpoint modulator is operably linked to a late promoter.

[0510] Embodiment 53. The oncolytic virus of any one of embodiments 20-52, wherein the second nucleic acid encoding the bispecific molecule is operably linked to a late promoter.

[0511] Embodiment 54. The oncolytic virus of embodiment 52 or 53, wherein the late promoter driving the expression of immune checkpoint modulator and/or bispecific molecule is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter.

[0512] Embodiment 55. The oncolytic virus of embodiment 54, wherein the late promoter is F17R.

[0513] Embodiment 56. The oncolytic virus of any one of embodiments 20-55, wherein the oncolytic virus further comprises a third nucleic acid encoding a cytokine.

[0514] Embodiment 57. The oncolytic virus of embodiment 56, wherein the cytokine is GM- CSF.

[0515] Embodiment 58. A pharmaceutical composition comprising the oncolytic virus of any one of embodiments 1-57, and a pharmaceutical acceptable carrier.

[0516] Embodiment 59. A method of treating a cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 58.

[0517] Embodiment 60. The method of embodiment 59, wherein the effective amount is about 10 ⁵ to about 10 ¹³ pfu.

[0518] Embodiment 61. The method of embodiment 60, wherein the effective amount is about 10 ⁹ pfu.

[0519] Embodiment 62. The method of any one of embodiments 59-61, wherein the pharmaceutical composition is administered systemically. [0520] Embodiment 63. The method of embodiment 62, wherein the pharmaceutical composition is administered intravenously.

[0521] Embodiment 64. The method of any one of embodiments 59-61, wherein the pharmaceutical composition is administered locally.

[0522] Embodiment 65. The method of embodiment 64, wherein the pharmaceutical composition is administered intratumorally.

[0523] Embodiment 66. The method of any one of embodiments 59-65, wherein the cancer is a solid tumor.

[0524] Embodiment 67. The method of embodiment 66, wherein the cancer is selected from the group consisting of colorectal cancer, liver cancer, and breast cancer.

[0525] Embodiment 68. The method of any one of embodiments 59-67, further comprising administering to the individual an additional cancer therapy.

[0526] Embodiment 69. The method of embodiment 68, wherein the additional cancer therapy is surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof.

[0527] Embodiment 70. The method of any one of embodiments 59-69, wherein the individual is a human.

[0528] Embodiment 71. An anti-PD-1 antibody comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising (1) a HVR- L1 comprising the amino acid sequence of SEQ ID NO: 4; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0529] Embodiment 72. The anti-PD-1 antibody of embodiment 71, wherein the antigen binding domain of the construct comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14. [0530] Embodiment 73. An anti-PD-1 antibody comprising a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (VL) comprising (1) a HVR- L1 comprising the amino acid sequence of SEQ ID NO: 10; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0531] Embodiment 74. The anti-PD-1 antibody of embodiment 73, wherein the antigen binding domain of the construct comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.

[0532] Embodiment 75. The construct of any one of embodiments 71-74, wherein the anti-PD- 1 antibody is a full length antibody.

[0533] Embodiment 76. The construct of any one of embodiments 71-74, wherein the anti-PD- 1 antibody is a scFv.

[0534] Embodiment 77. An isolated nucleic acid encoding the anti-PD-1 antibody of any one of embodiments 71-76.

[0535] Embodiment 78. The isolated nucleic acid of embodiment 77, comprising a first nucleic acid sequence of SEQ ID NO: 17, and a second nucleic acid sequence of SEQ ID NO: 18.

[0536] Embodiment 79. The isolated nucleic acid of embodiment 77, comprising a first nucleic acid sequence of SEQ ID NO: 19, and a second nucleic acid sequence of SEQ ID NO: 20.

[0537] Embodiment 80. The isolated nucleic acid of any one of embodiments 77-79, wherein the isolated nucleic acid is operably linked to a promoter.

[0538] Embodiment 81. The isolated nucleic acid of embodiment 80, wherein the promoter is a late promoter.

[0539] Embodiment 82. The isolated nucleic acid of embodiment 81, wherein the promoter is a vaccinia virus late promoter. [0540] Embodiment 83. The isolated nucleic acid of embodiment 82, wherein vaccinia virus late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P _7.5k early/late promoter, P _EL early/late promoter, P _11k late promoter, P _SEL synthetic early/late promoter, and P _SL synthetic late promoter.

[0541] Embodiment 84. The isolated nucleic acid of embodiment 83, wherein the vaccinia virus late promoter is F17R.

[0542] Embodiment 85. An isolated host cell comprising the isolated nucleic acid of any one of embodiments 77-84.

[0543] Embodiment 86. An oncolytic virus comprising the nucleic acid of any one of embodiments 77-84.

[0544] Embodiment 87. A pharmaceutical composition comprising the anti-PD-1 antibody of any one of embodiments 71-76, the isolated host cell of embodiment 85, or the oncolytic virus of embodiment 86, and a pharmaceutically acceptable carrier.

[0545] Embodiment 88. A method of treating a cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 87.

[0546] Embodiment 89. The method of embodiment 88, wherein the pharmaceutical composition is administered to the individual intravenously or intratumorally.

[0547] Embodiment 90. The method of embodiment 88 or 89, wherein the individual is a human.

[0548] Embodiment 91. A pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator, a second OV comprising a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, and a pharmaceutical acceptable carrier.

[0549] Embodiment 92. The pharmaceutical composition of embodiment 91, comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator of any one of embodiments 20-36, 52, 54, and 55, a second OV comprising a second nucleic acid encoding a bispecific molecule of any one of embodiments 20, 37-51, and 53-55, and a pharmaceutical acceptable carrier.

