CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Application Serial No. 62/771,928, filed November 27, 2018, and U.S. Provisional Application Serial No.

62/826,439, filed March 29, 2019 each of which is incorporated herein by reference.

SEQUENCE LISTING

[002] The Sequence Listing written in file 52263 l_SeqListing_ST25 is 143 kilobytes in size, was created November 22, 2019, and is hereby incorporated by reference.

BACKGROUND

[003] Cancer immunoediting is responsible for eliminating tumors and sculpting the immunogenic phenotypes of tumors that eventually form in immunocompetent hosts following tumor escape from immune destruction. Immune system-tumor interactions are postulated to occur in three continuous phases: elimination, equilibrium, and escape. Elimination entails the destructi on of tumor cells by T lymphocytes. In equilibrium, a population of immune-resistant tumor cells appears. During escape, the tumor has developed strategies to evade immune detection or destruction. Escape may occur through loss or ineffective presentation of tumor antigens, secretion of inhibitory cytokines, or downregulation of major histocompatibility complex molecules.

[004] Cancer immunotherapy aims to elicit successful T-cell response that leads to cancer regression. Various efforts have been made to activate effector T-cell responses, such as though presentation of tumor antigen by antigen presenting cells (APCs), prime T cells to successfully target and infiltrate tumors, and enhancing infiltrating T cells to bind to the MHCI-peptide complex to activate the cytotoxic T-cell response.

[005] Studies have shown a survival benefit associated with the presence of tumor infiltrating lymphocytes (TILs). There is evidence that immunostimulatory cytokines, such as IL-12, can increase the immune cell infiltrate in solid tumors. However, systemic administration of IL-12 has a narrow therapeutic index and is often accompanied by unacceptable levels of adverse events. The limitations of systemic administration of IL-12 can be overcome by therapies that result in local expression of IL-12, such as intratumoral electroporation of plasmid encoding IL-12.

[006] While IL-12 can increase the number of TILs, there remains a need to increase the presence and number of tumor-specific T cells in a tumor. CD3 (cluster of

differentiation 3) T cell co-receptor helps to activate both the cytotoxic T cell (CD8+ naive T cells) and also T helper cells (CD4+ naive T cells). Because of its role in activating T cell response, anti-CD3 antibodies have been explored for use as immunosuppressant therapies. Bispecific antibodies, including Bi-specific T-celi engagers (BiTEs), targeting CD3 and a cancer antigen (tumor marker) have been developed to target T cells to cancer cells.

SUMMARY

[007] Described are expression cassettes encoding CXCL9, CXCL9 plus IL-12, anti- CTLA-4 scFv, anti-CTLA-4 scFv plus IL-12, CD3 half-BiTE and CD3 half-BiTE plus IL- 12. The describe expression cassettes are useful in the treatment of cancer. Methods of using the described expression cassettes to treat tumors, including cancers and metastatic cancers, are also described. The described expression cassettes, when delivered to a tumor, such as by electroporation, result in local tumor expression of the encoded proteins, leading to T cell recruitment and anti-tumor activity. In some embodiments, the methods also result in abscopal effects, i.e., regression of one or more untreated tumors. In some embodiments, regression includes debulking of a solid tumor.

[008] Expression cassettes encoding CXCL9 are described. In some embodiments, an expression cassette encoding CXCL9 further encodes IL-12. The described CXCL9 expression cassettes can be delivered intratumorally, peritumorally, into a lymph node, intradermally, and/or intramuscularly. In some embodiments, the CXCL9 and IL12 coding sequences are expressed on a multi cistronic expression cassette from a single promoter and separated by an IRES or 2A translation modification element. In some embodiments, the 2A element is a P2A element. IL-12 is a heterodimeric cytokine having both IL-12A (p35) and IL-12B (p40) subunits. The encoded IL-12 can comprise a fusion construct encoding an IL-12 p35— I L- 12 p40 fusion protein (IL12 p70). In some embodiments, the IL-12 p35 and p40 coding sequences are expressed from a multi cistronic expression cassette from a single promoter and separated by an IRES or 2A element. In some embodiments, the 2A element is a P2A element. In some embodiments, multi cistronic expression cassettes are described, comprising CXCL9, IL12 p35, and IL-12 p40 coding regions separated by IRES or 2A elements. In some embodiments, the 2A element is a P2A element. [009] Expression cassettes encoding anti-CTLA-4 scFv’s are described. An anti- CTLA-4 scFv comprises an anti-CTLA-4 single-chain variable fragment. The described anti-CTLA-4 scFv expression cassettes can be delivered intratumorally, peritumorally, into a lymph node, intradermally, and/or intramuscularly. The lymph node can be a draining lymph node. An anti-CTLA-4 scFv expression cassette can also be delivered in a peritumoral region between the tumor and the draining lymph node. For each of intratumoral, peritumoral, lymph node, intradermal, and/or intramuscular delivery of an anti-CTLA-4 scFv expression cassette, the delivery can be facilitated by electroporation. Direct expression of an anti-CTLA-4 scFv expression cassette can result in fewer side effects and/or toxicity when compared to systemic administration of anti-CTLA-4 antibodies. The described anti-CTLA-4 scFv expression cassettes facilitate delivery of local yet efficacious dose of anti-CTLA-4.

[0010] CD3 half-BiTEs and expression cassettes encoding CD3 half-BiTEs are described. CD3 half-BiTEs comprise anti-CD3 single-chain variable fragment (scFv) fused to a transmembrane domain (TM). In some embodiments, an expression cassette encoding a CD3 half-BiTE further encodes a signal peptide. The encoded signal peptide can be operably linked to the 5' end of the anti-CD3 single-chain variable fragment coding sequence. In some embodiments, an expression cassette encoding a CD3 half-BiTE further encodes IL-12. The described CD3 half-BiTE expression cassettes can be delivered intratumorally, peritumorally, into a lymph node, intradermally, and/or intramuscularly. In some embodiments, the CD3 half-BiTE and IL12 coding sequences are expressed on a multi cistronic expression cassette from a single promoter and separated by an IRES or 2A translation modification element. In some embodiments, the 2A element is a P2A element. IL-12 is heterodimeric cytokine having both IL-12A (p35) and IL-12B (p40) subunits. The encoded IL-12 can contain a fusion construct encoding an IL-12 p35 IL-12 p40 fusion protein (IL12 p70). In some embodiments, the IL-12 p35 and p40 coding sequences are expressed from a multicistronic expression cassette from a single promoter and separated by an IRES or 2A translation modification element. In some embodiments, the 2A element is a P2A element. In some embodiments, multicistronic expression cassettes are described, comprising a CD3 half-BiTE, IL12 p35, and IL-12 p40 coding regions separated by IRES or 2A translation modification elements. In some embodiments, the 2A element is a P2A element.

[0011] Described are methods of treating a cancer comprising administering to a subject, by intratumoral electroporation (IT-EP), a composition comprising a

therapeutically effective amount one or more of the described expression cassettes. The composition is injected into a tumor, tumor microenvironment, and/or tumor margin tissue and electroporation therapy is applied to the tumor, tumor microenvironment, and/or tumor margin tissue. The electroporation therapy may be applied by any suitable electroporation system known in the art. In some embodiments, the electroporation is at a field strength of about 60 V/cm to about 1500 V/c , and a duration of about 10 microseconds to about 20 milliseconds. In some embodiments, the electroporation incorporates Electrochemical Impedance Spectroscopy (EIS). The subject can be a mammal. The mammal can be, but is not limited to, a human, canine, feline, or equine.