[0550] Embodiment 93. A pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator, a second OV comprising a second nucleic acid encoding a cytokine, and a pharmaceutical acceptable carrier.

[0551] Embodiment 94. The pharmaceutical composition of embodiment 93, comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator of any one of embodiments 20-36, 52, 54, and 55, a second OV comprising a second nucleic acid encoding a cytokine of embodiment 56 or 57, and a pharmaceutical acceptable carrier.

[0552] Embodiment 95. A pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator, a second OV comprising a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, a third OV comprising a third nucleic acid encoding a cytokine, and a pharmaceutical acceptable carrier.

[0553] Embodiment 96. The pharmaceutical composition of embodiment 95, comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator of any one of embodiments 20-36, 52, 54, and 55, a second OV comprising a second nucleic acid encoding a bispecific molecule of any one of embodiments 20, 37-51, and 53-55, a third OV comprising a third nucleic acid encoding a cytokine of embodiment 56 or 57, and a pharmaceutical acceptable carrier.

[0554] Embodiment 97. A method of treating a cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of any one of embodiments 91-96.

[0555] Embodiment 98. A method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator, and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, and a second pharmaceutical acceptable carrier.

[0556] Embodiment 99. The method of embodiment 98, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator of any one of embodiments 20-36, 52, 54, and 55, and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a bispecific molecule of any one of embodiments 20, 37-51, and 53-55, and a second pharmaceutical acceptable carrier.

[0557] Embodiment 100. A method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator, and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a cytokine, and a second pharmaceutical acceptable carrier.

[0558] Embodiment 101. The method of embodiment 100, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator of any one of embodiments 20-36, 52, 54, and 55, and a first pharmaceutical acceptable carrier, and an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a cytokine of embodiment 56 or 57, and a second pharmaceutical acceptable carrier.

[0559] Embodiment 102. A method of treating a cancer in an individual, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator, and a first pharmaceutical acceptable carrier, an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a bispecific molecule comprising a first antigen-binding domain specifically recognizing a tumor antigen and a second antigen-binding domain specifically recognizing a cell surface molecule on an effector cell, and a second pharmaceutical acceptable carrier, and an effective amount of a third pharmaceutical composition comprising a third OV comprising a third nucleic acid encoding a cytokine, and a third pharmaceutical acceptable carrier.

[0560] Embodiment 103. The method of embodiment 102, comprising administering to the individual an effective amount of a first pharmaceutical composition comprising a first OV comprising a first nucleic acid encoding an immune checkpoint modulator of any one of embodiments 20-36, 52, 54, and 55, and a first pharmaceutical acceptable carrier, an effective amount of a second pharmaceutical composition comprising a second OV comprising a second nucleic acid encoding a bispecific molecule of any one of embodiments 20, 37-51, and 53-55, and a second pharmaceutical acceptable carrier, and an effective amount of a third pharmaceutical composition comprising a third OV comprising a third nucleic acid encoding a cytokine of embodiment 56 or 57, and a third pharmaceutical acceptable carrier.

EXAMPLES

[0561] The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0562] In this disclosure, oncolytic vaccinia virus expressing the recombinant PD1-IgG4-Fc fusion (hereinafter referred to as“PD1-Ig-VV”), is used to block the co-inhibitory interaction between PD1 ligands on the cancer cell and PD1 receptor on the T cell, thereby resulting in an enhanced anti-tumor immune response. The PD1-IgG4-Fc recombinant protein (hereinafter referred to as“PD1-Ig”) is generated, with the extracellular domain of PD1 fused to the constant (Fc) domain of immunoglobulin G4, effectively creating an inhibitor of the interaction between PD1 and its ligands.

Example 1: Construction of PD1-Ig-VV, GPC3-CD3-VV, FAP-CD3-VV, and PD1-Ig-FAP- CD3-VV

[0563] The oncolytic vaccinia virus (VV) construct PD1-Ig-VV was generated to express a recombinant protein with the extracellular domain of PD-1 fused to the constant (Fc) domain of immunoglobin-G4 (IgG4). The extracellular domain of PD-1 comprises an amino acid sequence of SEQ ID NO: 25. GPC3-CD3 (also hereinafter referred to as GPC3-TE (GPC3 T-cell engager)) is a bispecific molecule targeting the hepatocellular carcinoma (HCC) tumor antigen Glypican-3 (GPC3) and CD3 on T cells. The oncolytic VV construct GPC3-CD3-VV (also hereinafter referred to as GPC3-TEA-VV) was generated to express secretory GPC3-scFv-human CD3-scFv (GPC3-CD3). FAP-CD3 (also hereinafter referred to as FAP-TE) is a bispecific molecule targeting the fibroblast activation protein (FAP) antigen on cancer associated fibroblast and CD3 on T cells. The oncolytic VV construct FAP-CD3-VV (hereinafter also referred to as FAP-TEA- VV) was generated to express secretory FAP-scFv-human CD3-scFv (FAP-CD3, or FAP-TE). The oncolytic VV construct PD1-Ig-FAP-CD3-VV (also hereinafter referred to as PD1-Ig-FAP- TEA-VV (PD1-Ig-FAP-T cell engager armed VV)) was generated to co-express secretory PD1- IgG4-Fc and secretory FAP-scFv-human CD3-scFv (FAP-CD3). The nucleic acid sequences encoding PD1-IgG4-Fc and FAP-CD3 were connected by a T2A self cleaving sequence. The anti-FAP scFv comprises a heavy chain variable region (VH) comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region (VL) comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41. The anti-FAP scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 42, and a VL comprising the amino acid sequence of SEQ ID NO: 43. The anti-FAP scFv comprises an amino acid sequence of SEQ ID NO: 44. The FAP-CD3 bispecific molecule comprises an amino acid sequence of SEQ ID NO: 45. The co-expressed PD1-IgG4-Fc comprises an extracellular domain of PD-1 comprising an amino acid sequence of SEQ ID NO: 25.

[0564] Vaccinia viruses (Western Reserve strain) encoding secretory PD1-IgG4-Fc (PD1-Ig- VV), GPC3-CD3 (GPC3-CD3-VV, or GPC3-TEA-VV), FAP-CD3 (FAP-CD3-VV, or FAP- TEA-VV), or co-expressing secretory PD1-IgG4-Fc and FAP-CD3 (PD1-Ig-FAP-CD3-VV, or PD1-Ig-FAP-TEA-VV) were generated by recombination of a version of pSEM-1 plasmid containing PD1-Ig, T-cell engagers (TE), or PD1-Ig and FAP-CD3 into the TK gene of the VSC20 strain of WR vaccinia virus (WR VV). Firstly, the shuttle vector pSEM-1 was constructed to contain the PD1-Ig, T-cell engagers, or both PD1-Ig and FAP-CD3 (FIG. 1). The inserted PD1-Ig, TE, or both PD1-Ig and FAP-CD3 were expressed under the transcriptional control of the F17R late promoter to allow for sufficient viral replication before T-cell activation. The VVs also expressed YFP-GFP marker to allow for virus selection. YFP-GFP was expressed under the transcriptional control of P7.5 promoter, and loxP sites in the same orientation flanking YFP-GFP, the selectable marker (FIG. 1). To construct the recombinant virus encoding PD1-Ig, TE, or both PD1-Ig and FAP-CD3, firstly, the shuttle vectors pSEM-1 were transfected into human 143 TK- cells. Cells were then infected with virus VSC20 at a multiplicity of infection (MOI) of 0.1. After five rounds of plaque selection and amplification to confirm the expression of PD1-Ig, TE, or both PD1-Ig and FAP-CD3, one of the clones was selected for amplification and purification. To remove the YFP-GFP cassette from the recombinant viruses, viruses were passaged on a U2OS cell line expressing a cytoplasmic form of Cre recombinase (U2OS-Cre). After five rounds of plaque selection and amplification to confirm the expression of PD1-Ig, TE, or both PD1-Ig and FAP-CD3, one of the YFP-GFP-negative clones was selected for amplification and purification.

Example 2: Expression of vaccinia virus encoded PD1-Ig, and test of its binding to PD-L1 on Huh7 cells

[0565] To test for expression of PD1-Ig from the PD1-Ig-VV, human osteosarcoma 143 TK- cells infected with PD1-Ig-VV (as described in Example 1) or VV-GFP control (vaccinia virus encoding GFP) were harvested and the supernatant was analyzed by Western blot. PD1-Ig was detected at 85-90 KDa (FIG.2A).

[0566] To test for binding of VV-expressed PD1-Ig to PD-L1 ligand on Huh7 cells, Huh7 cells were incubated with supernatant from VV-GFP or PD1-Ig-VV infected human 143 TK- cells for two hours. Cells were subjected to FACS analysis for APC-anti-Fc to detect PD1-IgG4-Fc (see below for FACS protocol). Huh7 cells incubated with PD1-Ig supernatant showed an increase in Fc-positive cells when compared to cells incubated with GFP, indicating that PD1-Ig binds to PD-L1 on Huh7 cells (FIG.2B)

FACS [0567] FACS was performed according to general protocols. Briefly, cells were collected and washed once with PBS containing 1% FBS (Sigma, St. Louis, MO; FACS buffer) before the addition of antibodies. Cells were then incubated for 30 minutes on ice in the dark, washed once, and incubated for 30 minutes on ice in the dark with fluorescein-conjugated secondary antibodies (if applicable), and washed once afterwards. The washed cells were subsequently fixed in 0.5% paraformaldehyde/FACS buffer before analysis. For each sample, 10,000 cells were analyzed using FACSCalibur instrument (BD, Becton Dickinson, Mountain View, CA) with the Cell Quest Software (Becton Dickinson) or with FCS Express software (De Novo Software, Los Angeles, CA).

Example 3: PD1-Ig enhanced GPC3-CD3-dependent killing of Huh7-GFP cells mediated by activated T cells

[0568] To test the ability of PD1-Ig to enhance the anti-tumor effects of bispecific T-cell engagers, PD1-Ig was co-expressed with GPC3-CD3, a bispecific molecule targeting the hepatocellular carcinoma (HCC) tumor antigen Glypican-3 (GPC3) and CD3 on T cells (GPC3- scFv-CD3-scFv; see Example 1 for construction). Huh7-GFP cells were infected with empty virus (-GPC3-CD3), vaccinia virus expressing GPC3-CD3 (+GPC3-CD3), or co-infected with vaccinia virus encoding GPC3-CD3 and vaccinia virus encoding PD1-Ig (GPC3-CD3+PD1-Ig) (FIG. 3A). Tumor cells were infected with VVs at a MOI of 1 in 2.5% FBS medium for two hours followed by culturing in complete medium. Unstimulated human PBMCs (see below for protocol) were incubated at 37 ^o C for two hours to remove adherent cells and non-adherent PBMCs were added to Huh7-GFP cell culture at an effector: target (E:T) ratio of 1:1 (FIG. 3A upper panels) or 5:1 (FIG. 3A lower panels). Cells were sorted by FACS for CD3-APC and Huh7-GFP, as described in Example 2, and Huh7 cell viability was assessed by GFP.