[0012] In some embodiments, the methods further comprise administering to the subject a therapeutically effect amount of an immunostimulatory cytokine. The

immunostimulatory cytokine can be an expression cassette encoding the

immunostimulatory cytokine delivered by IT-EP. The immunostimulatory cytokine can be, but is not limited to, IL-12. The immunostimulatory cytokine can be delivered prior to, subsequent to, or concurrent with one or more of the described CXCL9, CTLA-4 scFv and CDS half-BiTE expression cassettes.

[0013] In some embodiments, the methods further comprise administration of one or more additional therapies. The one or more additional therapies can be, but are not limited to, immune checkpoint therapy. Immune checkpoint therapy can be, but is not limited to, administration of one or more immune checkpoint inhibitors. "Immune checkpoint" molecules refer to a group of immune cell surface receptor/ligands which induce T cell dysfunction or apoptosis. These immune inhibitory targets attenuate excessive immune reactions and ensure self-tolerance. Tumor cells harness the suppressive effects of these checkpoint molecules. Immune checkpoint target molecules include, but are not limited to, Cytotoxic T Lymphocyte Antigen-4 (CTLA-4), Programmed Death 1 (PD-1),

Programmed Death Ligand 1 (PD-L1), Lymphocyte Activation Gene-3 (LAG-3), T cell Immunoglobulin Mucin-3 (TIM3), Killer Cell Immunoglobulin-like Receptor (MR), B- and T- Lymphocyte Attenuator (BTLA), Adenosine A2a Receptor (A2aR), and Herpes Virus Entry Mediator (HVEM). "Immune checkpoint inhibitors" include molecules that prevent immune suppression by blocking the effects of immune checkpoint molecules. Checkpoint inhibitors include, but are not limited to, antibodies and antibody fragments, nanobodies, diabodies, soluble binding partners of checkpoint molecules, small molecule therapeutics, and peptide antagonists. An immune checkpoint inhibitor can be, but is not limited to, a PD-1 and/or PD-Ll antagonist. A PD-i and/or PD-Li antagonist can be, but is not limited to, an anti-PD-1 or anti-PD-Ll antibody. Anti-PD-l/PD-Ll antibodies include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, and

atezolizumab.

[0014] Described are methods of treating a tumor in a subject comprising:

administering at least one treatment cycle to the subject, the cycle comprising:

administering to the tumor, by IT-EP, a composition comprising a therapeutically effective amount of one or more of the described CXCL9, CXCL9 plus IL-12 (i.e., IL12~CXCL9 ), anti-CTLA-4 scFv, anti-CTLA-4 scFv plus IL-12, CD3 half-BiTE, or CD3 half-BiTE plus IL-12 (i.e., CD3 half-BiTE~IL12) expression cassettes. In some embodiments, the cycle is a three week cycle. In some embodiments, the cycle is a four, five, or six week cycle. The composition can be administered by IT-EP on 1, 2, 3, 4, 5, or 6 days of a cycle. In some embodiments, the composition is administered by IT-EP on day 1 of each cycle. In some embodiments, the composition administered by IT-EP on days 1 and 5±2 of each cycle. In some embodiments, the composition is administered by IT-EP on days 1 and 8±2 of each cycle. In some embodiments, the composition is administered by IT-EP on days 1, 5±2, and 8±2 of each cycle. The cycles can be repeated as often as is necessary to treat the subject. In some embodiments, a cycle further comprises administration of an additional therapeutic. The additional therapeutic can be, but is not limited to, an immune checkpoint therapy. In some embodiments, the immune checkpoint therapy is administered to the subject on day 1, 2, or 3 of the cycle.

[0015] In some embodiments, a subject is treated with one of more cycles of IT-EP therapy with one or more of the described expression cassettes. Any of the above cycles can be repeated in subsequent cycles. The subsequent cycles can be consecutive cycles or alternating cycles. Alternating cycles can have one or more intervening cycles of no therapy of alternative therapy (e.g., immune checkpoint therapy). For example, any of the described expression cassettes can be administered on days 1, 5±2, and 8±2 of alternating cycles (e.g., cycles 1, 3, 5, etc. as needed) and an alternative therapy can be administered, e.g., on day 1, 2, or 3, of consecutive cycles (e.g., cycles 1, 2, 3, 4, 5, etc. as needed).

[0016] In some embodiments, a subject is administered alternating cycles of IT-EP of any of the described CXCL9, CTLA-4 scFv, and/or CD3 half-BiTE expression cassettes, with or without immune checkpoint inhibitor therapy, and immune checkpoint inhibitor therapy. In other words, a subject can be administered, by IT-EP, a composition comprising a therapeutically effective amount of one or more of the described CXCL9, CXCL9 plus IL-12, anti-CTLA-4 scFv, anti-CTLA-4 scFv plus IL-12, CD3 half-BiTE, or CD3 half-BiTE plus IL-12 expression cassettes and optionally administered immune checkpoint inhibitor therapy on odd numbered cycles (cycles 1 , 3, etc.) and administered immune checkpoint inhibitor therapy on even numbered cycles (cycles 2, 4, etc.).

Alternatively, a patient can be administered immune checkpoint inhibitor therapy on odd numbered cycles (cycles 1, 3, etc.) and administered, by IT-EP, a composition comprising a therapeutically effective amount of one or more of the described CXCL9, CXCL9 plus IL-12, anti-CTLA-4 scFv, anti-CTLA-4 scFv plus IL-12, CD3 half-BiTE, or CD3 half- BiTE plus IL-12 expression cassettes and optionally administered immune checkpoint inhibitor therapy on even numbered cycles (cycles 2, 4, etc.).

[0017] The expression cassettes and methods can be used to treat a subject having advanced, metastatic, treatment refractory tumor. A treatment refractory tumor can be, but is not limited to, an immune checkpoint inhibitor refractory tumor, a hormone refractory tumor, a radiation refractory tumor, and a chemotherapy refractory tumor. In some embodiments, the subject has failed to respond to at least one course of immune checkpoint inhibitor therapy. In some embodiments, the subject is progressing on or has progressed on one or more anti-cancer therapies, such as, but not limited to, checkpoint inhibitor therapy.

[0018] The expression cassettes and methods can be used to treat subjects having tumors predicted to be refractory to or not respond to one or more anti-cancer therapies. In some embodiments, the subject has low tumor infiltrating lymphocytes, low partially cytotoxic lymphocytes, or exhausted T cells. In some embodiments, the subject has advanced on one or more prior cancer therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 A. Illustrations of the expression constructs for mCXCL9~mCherry (mCXCL9-PTA-mCherry), mCXCL9, mIL12-2A (mIL-12 p35-P2A-mIL-12 p40), mIL 12~mCXCL9 (mIL-12 p35-P2A-mIL-12 p40-P2A-mCXCL9).

[0020] FIG. IB. Illustrations of expression constructs for hCXCL9, hIL12-2A (hIL-12 p35-P2A-hIL-12 p40), hIL12~hCXCL9 (hIL-12 p35-P2A-hIL-12 p40-P2A-hCXCL9).

[0021] FIG. 2. Graphs illustrating (A) mIL12p70 protein expression, and (B) mCXCL9 protein expression in HEK293 cells following transfection with mIL12-2A, mCXCL9, and mIL12~mCXCL9 expression vectors.

[0022] FIG. 3. Graph illustrating dose-response to mIL-12p70 from transiently transfected HEK293 cells with mouse IL-12 or mouse IL-12-CXC constructs. Both constructs encode biologically active IL-12.

[0023] FIG. 4A. Graph illustrating transfection-derived mouse CXCL9 induced chemotaxis of SIINFEKL-pulsed (24hr @ 1 pg/mL, 72hr recovery) OT-I splenocytes through polycarbonate membranes with 5.0-micron pores (Costar 3421). Migration index is defined as the number of observed chemotactic cells after 2.5 hours at 37°C, normalized to the number of cells that passively migrated through the membrane in the OptiMEM negative control. Abrogation of chemotaxis was observed with the pre-incubation of anti- mCXCL9 neutralizing monoclonal antibody (BioXCell BE0309).

[0024] FIG. 4B. Graph illustrating transfection-derived (HEK293) human CXCL9- induced chemotaxis of SIINFEKL-pulsed (24hr @ 1 pg/mL, 72hr recovery) OT-I splenocytes through polycarbonate membranes with 5.0-micron pores (Costar 3421). Migration index is defined as the number of observed chemotactic cells after 2 hours at 37°C, normalized to the number of cells that passively migrated through the membrane towards the OptiMEM negative control.

[0025] FIG. 4C. Graph illustrating transfection-derived (HEK293) human CXCL9- induced chemotaxis of human peripheral mononuclear cells (thawed from

cryopreservation, rested for 24hr in X-VIV015 medium) through polycarbonate membranes with 5.0-micron pores (Costar 3421). Migration index is defined as the number of observed chemotactic cells after 2 hours at 37°C, normalized to the number of cells that passively migrated through the membrane towards the OptiMEM negative control.

[0026] FIG. 5. Graph illustrating intratumoral expression of mCXCL9 using ELISA for mCXCL9 (DuoSet ELISA DY392) 48hrs post-electroporation in tumor lysates from mice bearing CT26 tumors (n=3; * P<0.05; T test with Welch correction).

[0027] FIG. 6. Graphs illustrating Kaplan-Meir curves in untreated mice and mice treated with control vector, IT-EP IL12-2A alone, or IT-EP IL12-2A in combination with IT-EP CXCL9 (** P<0.005 ; log-rank (Mantel-Cox) test).

[0028] FIG. 7. Graphs illustrating (A) decreased tumor volume, and (B) decreased contralateral (untreated) tumor volume, in tumor bearing mice treated with IT-EP therapy with mIL12-2A plus mCXCL9 compared to IL-12 therapy alone on control plasmid.

[0029] FIG. 8. Flow cytometric analysis of splenocytes from mice treated with IT-EP pUMCV3 or IL12-2A on day 0 and IT-EP pUMVC3 or mCXCL9 on days 4 and 7

[0030] FIG. 9. Graph illustrating fold increase in the number of AH1+ CD8+ T cells in mice tumors treated with control vector (pUMC3), IT-EP IL12 (IL-12 p35 - P2A - IL-12 p40), or IT-EP IL12 plus IT-EP CXCL9. N=2 independent experiments with 3-5 animals/group; * P<0.05, ** P<0.005; One way ANOVA.

[0031] FIG. 10. Graphs illustrating (A) hIL-12 protein expression in HEK293 cells transfected with hlT/l 2-2 A and hIL12~hCXCL9 expression vectors and (B) hCXCL9 protein expression in HEK293 cells transfected with hCXCL9 and hIL12~hCXCL9 expression vectors.

[0032] FIG. 11. Graph illustrating activation of STAT4 pathway in HEK-Blue IL-12 cells using recombinant human IL-12 (rhIL12, positive control), or hIL12 produced from cells expressing an hIL12-2A expression vectors.

[0033] FIG. 12 A. Illustrations of the mouse CD3 half-BiTE expression cassettes for HA-2C11-Myc scFv, HA-2C11 scFv, 2C11 scFv, and 2C11 scFv~hIL12.

[0034] FIG. 12B. Illustrations of human CD3 half-BiTE expression cassettes for HA- OKT3-Myc scFv, HA-OKT3 scFv, OKT3 scFv, HA-OKT3 scFv~hIL12, and OKT3 scFv~hIL12.

[0035] FIG. 13. Western blots showing: (A) expression of anti-CD3 scFv in HEK293 cells transfected with HA-OKT3 scFv and HA-2C11 scFv CD3 half-BiTE expression vectors, and (B) expression of CD3 half-BiTE in B16-F10 cells transfected with HA-2C11 scFv and HA-2C11 scFv~mIL12 expression vectors.

[0036] FIG. 14A-C. Flow cytometry showing the surface expression of anti-CD3 scFv in HEK 293 cells transfected with HA-OKT3 scFv and HA-OKT3 scFv~hIL12 expression vectors.

[0037] FIG. 14D-E. (D) Flow cytometry showing the surface expression of anti-CD3 scFv in B16-F10 cells transfected with HA-2C11 scFv and HA-2C11 scFv~mIL12 expression vectors. (E) Graph illustrating IL12p70 expression in B16-F10 cells following transfection with mIL12-2A, HA-2C11 scFv~mIL12 expression vector. [0038] FIG. 15. Graph illustrating IL12p70 expression in HEK293 cells following transfection with hIL12-2A, HA-OKT3 scFv~hIL12, and OKT3 scFv~hIL12expression vectors.

[0039] FIG. 16A-B. (A) Western blot showing expression of CD3 scFv in B16F10 melanoma or 4T1 breast cancer cells in vivo following intratumoral electroporation of HA-2C11 scFv. (B) Flow analysis of surface expression of CD3 scFv on 4T1 breast cancer cells in vivo following intratumoral electroporation of HA-2C11 scFv.

[0040] FIG. 16C. Graph illustrating IL12p70 expression in B16-F10 cells following intratumoral electroporation of mIL12-2A and HA-2C11 scFv~mIL12 expression vectors.

[0041] FIG. 17. Graph illustrating induction of INFy expression following co-culture of naive mouse splenocytes with B16F10 cells transfected in vitro with control vector (EV control), 2C11 scFv expression vector with or without recombinant mouse IL12, or with plate bound anti-CD3 (positive control).

[0042] FIG. 18. Graphs illustrating FACS analyses of proliferation of CFSE labeled CD3+CD45+ T cells following co-culture of naive mouse splenocytes with B16F10 cells transfected in vitro with control vector (Tfx control), 2C11 scFv expression vector with or without recombinant mouse IL12, or with plate bound anti-CD3 (positive control).

[0043] FIG. 19. Graph illustrating in vivo OT-1 and polyclonal T cell proliferation in DLN in B16-OVA tumor model mice treated with 2C11 scFv IT-EP or negative control.

[0044] FIG. 20. Graphs illustrating an increased CD8+ T cells in CD45.1+ live cells in TILs in B 16-OVA tumor model mice treated with 2C11 scFv IT-EP or negative control.

[0045] FIG. 21. Graph illustrating an increased antigen specific(SIINFEKL+) CD8+ T cells in TILs in B 16-OVA tumor model mice treated with 2C11 scFv IT-EP or negative control.

[0046] FIG. 22. FACS analysis of scan CFSE cells displaying (Hi) or not displaying (Lo) OVA257-264 peptide showing increase lysis of OVA257-264 peptide-displaying CFSE cells in B16-OVA tumor containing mice treated with 2C11 scFv IT-EP compare with negative transfected control.

[0047] FIG. 23. Graph illustrating increase in lysis of adoptive transferred OVA257-264- dispfaying CFSE cells in B 16-OVA tumor containing mice treated with IT-EP CD3 half- BiTE. The increased T cell killing ability observed in both spleen and driven lymph node. [0048] FIG. 24. FACS analysis of CFSE cells showing increase tumor-specific killing of OVA expressing cells in mice treated with IT-EP CD3 half-BiTE.

[0049] FIG. 25. Graph illustrating tumor progression of treated tumors in melanoma model mice treated with control, IL-12, or IL-12 plus CD3 half-BiTE IT-EP therapy.