Peripheral Blood Mononuclear Cells (PBMC)

[0569] To generate PBMC populations, blood samples from healthy donors were obtained in accordance to protocols approved by the Institutional Review Board of Baylor College of Medicine. Peripheral blood was processed over Ficoll gradients, and the resulting PBMCs were cultured in Roswell Park Memorial Institute 1640 (Thermo Scientific HyClone, Waltham, MA; Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated FCS and 2 mmol/L GLUTAMAX.

[0570] As can be seen from FIG. 3A, there was a decrease in the number of viable Huh7-GFP cancer cells expressing GPC3-CD3 at both E:T ratios when compared to negative control cells (- GPC3-CD3). There was a further decrease in GFP signal in Huh7 cells expressing both PD1-Ig and GPC3-CD3 (GPC3-CD3+PD1-Ig). This indicates that PD1-Ig enhanced GPC3-CD3 mediated Huh7 tumor cell lysis by T cells, and the effect was further augmented at a higher E:T ratio (compare FIG. 3A bottom panels with upper panels). The enhanced tumor lysis effect induced by PD1-Ig in the presence of T cells was also visualized as a decrease in GFP signal using fluorescence microscopy (FIG.3B).

[0571] To further illustrate that PD1-Ig can enhance the effect of GPC3-CD3 mediated tumor cell killing by T cells, VV-infected Huh7 cells were assayed using FACS plotting with apoptotic markers annexin-V and propidium iodide (PI). Briefly, VV-infected Huh7 cells co-cultured with PBMC at a E:T of 5:1 were harvested and incubated with recombinant annexin-V. Just before analysis by flow cytometry, PI was added to all samples. Cells were analyzed for annexin-V binding as well as PI uptake and the percentages of cells that are annexin-V positive, PI positive, or double positive are indicated (FIG.3C).

[0572] Huh7 cells infected with control virus (-GPC3-CD3) had only 8.94% PI/annexin-V double positive cells (FIG. 3C left panel), whereas cells expressing GPC3-CD3 had 24.1% double positive cells (FIG. 3C mid panel). Co-infecting with VV encoding PD1-Ig and VV encoding GPC3-CD3 augmented the percent of annexin-V/PI double positive Huh7 tumor cells to 44.2% (FIG.3C right panel), indicating an increase in tumor lysis. These data indicate that co- expressing PD1-Ig in Huh7 human hepatocarcinoma cells can augment the effect of GPC3-CD3- mediated tumor lysis in the presence of T cells.

Example 4: PD1-Ig did not affect T cell phenotypes in response to GPC3-CD3

[0573] To characterize the effects of PD1-Ig on T cells, unstimulated human PBMCs (1×10 ⁶ /well) were cultured alone or co-incubated with VV-infected Huh7 cells (0.4×10 ⁶ /well) in 24 well plates for two days. Cells were then harvested and stained with fluorescein-conjugated antibodies against cell surface markers CD3 and CD69 (CD69 is a T cell activation marker) (FIG. 4A) or CD45RA and CCR7 (FIG. 4B) followed by FACS analysis, as described in Example 2. The cell population percentages are shown. Antibodies against CD45RA and CCR7 (markers to distinguish CD4+ and CD8+ T cells) were used as a negative control, as PD-1 is expressed primarily on CD4-/CD8- negative T cells (FIG. 4B). PBMC were cultured alone (FIGS. 4A and 4B, left panels) or co-cultured with Huh7 tumor cells infected with empty virus (second panels from left, top and bottom, -GPC3-CD3), VV encoding GPC3-CD3 (second panels from right, GPC3-CD3/medium), or co-infected with vaccinia virus encoding GPC3-CD3 and vaccinia virus encoding PD1-Ig (right panels, GPC3-CD3/PD1-Ig).

[0574] When PBMC were co-cultured with Huh7 cells expressing GPC3-CD3, there was a dramatic increase in CD3+/CD69+ cells to 57.9% (FIG. 4A). The percentage of double positive cells increased mildly to 59.7% in PBMCs co-cultured with Huh7 cells expressing both GPC3- CD3 and PD1-Ig (FIG. 4A, right panel). Thus, FACS detection of CD69, a lymphoid activation antigen, showed a marked increase in T cell activation in PBMCs incubated with GPC3-CD3 expressing Huh7 cells; however, this number did not significantly increase upon addition of PD1-Ig, indicating that PD1-Ig does not affect T cell phenotype.

Example 5: PD1-Ig augmented GPC3-CD3-dependent increase in cytokine production by T cells

[0575] To investigate if the presence of PD1-Ig increases cytokine production by T cells, PBMC were co-cultured with Huh7 cells expressing GPC3-CD3 alone or together with PD1-Ig. For co-expression of GPC3-CD3 and PD1-Ig in Huh7 cells, Huh7 cells were co-infected with VV encoding GPC3-CD3 (GPC3-CD3-VV) and VV encoding PD1-Ig (PD1-Ig-VV). Huh7 cells were infected with VV at a MOI of 1, PBMC were added as described above, and 24h-48h post virus infection cell culture was collected and enzyme-linked immunosorbent assay (ELISA) was used to assay for the presence of pro-inflammatory cytokines IFNȖ, TNFĮ, and IL-2 (FIGS. 5A, 5B, and 5C, respectively). T cells cultured with GPC3-CD3-expressing Huh7 cells produced higher levels of IFNȖ, TNFĮ, and IL-2 when compared to T cells cultured with control virus- infected Huh7 (FIGS. 5A-5C, respectively,“Medium” vs.“-GPC3-CD3”). The release of IFNȖ, TNFĮ, and IL-2 was further amplified when T cells were cultured with Huh7 cells expressing both GPC3-CD3 and PD1-Ig (FIGS. 5A-5C, respectively,“PD1-Ig”). The amount of IFNȖ, TNFĮ, and IL-2 release in the presence of GPC3-CD3 and PD1-Ig was even greater than that in the presence of GPC3-CD3 and anti-PD-1 antibody (FIGS. 5A-5C, respectively,“ĮPD1”; Cat#10377-mhT28-200, Sino Biological) or GPC3-CD3 and anti-PD-L1 antibody (FIGS.5A-5C, respectively,“ĮPD-L1”; Clone 29E.2A3, BXCell). This indicates that PD1-Ig can augment the ability of GPC3-CD3 to induce cytokine release by T cells.