[0050] FIG. 26. (A) Graph illustrating tumor progression in breast cancer model mice treated with control, IL-12, or IL-12 plus 2C1 1 IT-EP therapy (B) Graph illustrating lung metastasis nodules in 4T1 breast cancer model mice treated with control, IL12-2A or IL12-2A plus 2C1 1 IT-EP therapy. (C) Graph illustrating the absolute number of effector T cells(CD127-CD62L-CD3+) per iiL peripheral blood in 4T1 breast cancer model mice treated with control, IL12-2A or ILI2-2A plus 2C1 1 IT-EP therapy.

[0051] FIG. 27. Graphs illustrating (A) hIL12p70 protein secretion, and (B) hCXCL9 protein secretion in HEK293 cells following transfection with hIL12-2A, hCXCL9, and hIL12~hCXCL9 expression vectors. Protein detected by ELISA, n = 5.

[0052] FIG. 28A. Volcano plots displaying p-values and log2 fold change for the indicated genes. Differential gene expression was examined in mice treated with mCXCL9 alone (top panel) and mice treated with mCXCL9 in combination with IL12 (bottom panel). Horizontal lines indicate False Discovery Rate (FDR) thresholds.

[0053] FIG. 28B. Graph illustrating‘Cytotoxic immune cells’ cell type scores. Each cell type’s score (Log 2 scale) has been centered to have mean 0.

[0054] FIG. 29A. Graph illustrating IL12 p70 expression in 48 hr post electroporation in tumor lysates from mice bearing B16.F10 tumors after treatment with 10 pg or 100 pg of IL 12-2 A (TAVO) on days 1, 5, and 8, or 100 pg of IL12-CXCL9 or CD3 half- BiTE~IL12 (SPARK) on each of days 1, 5, and 8 (n=8 animals; DuoSet ELISA DY419).

[0055] FIG. 29B-C Graph illustrating primary (B) and secondary (C) tumor growth in mice bearing B16.F10 tumors after treatment with 10 pg or 100 pg of IL12-2A (TAVO) on days 1, 5, and 8, or 100 pg of IL12-CXCL9 or CD3 half-BiTE~IL12 (SPARK) on each of days 1, 5, and 8. (From left to right for each of days 0 and 12: 10 pg IL12-2A, SPARK, 100 pg of IL12-2A).

[0056] FIG. 30. Graph illustrating: (A) anti-CTLA4 scFv transfection supernatant binding to recombinant mCTLA-4/Fc, and (B) detection of anti-CLTA-4 scFv on RENCA tumor lysates. DETAILED DESCRIPTION

I. Definitions

[0057] A "nucleic acid" includes both R A and DNA. RNA and DNA include, but are not limited to, cDNA, genomic DNA, plasmid DNA, condensed nucleic acid, nucleic acid formulated with cationic lipids, nucleic acid formulated with peptides or cationic polymers, RNA and mRNA Nucleic acid also includes modified RNA or DNA.

[0058] An“expression cassette” refers to an RNA or DNA coding sequence or segment of RNA or DNA that codes for an expression product (e.g., peptide(s) (i.e., polypeptide(s) or protein(s)) or RNA). An expression cassette can be present in a plasmid. An expression cassette is capable of expressing one or more polypeptides in a cell, such a mammalian cell. The expression cassette may comprise one or more sequences necessary for expression of the encoded expression product. The expression cassette may comprise one or more of an enhancer, a promoter, a terminator, and a polyA signal operably linked to the DNA coding sequence.

[0059] The term "plasmid" refers to a nucleic acid that includes at least one sequence encoding a polypeptide (such as any of the described expression cassettes) that is capable of being expressed in a mammalian cell. A plasmid can be a closed circular DNA molecule. A variety of sequences can be incorporated into a plasmid to alter expression of the coding sequence are to facilitate replication of the plasmid in a cell. Sequences can be used that influence transcription, stability of a messenger RNA (mRNA), RNA processing, or efficiency of translation. Such sequences include, but are not limited to, 5' untranslated region (5* UTR), promoter, introns, and 3 ^f untranslated region (3' UTR). Plasmids can be manufactured in large scale quantities and/or in high yield. Plasmids can further be manufacture using cGMP manufacturing. Plasmids can be transformed into bacteria, such as E. coli. The DNA plasmids are can be formulated to be safe and effective for injection into a mammalian subject.

[0060] “Protein,”“peptide,” or“polypeptide” includes a contiguous string of two or more amino acids. A“protein sequence,”“peptide sequence,”“polypeptide sequence,” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide or polypeptide.

[0061] The terms“express” and“expression” mean allowing or causing the information in a gene, RNA or DNA sequence to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene A DNA sequence is expressed in or by a cell to form an expression product such as an RNA (e.g., mRNA) or a protein. The expression product itself may also be said to be expressed by the cell.

[0062] “Operably linked” refers to the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter operably linked to a coding sequence will direct RNA polymerase mediated transcription of the coding sequence into RNA, including mRNA, which may then be spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence. A coding sequence can be“operably linked” to one or more transcriptional or translational control sequences. A terminator/polyA signal operably linked to a gene terminates transcription of the gene into RNA and directs addition of a polyA signal onto the RNA.

[0063] A“promoter” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter may comprise one or more additional regions or elements that influence transcription initiation rate, including, but not limited to, enhancers. A promoter can be, but is not limited to, a constitutively active promoter, a conditional promoter, an inducible promoter, or a cell- type specific promoter. Examples of promoters can be found, for example, in WO

2013/176772. The promoter can be, but is not limited to, CMV promoter, IgK promoter, mPGK, SV40 promoter, b-actin promoter, a-actin promoter, SRa promoter, herpes thymidine kinase promoter, herpes simplex virus (HSV) promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), rous sarcoma virus (RSV) promoter, and EFla promoter. The CMV promoter can be, but is not limited to, CMV immediate early promoter, human CMV promoter, mouse CNV promoter, and simian CMV promoter.

[0064] A“translation modification element” enables translation of two or more genes from a single transcript. Translation modification elements include Internal Ribosome Entry Sites (IRES), which allow for initiation of translation from an internal region of an mRNA, and 2A peptides, derived from picomavirus, which cause the ribosome to skip the synthesis of a peptide bond at the C-terminus of the el ement. Incorporation of a transl ation modulating element results in co-expression of two or more polypeptide from a single polycistronic mRNA 2A modulators include, but are not limited to, P2A, T2A, E2A or F2A. 2A modulators contain a PG/P cleavage site.

[0065] A‘'homologous” sequence (e.g., nucleic acid sequence or amino acid sequence) refers to a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Sequence identity can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated the window of comparison between two sequences is defined by the entire length of the shorter of the two sequences

[0066] "Immunostimulatory cytokine" includes cytokines that mediate or enhance the immune response to a foreign antigen, including viral, bacterial, or tumor antigens.

Immunostimulatory cytokines can include, but are not limited to: TNFa, IL-1, IL-10, IL- 12, IL-12 p35, IL-12 p40, IL-15, IL-15Ra, IL-23, IL-27, IFNa, IFNp, IFNy, IL-2, IL-4, IL-5, IL-7, IL-9, IL-21, and TGFp.

[0067] The term "cancer" includes a myriad of diseases generally characterized by inappropriate cellular proliferation, abnormal or excessive cellular proliferation. Examples of cancer include, but are not limited to, breast cancer, triple negative breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, ovarian cancer, kidney cancer, brain cancer, or sarcomas.