Example 6: PD1-Ig-VV inhibits SK-BR-3 breast cancer tumor growth in vivo

[0576] The in vivo efficacies of PD1-Ig-VV were evaluated in a mouse xenograft model, in which human tumor cells were implanted. To establish a mouse xenograft model of breast cancer, 4×10 ⁶ SK-BR-3 human breast cancer cells were inoculated subcutaneously into the right flank of NSG mice. This was followed by injection of PBS, 1×10 ⁸ pfu of control-VV, or PD1-Ig- VV into the right flank tumor on day 8, and i.v. implantation of 2×10 ⁷ unactivated human PBMC cells on day 11 (FIG. 6A). Mice received PBS/PBMC only served as controls. In an analysis of tumor volume, mice received PBS/PBMC developed a tumor volume of approximately 3600 mm ³ by day 21 (FIG. 6B and FIG. 6C left panel). Mice received control-VV had moderately reduced tumor volume in the presence of PBMC (FIG. 6B and FIG. 6C mid panel). Mice injected with PD1-Ig-VV showed a significantly lower tumor volume at 21 days in the presence of PBMC (FIG. 6B and FIG. 6C right panel), indicating that PD1-Ig-VV can inhibit SK-BR-3 breast cancer tumor growth in vivo.

Example 7: PD1-Ig-VV inhibits HT-29 colorectal adenocarcinoma tumor growth in vivo

[0577] To extend the ability of PD1-Ig-VV in inhibiting tumor growth to another tumor model, the HT-29 colorectal cancer xenograft model was employed. To establish this tumor model, 4×10 ⁶ HT-29 colorectal adenocarcinoma cells were inoculated subcutaneously into the right flank of NSG mice, followed by injection of PBS, 1×10 ⁸ pfu of control-VV, or PD1-Ig-VV into the right flank tumor on day 8, followed by i.v. implantation of 2×10 ⁷ unactivated human PBMC cells on day 11 (FIG. 7A). An examination of tumor volume over the course of 24 days showed that control mice injected with PBS alone or PBS in the presence of PBMC developed tumors of approximately an average of 1000 mm ³ at day 24 (FIG.7B). Mice injected with the control virus GFP-VV and implanted with PBMC had a somewhat reduced tumor volume (approximately average of 350 mm ³ ). However, mice injected with PD1-Ig-VV and implanted with PBMC showed a dramatic inhibition of tumor growth, with an average tumor size of close to 0 mm ³ . These results suggest that PD1-Ig-VV can strongly inhibit in vivo tumor growth in multiple mouse xenograft models.

Example 8: Vaccinia virus co-expressing PD1-Ig and FAP-CD3, and test of their binding to PD-L1 and FAP on U87 cells

[0578] To test for the co-expression of PD1-Ig and FAP-CD3 from PD1-Ig-FAP-CD3-VV, human osteosarcoma 143 TK- cells infected with PD1-Ig-FAP-CD3-VV (as described in Example 1) or VV-GFP control (VV encoding GFP) are harvested and the supernatant is analyzed by Western blot.

[0579] To test for the binding of VV-expressed PD1-Ig to PD-L1 ligand on U87 (human glioblastoma) cells, U87 cells are incubated with supernatant from VV-GFP or PD1-Ig-FAP- CD3-VV infected human 143 TK- cells for two hours. Cells are subjected to FACS analysis for APC-anti-Fc to detect PD1-IgG4-Fc (see Example 2 for FACS protocol). U87 cells incubated with PD1-Ig supernatant are expected to show an increase in Fc-positive cells when compared to cells incubated with GFP supernatant, indicating that PD1-Ig binds to PD-L1 on U87 cells.

[0580] To test for the binding of VV-expressed FAP-CD3 to FAP antigen on FAP-positive U87 cells, U87 cells are incubated with supernatant from VV-GFP or PD1-Ig-FAP-CD3-VV infected human 143 TK- cells for two hours. Cells are subjected to FACS analysis for anti-CD3 scFv antibody to detect FAP-CD3 (FAP-TE) (see Example 2 for FACS protocol). U87 cells incubated with FAP-CD3 supernatant are expected to show an increase in FAP-CD3-positive cells when compared to cells incubated with GFP supernatant, indicating that FAP-CD3 binds to FAP on U87 cells. Example 9: PD1-Ig enhances FAP-CD3-dependent killing of U87 cells mediated by activated T cells

[0581] To test the ability of PD1-Ig to enhance the anti-tumor effects of bispecific T-cell engagers, U87-GFP cells are infected with empty virus, vaccinia virus encoding FAP-CD3 only (FAP-CD3-VV), or VV co-expressing FAP-CD3 and PD1-Ig (PD1-Ig-FAP-CD3-VV). See Example 1 for construction. Tumor cells are infected with VVs at a MOI of 1 in 2.5% FBS medium for two hours followed by culturing in complete medium. Unstimulated human PBMCs (see Example 3 for protocol) are incubated at 37 ^o C for two hours to remove adherent cells, and non-adherent PBMCs are added to U87-GFP cell culture at an effector: target (E:T) ratio of 1:1 or 5:1. Cells are sorted by FACS for CD3-APC and GFP, as described in Example 2, and U87- GFP cell viability is assessed by GFP.