[0068] A“treatment-refractory cancer” is a cancer that does not respond, or has not responded, to at least one prior medical treatment. In some embodiments, a treatment- refractory, with respect to a treatment, indicates an inadequate response to a treatment or the lack of a partial or complete response to the treatment. For example, patients may be considered refractory to a treatment, ( e.g ., checkpoint inhibitor therapy such as a PD-1 or PD-L1 inhibitor therapy) if they do not show at least a partial response after receiving at least 2 doses of the treatment.

[0069] The“tumor microenvironment” refers to the environment around a tumor and includes the non-malignant vascular and stromal tissue that aid in growth and/or survival of a tumor, such as by providing the tumor with oxygen, growth factors, and nutrients, or inhibiting immune response to the tumor. A tumor microenvironment includes the cellular environment in which the tumor exists, including surrounding blood vessels, immune ceils, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix.

[0070] The“tumor margin” or“margin tissue” is the visually normal tissue

immediately near or surrounding a tumor. Typically, the margin tissue is the visually normal tissue within 0.1-2 cm of the tissue. Tumor margin tissue is often removed when a tumor is surgically resected.

[0071] The term "treatment" includes, but is not limited to, a medicament or therapy for inhibition or reduction of proliferation of cancer cells, destruction of cancer cells, prevention of proliferation of cancer cells, prevention of initiation of malignant cells, arrest or reversal of the progression of transformed premalignant cells to malignant disease, or amelioration of the disease.

[0072] The term "electroporation" refers to the use of an electroporative pulse to facilitate entry of biomolecules such as a plasmid, nucleic acid, or drug, into a cell.

[0073] A“draining lymph node” is a lymph node that filters lymph from a particular region or organ. In context of tumors and tumor treatment, a draining lymph node lies immediately downstream of the tumor.

[0074] An“epitope tag” is a short amino acid sequence (or nucleic acid sequence encoding the short amino acid sequence) to which a high affinity antibody binds.

Exemplary epitope tags include, but are not limited to, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag and NE-tag. Epitope tags can be used to facilitate immunodetection. II. CXCL9

[0075] C-X-C Motif Chemokine ligand 9 (CXCL9) is a small cytokine belonging to the CXC chemokine family. CXCL9 is also known as Monokine Induced by Gamma interferon (MIG). CXCL9 is a T-cell chemoattractant, and facilitates chemotactic recruitment of tumor infiltrating lymphocytes (TIL). The mouse and human CXCL9 amino acid sequences are represented by SEQ ID NO: 35 and SEQ ID NO: 58, respectively. In some embodiments, a CXCL9 comprises: (a) the amino acid sequence of SEQ ID NO: 35 or 58 or a functional equivalent thereof; or (b) an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identify to the amino acid sequence of SEQ ID NO: 35 or 58.

III. anti-CTLA-4 scFv

[0076] An anti-CTLA-4 scFv comprises an anti-CTLA-4 single-chain variable fragment (scFv) having affinity for an extracellular domain of CTLA-4 and/or inhibiting CTLA-4 signaling. An scFv comprises a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide. Exemplary mouse anti-CTLA-4 heavy chain variable region amino acid sequences are represented by SEQ ID NO: 39 and 43. Exemplary mouse anti-CTLA-4 light chain variable region amino acid sequences are represented by SEQ ID NO: 37 and 41.

[0077] An anti-CTLA-4 scFv can be identified from phage display. An anti-CTLA-4 scFv can also be generated by subcloning the VH and VL from a known anti-CTLA-4 antibody, such as from a hybridoma. Known anti-CTLA-4 antibodies have been described, for instance in 20190048096, 20130136749, 20120148597, 20140099325, 20150104409, 20110296546, and 20080233122, among others. Known anti-CTLA-4 antibodies include, but are not limited to, ipilimumab and tremelimumab. In some embodiments, the VH and or VL domains of an anti-CTLA-4 scFv can be humanized. A humanized antibody (or antibody fragment or domain) is an antibody from a non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. In some embodiments, humanized antibodies can be made by inserting the relevant complementarity-determining regions (CDRs, also termed hypervariable regions (HVRs)) of an anti-CTLA-4 antibody into human VH and VL domain scaffolds. [0078] An anti-CTLA-4 scFv can be formed by linking the C-terminus of the VH chain with the N-terminus of the VL. Alternatively, the C-terminus of the VL can be linked to the N-terminus of the VH. The peptide linker can be about 10 to about 25 amino acids. In some embodiments, the scFv peptide linker is rich in glycine. An scFv peptide linker can be, but is not limited to, (G4S) _X where x is an integer from 2 to 5 (inclusive). In some embodiments, the scFv peptide linked comprises Gly-Gly-Gly-Gly-Gly-Ser-Gly- Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (i.e., also termed [(Gly) ₄ Ser] ₃ , (G ₄ S) ₃ or

G4S ( ^x 3)). In some embodiments, the scFv peptide linker consists of G4S ( ^x 3). In some embodiments, the encoded anti-CTLA-4 scFv polypeptide includes a signal peptide such as an IgK signal peptide. Exemplary anti-CTLA-4 scFv amino acid sequences are represented by SEQ ID NO: 70 and 72. In some embodiments, an anti-CTLA-4 scFv comprises: (a) the amino acid sequence of SEQ ID NO: 70 or 72 or a functional equivalent thereof; or (b) an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identify to the amino acid sequence of SEQ ID NO: 70 or 72.

IV. CD3 half-BiTE

[0079] A CD3 half-BiTE comprises an anti-CD3 single-chain variable fragment (scFv) fused to a transmembrane domain (TM). An scFv comprises a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide. Exemplary anti-CD3 heavy chain variable region amino acid sequences are represented by SEQ ID NO: 8 and 47. Exemplary mouse anti-CD3 light chain variable region amino acid sequences are represented by SEQ ID NO: 1 1 and 50.

[0080] An anti-CD3 scFv can be identified from phage display. An anti-CD3 scFv can also be generated by subcloning the VH and VL from a known anti-CD3 antibody, such as from a hybridoma. Known anti-CD3 antibodies have been described, for instance in US20180117152, US20140193399, US20100183554, and US20060177896. Known anti- CD3 antibodies also include, but are not limited to, OKT3 (Muromonab-CD3), 145-2C1 1, 17A2, SP7, and UCHTl. hi some embodiments, the VH and or VL domains of an anti - CD3 scFv can be humanized. A humanized antibody (or antibody fragment or domain) is an antibody from a non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. In some embodiments, humanized antibodies can be made by inserting the relevant complementarity-determining regions (CDRs, also termed hypervariable regions (HVRs)) of an anti-CD3 antibody into human VH and VL domain scaffolds

[0081] An anti-CD3 scFv can be formed by linking the C-terminus of the VH chain with the N-terminus of the VL. Alternatively, the C-terminus of the VL can be linked to the N-terminus of the VH. The peptide linker can be about 10 to about 25 amino acids. In some embodiments, the scFv peptide linker is rich in glycine. An scFv peptide linker can be, but is not limited to, (G4S) _X where x is an integer from 2 to 5 (inclusive). In some embodiments, the scFv peptide linker comprises Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly- Gly-Ser-Gly-Gly-Gly-Gly-Ser (i.e., also termed [(Gly)4Ser]3, (G4S)3 or G4S ( ^x 3)). In some embodiments, the scFv peptide linker consists of GiS ( ^x 3).

[0082] A transmembrane domain (TM) comprises a polypeptide capable of being inserted into a biological lipid bi layer (membrane) and anchoring the CD3 half-BiTE to the membrane. TMs are known in the art and typically consist predominantly of nonpolar amino acids. The transmembrane domain can be, but is not limited to, a PDGFRP transmembrane domain or a PDGFRa transmembrane domain (PDGFR is Platelet-derived growth factor receptor). In some embodiments, a spacer is included between the anti-CD3 scFv and the transmembrane domain. In some embodiments, the TM domain comprises an amino acid sequence selected from the group comprising:

VGQDTQE VIWPHSLPFK W VIS AIL ALVVLTIISLIILIMLWQKKPR (SEQ ID NO : 25), A VGQ D TQ E V 1 V V P H S LP F K V V V 18 A 1 LA LV VLT 11 S Lll LI M LW 0 K K P R (SEQ ID NO: 27), PDGFR : WISAILALWLTVISLIILI (SEQ ID NO: 83), PDGFR [3:

V VI S A I LA L V V LTIISL1 ILL (SEQ ID NO: 84), PDGFRa: AAVLVLLVIVIISLIVLWIW (SEQ ID NO: 85), and PDGFRa: AAVLVLLVIVIVSLIVLWIW (SEQ ID NO: 86). In some embodiments, the TM domain is encoded by a nucleic acid sequence selected from the group comprising:

gtgggccaggacacgcaggaggtcatcgtggtgccacactccttgccctttaaggtg gtggtgatctcagccatcc tggccctggtggtgctcaccatcatctcccttatcatcctcatcatgctttggcagaaga agccacgt (SEQ ID NO: 24), gctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttgccctttaaggtg gtggtgatctcagccatcctggccct gg tggtgctcaccatcatctcccttatcatcctcatcatgctttggcagaagaagccacgt (SEQ ID NO: 26), PDGFRj tggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctca tc (SEQ ID NO: 87), PDGFRfl: gtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctc atc (SEQ ID NO: 88), PDGFRa: gctgcagtcctggtgctgttggtgattgtgatcatctcacttattgtcctggttgtcatt tggaa (SEQ ID NO: 89). [0083] In some embodiments, the encoded anti-CD3 half-BiTE polypeptide includes a signal peptide such as an IgK signal peptide.

[0084] Exemplary CD3 half-BiTE amino acid sequences are represented by SEQ ID NO: 60, 62, 74, and 76. In some embodiments, a CD3 half-BiTE comprises: (a) the amino acid sequence of SEQ ID NO: 60, 62, 74, or 76 or a functional equivalent thereof; or (b) an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identify to the amino acid sequence of SEQ ID NO: 60, 62, 74, or 76.

V. Expression cassettes

[0085] Any of the described polypeptides, CXCL9, CD3 half-BiTE, anti-CTLA4 scFv, and IL-12, may be encoded on a nucleic acid. The nucleic acid can be, but is not limited an expression cassette. The expression cassette can be on a plasmid. The term “plasmid” includes any nucleic acid vector including a bacterial vector, a viral vector, an episomal plasmid, an integrative plasmid, or a phage vector. As used herein, delivery of an expression cassette includes deliver} of a plasmid or nucleic acid vector (as termed “expression vector” or“vector”) containing the expression cassette.

[0086] An encoded polypeptide may be linked, in an expression cassette, to a sequence encoding a second polypeptide. In some embodiments, an expression cassette encodes a fusion protein. The term“fusion protein” refers to a protein comprising two or more polypeptides linked together by peptide bonds or other chemical bonds. In some embodiments, a fusion protein is recombinantly expressed as a single-chain polypeptide containing the two polypeptides. The two or more polypeptides can be linked directly or via a linker comprising one or more amino acids

[0087] An expression cassette or plasmid may contain a multi cistronic expression cassette. Multi cistronic expression cassettes express two or more separate proteins from the same mRNA and contain one or more translation modification elements.

[0088] In some embodiments, the described expression cassettes encode two or three polypeptides expressed from a single promoter, with one or more translation modification elements to allow the two or three polypeptides to be expressed from a single mRNA. In some embodiments, the expression cassettes comprise:

a) P-A-T-B,

b) P-B-T-A, c) P-B-T-B'

c) P-A-T-B-T-B' or

d) P-B-T-B'-T-A

wherein P is a promoter, A encodes CXCL9 or a CD3 half-BiTE, B and B' encode cytokines or cytokine subunits, and T is a translation modification element.

[0089] A promoter can be, but is not limited to, a constitutively active promoter, a conditional promoter, an inducible promoter, or a cell-type specific promoter. Examples of promoters can be found, for example, in WO 2013/176772. The promoter can be, but is not limited to, a CMV promoter, a IgK promoter, a mPGK, a SV40 promoter, a b-actin promoter, an a-actin promoter, a SRa promoter, a herpes thymidine kinase promoter, a herpes simplex virus (HSV) promoter, a mouse mammary tumor virus long terminal repeat (LTR) promoter, an adenovirus major late promoter (Ad MLP), a rous sarcoma virus (RSV) promoter, and an EFltx promoter. A CMV promoter can be, but is not limited to, a CMV immediate early promoter, a human CMV promoter, a mouse CNV promoter, and a simian CMV promoter.

[0090] In some embodiments, T is an internal ribosome entry site (IRES) element or a ribosomal skipping modulator. A ribosome skipping modulator can be, but is not limited to, a 2A element (also termed 2A peptide or 2A self-cleaving peptide). The 2A element can be, but is not limited to, a P2A (SEQ ID NO: 29), T2A, E2A or F2A element.

[0091] The CXCL9 can be, but is not limited to, mouse CXCL9 and human CXCL9, or a functional equivalent thereof.

[0092] The CD3 half-BiTE can be, but is not limited to: anti-CD3

sc Fv - trail sm c mb ran e domain (TM), epitope tag (ET) anti-CD3 scFv-ET-TM, ET anti- CD3 scFv-TM, anti-CD3, scFv-ET-TM, HA anti-CD3 scEv-Myc-TM, FLA-anti-CDS scFv-TM, anti-CD3, scFv-Myc-TM, anti-CD3 scFv-TM, or anti-CD3 scFv-TM. The anti-CD3 scFv can be an anti-mouse CD3 scFv or an anti-human CD3 scFv. Each of these can include a signal peptide. The signal peptide can be, but is not limited to, an IgK signal peptide. The TM can be, but is not limited to, a PDGFR TM. The anti-CD3 scFv can be, but is not limited to, 201 or OKT3.

[0093] In some embodiments, the cytokine is an immunostimulatory cytokine. In some embodiments, the immunostimulatory cytokine is an interleukin. Cytokines include, but are not limited to, IL-1, IL-2, IL-10, IL-12, 1 L- 15, IL-23, IL-27, IL-35, IFN-a, IFN-b, IFN-g, and TGF-b. In some embodiments, B and/or B' encode an IL-12, IL-12 p35-TL-12 p40 fusion, IL-12 p70, IL-12 p35, or IL-12 p40 polypeptide. The IL-12, IL-12 p35-TL-12 p40 fusion, IL-12 p7G, IL-12 p35, or IL-12 p40 polypeptide may be, but are not limited to, a mouse or human IL-12, IL-12 p35-IL-12 p40 fusion, IL-12 p7Q, IL-12 p35, or IL-12 p40 polypeptide. In some embodiments, B encodes IL-12 p35 and B ^f encodes IL-12 p40.

[0094] In some embodiments P is a CMV promoter, A encodes CXCL9, T is a P2A element, B encodes IL-12 p35 and B' encodes IL-12 p40.

[0095] In some embodiments P is a CMV promoter, A encodes a human CXCL9, T is a P2A element, B encodes IL-12 p35 and B' encodes IL-12 p4Q.

[0096] In some embodiments P is a CMV promoter, A encodes a mouse CXCL9, T is a P2A element, B encodes IL-12 p35 and B ^f encodes IL-12 p40.