[0582] There is expected to be a decrease in the number of viable U87-GFP cancer cells expressing FAP-CD3 at both E: T ratios when compared to negative control cells (empty virus). There is expected to be a further decrease in GFP signals in U87-GFP cells co-expressing PD1- Ig and FAP-CD3 (PD1-Ig-FAP-CD3-VV). This indicates that co-expressing PD1-Ig and FAP- CD3 can enhance FAP-CD3 mediated U87 tumor cell lysis by T cells, and the effect is expected to further augment at a higher E: T ratio. The enhanced tumor lytic effect induced by PD1-Ig in the presence of T cells is also visualized as a decrease in GFP signal using fluorescence microscopy. U87-GFP cells infected by PD1-Ig-FAP-CD3-VV are expected to have the weakest GFP signal compared to those infected by FAP-CD3-VV or empty virus.

[0583] To further illustrate that co-expressing PD1-Ig and FAP-CD3 can enhance the effect of FAP-CD3 mediated tumor cell killing by T cells, VV-infected U87 cells are assayed using FACS plotting with apoptotic markers annexin-V and propidium iodide (PI). Briefly, VV-infected U87 cells co-cultured with PBMC at a E:T of 5:1 are harvested and incubated with recombinant annexin-V. Just before analysis by flow cytometry, PI is added to all samples. Cells are analyzed for annexin-V binding as well as PI uptake and the percentages of cells that are annexin-V positive, PI positive, or double positive are indicated.

[0584] U87 cells infected with control virus are expected to have the lowest percentage of PI/annexin-V double positive cells, whereas cells infected by FAP-CD3-VV have the medium level of double positive cells. Co-expressing PD1-Ig and FAP-CD3 (PD1-Ig-FAP-CD3-VV) is expected to augment the percent of annexin-V/PI double positive tumor cells to a higher level, indicating an increase in tumor lysis. These data may indicate that co-expressing PD1-Ig and FAP-CD3 in U87 tumor cells can augment the effect of FAP-CD3-mediated tumor lysis in the presence of T cells.

Example 10: PD1-Ig and FAP-CD3 co-expression does not affect T cell phenotypes

[0585] To characterize the effects of co-expressed PD1-Ig and FAP-CD3 (PD1-Ig-FAP-CD3- VV) on T cells, unstimulated human PBMCs (1×10 ⁶ /well) are cultured alone or co-incubated with VV-infected U87 cells (0.4×10 ⁶ /well) in 24 well plates for two days. Cells are then harvested and stained with fluorescein-conjugated antibodies against cell surface markers CD3 and CD69 (CD69 is a T cell activation marker), or CD45RA and CCR7 (markers to distinguish CD4+ and CD8+ T cells) followed by FACS analysis, as described in Example 2. Antibodies against CD45RA and CCR7 are used as a negative control, as PD-1 is expressed primarily on CD4-/CD8- negative T cells. PBMC are cultured alone or co-cultured with U87 cells infected with empty virus, VV encoding FAP-CD3 (FAP-CD3-VV), or VV co-expressing FAP-CD3 and PD1-Ig (PD1-Ig-FAP-CD3-VV).

[0586] When PBMC are co-cultured with U87 cells expressing FAP-CD3, there is expected to be a dramatic increase in CD3+/CD69+ cells. The percentage of double positive cells in PBMCs co-cultured with U87 cells co-expressing both FAP-CD3 and PD1-Ig (PD1-Ig-FAP-CD3-VV) is expected to be similar to that when FAP-CD3 is expressed alone. Thus, FACS detection of CD69, a lymphoid activation antigen, is expected to show a marked increase in T cell activation in PBMCs incubated with FAP-CD3 expressing U87 cells; and this number may not significantly increase upon co-expressing PD1-Ig with FAP-CD3, indicating that PD1-Ig may not affect T cell phenotype.

Example 11: PD1-Ig augments FAP-CD3-dependent increase in cytokine production by T cells

[0587] To investigate if co-expressing PD1-Ig and FAP-CD3 increases cytokine production by T cells, PBMC is co-cultured with U87 cells infected with PD1-Ig-FAP-CD3-VV, empty virus, or FAP-CD3-VV, or U87 cells treated with anti-PD-1 or anti-PD-L1 antibodies. U87 cells are infected with VV at a MOI of 1, PBMC is added as described above, and 24h-48h post virus infection cell culture is collected and ELISA is used to assay for the presence of pro- inflammatory cytokines IFNȖ, TNFĮ, and IL-2. T cells cultured with FAP-CD3-VV infected U87 cells are expected to produce higher levels of IFNȖ, TNFĮ, and IL-2 when compared to T cells cultured with control virus-infected cancer cells. The release of IFNȖ, TNFĮ, and IL-2 is expected to be further amplified when T cells are cultured with U87 cells co-expressing FAP3- CD3 and PD1-Ig (PD1-Ig-FAP-CD3-VV). The amount of IFNȖ, TNFĮ, and IL-2 release in the presence of FAP-CD3 and PD1-Ig is compared to that in the presence of FAP-CD3 and anti-PD- 1 antibody (Cat#10377-mhT28-200, Sino Biological) or FAP-CD3 and anti-PD-L1 antibody (Clone 29E.2A3, BXCell). These results may indicate that co-expressing PD1-Ig and FAP-CD3 using PD1-Ig-FAP-CD3-VV can augment the ability of FAP-CD3 to induce cytokine release by T cells.