[0097] In some embodiments P is a CMV promoter, A encodes an IgK-HA-anti-CD3 scFv-PDGFR TM CD3 half-BiTE, T is a P2A element, B encodes IL-12 p35 and B' encodes IL-12 p40.

[0098] In some embodiments P is a CMV promoter, A encodes an IgK-anti-CD3 scFv PDGFR TM CDS half-BiTE, T is a P2A element, B encodes IL-12 p35 and B' encodes IL-12 p40.

[0099] In some embodiments P is a CMV promoter, A encodes an

IgK -HA-2 Cl l-PDGFR TM CD3 half-BiTE, T is a P2A element, B encodes IL-12 p35 and B' encodes IL-12 p4Q.

[00100] In some embodiments P is a CMV promoter, A encodes an IgK-2C 1 l-PDGFR TM CD3 half-BiTE, T is a P2A element, B encodes IL-12 p35 and B' encodes IL-12 p40.

[00101] In some embodiments P is a CMV promoter, A encodes an

IgK HA-OCT3-PDGFR TM CD3 half-BiTE, T is a P2A element, B encodes IL-12 p35 and B' encodes IL-12 p40.

[00102] In some embodiments P is a CMV promoter, A encodes an

IgK OKT3 PDGFR TM CD3 half-BiTE, T is a P2A element, B encodes IL-12 p35 and B ^f encodes IL-12 p4Q

[00103] In some embodiments, B encodes IL-12 p35, T is a P2A element, and B' encodes IL-12 p40. In some embodiments, B encodes IL-12 p35, T is an IRES element, and B' encodes IL-12 p40. The promoter can be, but is not limited to, a CMV promoter. [00104] In some embodiments, we describe expression cassettes encoding a

polypeptide comprising the amino acid sequence of SEQ ID NO: 60, 62, 74, or 76 or a polypeptide having at least 70% identity to the amino acid sequence of SEQ ID NO: 60, 62, 74, or 76 In some embodiments, an expression cassette encodes a polypeptide comprising an amino acid sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 60, 62, 74, or 76, wherein the encoded polypeptide retains the functional activity of an CD3 half-BiTE polypeptide

[00105] In some embodiments, we describe expression cassettes encoding a

polypeptide comprising the amino acid sequence of SEQ ID NO: 64, 66, 78, or 70, or a polypeptide having at least 70% identity to the amino acid sequence of SEQ ID NO: 64, 66, 78, or 70. In some embodiments, an expression cassette encodes a polypeptide comprising an amino acid sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 64, 66, 78, or 70, wherein encoded the polypeptides retain the functional activity of an CD3 half-BiTE polypeptide and an IL-12 polypeptide.

[00106] In some embodiments, we describe expression cassettes encoding a

polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or 58, or a

polypeptide having at least 70% identity to the amino acid sequence of SEQ ID NO: 35 or 58. In some embodiments, an expression cassette encodes a polypeptide comprising an amino acid sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 35 or 58, wherein the encoded polypeptide retains the functional activity of a CXCL9 polypeptide.

[00107] In some embodiments, we describe expression cassettes encoding a

polypeptide comprising the amino acid sequence of SEQ ID NO: 68 or 82, or a

polypeptide having at least 70% identity to the amino acid sequence of SEQ ID NO: 68 or 82. In some embodiments, an expression cassette encodes a polypeptide comprising an amino acid sequence having greater than 70%, 72 %, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88% _> , 90%, 92%, 93%, 95%, 96%, 97%, 98% , or 99% identity to the amino acid sequence of SEQ ID NO: 68 or 82, wherein encoded the polypeptides retain the functional activity of a CXCL9 polypeptide and an IL-12 polypeptide. [00108] In some embodiments, we describe expression cassettes encoding a

polypeptide comprising the amino acid sequence of SEQ ID NO: 70 or 72 or a polypeptide having at least 70% identity to the amino acid sequence of SEQ ID NO: 70 or 72. In some embodiments, an expression cassette encodes a polypeptide comprising an amino acid sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 70 or 72, wherein the encoded polypeptide retains the functional activity of an anti-CTLA-4 scFv polypeptide.

[00109] In some embodiments, we describe expression cassettes comprising the nucleotide sequence of SEQ ID NO: 59, 61 , 73, or 75, or a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 59, 61, 73, or 75. In some embodiments, an expression cassette comprises a sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 59, 61, 73, or 75 and encodes a polypeptide having the functional activity of an CD3 half-BiTE polypeptide. In some embodiments, the nucleotide sequence of SEQ ID NO: 59, 61, 73, or 75 or the nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 59, 61, 73, or 75 is operably linked to a CMV promoter.

[00110] In some embodiments, we describe expression cassettes comprising the nucleotide sequence of SEQ ID NO: 63, 65, 77, or 79, or a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 63, 65, 77, or 79. In some embodiments, an expression cassette comprises a sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 63, 65, 77, or 79, and encode polypeptides having the functional activity of an CD3 half-BiTE polypeptide and an IL-12 polypeptide. In some embodiments, the nucleotide sequence of SEQ ID NO: 63, 65, 77, or 79 or the nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 63, 65, 77, or 79 is operably linked to a CMV promoter.

[00111] In some embodiments, we describe expression cassettes comprising the nucleotide sequence of SEQ ID NO: 34 or 57, or a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 34 or 57. In some embodiments, an expression cassette comprises a sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 34 or 57, and encodes a polypeptide having the functional activity of a CXCL9 polypeptide. In some embodiments, the nucleotide sequence of SEQ ID NO: 34 or 57 or the nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 34 or 57 is operably linked to a CMV promoter

[00112] In some embodiments, we describe expression cassettes comprising the nucleotide sequence of SEQ ID NO: 67 or 81 or a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 67 or 81. In some embodiments, an expression cassette comprises a sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 67 or 81, and encodes polypeptides having the functional activity of a CXCL9 polypeptide and an IL-12 polypeptide. In some

embodiments, the nucleotide sequence of SEQ ID NO: 67 or 81 or the nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 67 or 81 is operably linked to a CMV promoter.

[00113] In some embodiments, we describe expression cassettes comprising the nucleotide sequence of SEQ ID NO: 69 or 71, or a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 69 or 71. In some embodiments, an expression cassette comprises a sequence having greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 69 or 71, and encodes a polypeptide having the functional activity of an anti-CTLA-4 scFv polypeptide. In some embodiments, the nucleotide sequence of SEQ ID NO: 69 or 71 or the nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 69 or 71 is operably linked to a CMV promoter.

VI. Methods of Treatment

[00114] Described are methods for treatment of a tumor in a subject comprising, administering a composition comprising an effective dose of one or more of the described CXCL9, CD3 half-BiTE, and or CTLA-4 scFv expression cassettes to the tumor, tumor microenvironment, and/or a tumor margin tissue and administering electroporation therapy to the tumor, tumor microenvironment, and/or a tumor margin tissue (IT-EP therapy). The CXCL9 or CD3 half-BiTE expression cassette may further encode IL-12. [00115] The treated tumor can be a cutaneous tumor, a subcutaneous tumor, or a visceral tumor. The tumor can be cancerous or non-cancerous. The tumor can be, but is not limited to, a solid tumor, a surface lesion, a non-surface lesion, a lesion within 15 cm of body surface, or a visceral lesion. In some embodiments, the described methods and expression vectors can be used to treat primary tumors as well as distant (z.e., untreated) tumors and metastases. In some embodiments, the described methods provide for reducing the size of or inhibiting the grow of a tumor, inhibiting the growth of cancer cells, inhibiting or reducing metastasis, reducing or inhibiting the development of metastatic cancer, and/or reducing recurrence of cancer in a subject suffering from cancer. The tumor is not limited to a specific type of tumor or cancer.