Example 12: PD1-Ig-FAP-CD3-VV inhibits U87 cancer tumor growth in vivo

[0588] The in vivo efficacies of PD1-Ig-FAP-CD3-VV are evaluated in a mouse xenograft model, in which U87 human cancer cells are implanted. To establish a mouse xenograft model of U87 cancer, 4×10 ⁶ U87 human cancer cells are inoculated subcutaneously into the right flank of NSG mice. This is followed by injection of PBS, 1×10 ⁸ pfu of control-VV, PD1-Ig-VV, FAP- CD3-VV, or PD1-Ig-FAP-CD3-VV into the right flank tumor on day 8, and i.v. implantation of 2×10 ⁷ unactivated human PBMC cells on day 11. Mice received PBS/PBMC only serves as controls. In an analysis of tumor volume on day 21, mice received PBS/PBMC only is expected to develop tumor with the highest volume. Mice received control-VV are expected to have moderately reduced tumor volume in the presence of PBMC. Mice injected with PD1-Ig-VV or FAP-CD3-VV are expected to show an even lower tumor volume at 21 days in the presence of PBMC, while mice injected with PD1-Ig-FAP-CD3-VV are expected to have the smallest tumor volume, likely due to the destruction of tumor stroma. These results may indicate that co- expressing PD1-Ig and FAP-CD3 can inhibit U87 tumor growth in vivo, possibly even better than PD1-Ig-VV or FAP-CD3-VV alone. SEQUENCE LISTING

SEQ ID NO: 1 (1H7e3 HVR-H1 amino acid sequence)

DYYIN SEQ ID NO: 2 (1H7e3 HVR-H2 amino acid sequence)

WIYPGSGNTKYNEKFKG SEQ ID NO: 3 (1H7e3 HVR-H3 amino acid sequence)

AFYRYDVGAY SEQ ID NO: 4 (1H7e3 HVR-L1 amino acid sequence)

KASQNVGTNVA SEQ ID NO: 5 (1H7e3 HVR-L2 amino acid sequence)

SASYRYS SEQ ID NO: 6 (1H7e3 HVR-L3 amino acid sequence)

QQYNSYPWT SEQ ID NO: 7 (4F11C3 HVR-H1 amino acid sequence)

DYYIN SEQ ID NO: 8 (4F11C3 HVR-H2 amino acid sequence)

WIYPGTGNTKYNEKFKG SEQ ID NO: 9 (4F11C3 HVR-H3 amino acid sequence)

SYYRYDVGAY SEQ ID NO: 10 (4F11C3 HVR-:L1 amino acid sequence)

KASQNVGTNVA SEQ ID NO: 11 (4F11C3 HVR-L2 amino acid sequence)

SASYRYS SEQ ID NO: 12 (4F11C3 HVR-L3 amino acid sequence)

QQYNIYPWT SEQ ID NO: 13 (1H7e3 VH amino acid sequence: HVRs are underlined)

QIQLQQSGPELVKPGASVKISCKASGYTFTDYYINWVKQKPGQGLEWIGWIYPGSGN TKYNEKFKGKATLT VDTSSSTAYMQLSSLTSEDTAVYFCEGAFYRYDVGAYWGQGTLVTVSA SEQ ID NO: 14 (1H7e3 VL amino acid sequence: HVRs are underlined)

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSG VPDRFTGSGSGTD FTLTISNVQSEDLAEYFCQQYNSYPWTFGGGTKLEIK SEQ ID NO: 15 (4F11C3 VH amino acid sequence: HVRs are underlined)

QIQLQQSGPELVKPGASVKISCKASGYTFTDYYINWVKQKPGQGLEWIGWIYPGTGN TKYNEKFKGKATL TVDTSSSTAYMQLSSLTSEDTAVYFCEGSYYRYDVGAYWGQGTLVTVSA SEQ ID NO: 16 (4F11C3 VL amino acid sequence: HVRs are underlined)

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSG VPDRFTGSGSGTD FTLTISNVQSEDLAEYFCQQYNIYPWTFGGGTKLEIK SEQ ID NO: 17 (nucleic acid encoding anti-PD-11H7e3 VH: HVR sequences are underlined)

CAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAG ATATCCTGCAA GGCTTCTGGCTACACCTTCACTGACTACTATATAAACTGGGTGAAGCAGAAGCCTGGACA GGGACTTG AGTGGATTGGATGGATTTATCCTGGAAGCGGTAATACTAAGTACAATGAGAAGTTCAAGG GCAAGGC CACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATC TGAGGACA CTGCTGTCTATTTCTGTGAGGGGGCCTTCTATAGGTACGACGTTGGAGCTTACTGGGGCC AAGGGACTC TGGTCACTGTCTCTGCA SEQ ID NO: 18 (nucleic acid encoding anti-PD-11H7e3 VL: HVR sequences are underlined)

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTC AGCGTCACCTG CAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATC TCCTAAA GCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGT GGATCTGG GACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTG TCAGCAAT ATAACAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA SEQ ID NO: 19 (nucleic acid encoding anti-PD-14F11C3 VH: HVR sequences are underlined) CAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATA TCCTGCAA GGCTTCTGGCTACACCTTCACTGACTACTATATAAACTGGGTGAAGCAGAAGCCTGGACA GGGACTTG AGTGGATTGGATGGATTTATCCTGGAACCGGTAATACTAAGTACAATGAGAAGTTCAAGG GCAAGGCC ACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCT GAGGACAC TGCTGTCTATTTCTGTGAGGGGTCCTACTATAGGTACGACGTTGGAGCTTACTGGGGCCA AGGGACTCT GGTCACTGTCTCTGCA SEQ ID NO: 20 (nucleic acid encoding anti-PD-14F11C3 VL: HVR sequences are underlined)

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTC AGCGTCACCTG CAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATC TCCTAAA GCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGT GGATCTGG GACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTG TCAGCAAT ATAACATCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA SEQ ID NO: 21 (amino acid sequence of 1H7e3, 4F11C3 VH signal peptide)

MGWSWIFLFLLSGTAGVHC SEQ ID NO: 22 (nucleic acid encoding 1H7e3, 4F11C3 VH signal peptide)

ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTGTCCATTGC SEQ ID NO: 23 (amino acid sequence of 1H7e3, 4F11C3 VL signal peptide)

MGIKMESQTQVFVYMLLWLSGVDG SEQ ID NO: 24 (nucleic acid encoding 1H7e3, 4F11C3 VL signal peptide)

ATGGGCATCAAGATGGAGTCACAGACTCAGGTCTTTGTATACATGTTGCTGTGGTTG TCTGGTGTTGAT GGA SEQ ID NO: 25 (PD-1 extracellular domain amino acid sequence)

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS NTSESFVLNWYR MSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLA PKAQIKESLRA ELRVTERRAEVPTAHPSPSPRPAGQFQ SEQ ID NO: 26 (PD-1 extracellular domain nucleic acid sequence)

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGG CGGCCAGGATG GTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTTCCCAGCCCTGCTCGT GGTGACCGA AGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAA CTGGTACC GCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGC CCGGCCA GGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGT CAGGGCCC GGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGA TCAAAGAG AGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCC AGCCCCT CACCCAGGCCAGCCGGCCAGTTCCAA SEQ ID NO: 27 (TMIGD2 extracellular domain amino acid sequence)

LSVQQGPNLLQVRQGSQATLVCQVDQATAWERLRVKWTKDGAILCQPYITNGSLSLG VCGPQGRLSWQA PSHLTLQLDPVSLNHSGAYVCWAAVEIPELEEAEGNITRLFVDPDDPTQNRNRIASFPG SEQ ID NO: 28 (signal peptide amino acid sequence of TMIGD2 extracellular domain-Fc fusion protein) MGSPGMVLGLLVQIWALQEASS SEQ ID NO: 29 (SIRPĮ extracellular domain amino acid sequence)

EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHF PRVTTVSDLTKRNNM DFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATP QHTVSFTCESHG FSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVT LQGDPLRGTAN LSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTEN KDGTYNWMS WLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY SEQ ID NO: 30 (CXCL12 N terminal sequence)

KRYSLSVGK SEQ ID NO: 31 (amino acid sequence of linker (IgG1 hinge) between SIRPĮ extracellular domain and CXCL12) EPKSCDKTHTCPPCP SEQ ID NO: 32 (signal peptide amino acid sequence of SIRPĮ-CXCL12-Fc fusion protein)

MEPAGPAPGRLGPLLCLLLAASCAWSGVAG SEQ ID NO: 33 (VH-VL linker amino acid sequence)

GGGGSGGGGSGGGGS SEQ ID NO: 34 (VH-VL linker amino acid sequence)

GGGGSGGGGSGGSA SEQ ID NO: 35 (scFv-scFv linker amino acid sequence)

GGGGS SEQ ID NO: 36 (FAP HVR-H1 amino acid sequence)

ENIIH SEQ ID NO: 37 (FAP HVR-H2 amino acid sequence)

WFHPGSGSIKYNEKFKD SEQ ID NO: 38 (FAP HVR-H3 amino acid sequence)

HGGTGRGAMDY SEQ ID NO: 39 (FAP HVR-L1 amino acid sequence)

RASKSVSTSAYSYMH SEQ ID NO: 40 (FAP HVR-L2 amino acid sequence)

LASNLES SEQ ID NO: 41 (FAP HVR-L3 amino acid sequence)

QHSRELPYT SEQ ID NO: 42 (FAP VH amino acid sequence: HVR sequences are underlined)

QVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWVKQRSGQGLEWIGWFHPGSGS IKYNEKFKDKATLT ADKSSSTVYMELSRLTSEDSAVYFCARHGGTGRGAMDYWGQGTSVTVSS SEQ ID NO: 43 (FAP VL amino acid sequence: HVR sequences are underlined)

QILMTQSPASSVVSLGQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASN LESGVPPRFSGSGSG TDFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKRA SEQ ID NO: 44 (FAP scFv amino acid sequence: HVR sequences are underlined, linker sequence is bolded) QVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWVKQRSGQGLEWIGWFHPGSGSIKY NEKFKDKATLT ADKSSSTVYMELSRLTSEDSAVYFCARHGGTGRGAMDYWGQGTSVTVSSGGGGSGGGGSG GSAQILMT QSPASSVVSLGQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASNLESGVPPR FSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKRA SEQ ID NO: 45 (full length FAP-human CD3 TE amino acid sequence: HVR sequences are underlined, VH-VL linker sequences are bolded, scFv-scFv linker sequence is bolded and italicized)

QVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWVKQRSGQGLEWIGWFHPGSGS IKYNEKFKDKATLT ADKSSSTVYMELSRLTSEDSAVYFCARHGGTGRGAMDYWGQGTSVTVSSGGGGSGGGGSG GSAQILMT QSPASSVVSLGQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASNLESGVPPR FSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKRAGGGGSDIKLQQSGAELARPGASVK MSCKTSGYTFT RYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSED SAVYYCARY YDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRA SSSVSYMN WYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSN PLTFGAGTKLE LKS