[00116] In some embodiments, the methods further comprise administering an effective dose of an immunostimulatory cytokine. The immunostimulatory cytokine can be administered by IT-EP of an expression cassette encoding the cytokine. In some embodiments, the cytokine is encoded on the expression cassette encoding the CXCL9 or CD3 half-BiTE. In some embodiments, the cytokine is encoded on a second expression vector and delivered to the cancerous tumor by IT-EP. In some embodiments, the cytokine is IL-12. In some embodiments, the expression cassette comprises B-T-B', wherein B encodes IL-12 p35, T is a P2A element, and B' encodes IL-12 p40. The cytokine may be administered prior to, concurrent with, or subsequent to IT-EP CXCL9 therapy or IT-EP CD3 half-BiTE therapy.

[00117] IT-EP CXCL9 therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective does of a described expression cassette encoding CXCL9 and administering electroporation therapy to the tumor.

[00118] IT-EP IL12~CXCL9 therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective does of a described expression cassette encoding CXCL9 and IL-12 and administering electroporation therapy to the tumor.

[00119] IT-EP CD3 half-BiTE therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective does of a described expression cassette encoding a CD3 half-BiTE and administering electroporation therapy to the tumor. [00120] IT-EP CD3 half-BiTE~IL-12 or treatment therapy comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective does of a described expression cassette encoding CD3 haif-BiTE and IL-12 and administering electroporation therapy to the tumor.

[00121] IT-EP anti-CTLA-4 scFv therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective does of a described expression cassette encoding anti-CTLA-4 scFv and administering

electroporation therapy to the tumor.

[00122] IT-EP IL12 therapy or treatment comprises injecting a tumor, tumor microenvironment, and/ or tumor margin tissue with an effective does of an expression cassette encoding IL-12 and administering electroporation therapy to the tumor. In some embodiments the expression cassette encoding IL-12 comprises IL12-2A (mlL12-2A and hIL12-2A; FIG. 1).

[00123] In some embodiments, the described expression cassettes, plasmids containing the described expression cassettes, and methods can be used to treat one or more tumors, tumor cells, or tumor lesions. The tumor cells can be, but are not limited to cancer cells. The term "cancer" includes a myriad of diseases generally characterized by inappropriate cellular proliferation, abnormal or excessive cellular proliferation. The cancer can be, but is not limited to, solid cancer, sarcoma, carcinoma, and lymphoma. The cancer can also be, but is not limited to, pancreas, skin, brain, liver, gall bladder, stomach, lymph node, breast, lung, head and neck, larynx, pharynx, lip, throat, heart, kidney, muscle, colon, prostate, thymus, testis, uterine, ovary, cutaneous, and subcutaneous cancers. Skin cancer can be, but is not limited to, melanoma and basal cell carcinoma. Breast cancer can be, but is not limited to, ER positive breast cancer, ER negative breast cancer, and triple negative breast cancer. In some embodiments, the described methods can be used to treat cell proliferative disorders. The term "cell proliferative disorder" denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue both morphologically and genotypically. In some embodiments, the described methods can be used to treat a human. In some embodiments, the described methods can be used to treat non-human animals or mammals. A non-human mammal can be, but is not limited to, mouse, rat, rabbit, dog, cat, pig, cow, sheep and horse.

[00124] The described expression cassettes and methods are contemplated for use in subjects afflicted with cancer or other non-cancerous (benign) growths. Tumors treated with the methods of the present embodiment may be any of noninvasive, invasive, superficial, papillary, flat, metastatic, localized, unicentric, multicentric, low grade, and high grade tumors. These growths may manifest themselves as any of a lesion, polyp, neoplasm (e.g. papillary urothelial neoplasm), papilloma, malignancy, tumor (e.g. Klatskin tumor, hilar tumor, noninvasive papillary urothelial tumor, germ cell tumor, Ewing's tumor, Askin's tumor, primitive neuroectodermal tumor, Leydig cell tumor, Wilms' tumor, Sertoli cell tumor), sarcoma, carcinoma (e.g. squamous cell carcinoma, cloacogenic carcinoma, adenocarcinoma, adenosquamous carcinoma, cholangiocarcinoma,

hepatocellular carcinoma, invasive papillary urothelial carcinoma, flat urothelial carcinoma), lump, or any other type of cancerous or non-cancerous growth. The expression cassettes and methods can be used to treat advanced, metastatic, or treatment refractory cancer.

[00125] The expression cassettes and methods described herein are contemplated for use in, e.g., adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer) bladder cancer, benign and cancerous bone cancer (e.g. osteoma, osteoid osteoma, osteoblastoma, osteochrondroma,

hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astrocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma,

ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous

adnocarcinoma, clear cell) esophagus cancer, gallbladder cancer (mucinous

adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g.

choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer, both melanoma and non-melanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly

differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).

[00126] in some embodiments, the subject has low tumor infiltrating lymphocytes (TILs) and/or impaired tumoral IFNy signaling.

[00127] The described methods can be used to cause one or more of the following: inflame a tumor, induce T cell infiltration to the tumor or tumor microenvironment (increase the number of tumor infiltrating lymphocytes (TILs)), enhance systemic T cell response, induce activation of tumor-specific T cells, increase antigen-specific T cell response, increase proliferation of antigen-specific T cells, increase polyclonal T cells response, enhance an immune response against treated and/or untreated tumors, decrease T cell exhaustion, increase lymphocyte and monocyte cell surface markers in one or more treated or untreated tumors, increase intratumoral levels of INFy regulated genes in one or more treated or untreated tumors, increase proliferating effector memory T cells in the subject’s blood, increase short-lived effector cells in the subject’s blood, increase expression of genes present in activated natural killer cells in a cancerous tumor, increase expression of genes that function in antigen presentation in a cancerous tumor, increase expression of genes that function in T cell survival and T cell mediated cytotoxicity in a cancerous tumor, induce regression of treated and/or untreated tumors, induce debulking of a treated and/or untreated tumor, and improve response to a second therapy, such as, but not limited to, immune checkpoint inhibitor therapy. In some embodiments, enhancement of immune reaction to the tumor leads to increased survival of the subject.

[00128] In some embodiments, the described methods comprise treating a subject having a cancerous tumor comprising: injecting the cancerous tumor with an effective dose of a plasmid encoding CXCL9, and administering electroporation therapy to the tumor. In some embodiments, the described methods comprise treating a subject having a cancerous tumor comprising: injecting the cancerous tumor with an effective dose of a plasmid encoding CD3 half-BiTE, and administering electroporation therapy to the tumor. In some embodiments, the described methods comprise treating a subject having a cancerous tumor comprising: injecting the cancerous tumor with an effective dose of a plasmid encoding an anti-CTLA-4 scFv, and administering electroporation therapy to the tumor. In some embodiments, the plasmid is administered substantially

contemporaneously with the electroporation treatment. The term "substantially

contemporaneously" means that the molecule and the electroporation treatment are administered reasonably close together with respect to time, i.e., before the effect of the electrical pulses on the cells diminishes.

[00129] In some embodiments, the described methods result in increased NK cells and T cell populations in a tumor or tumor microenvironment. IT-EP of CXCL9,

IL12~CXCL9, CD3 half-BiTE~IL12, and/or CD3 half-BiTE increases homing of tumor- specific T cells to tumors, increases activation and/or proliferation of tumor-specific T cells, and/or increases recruitment of CD8+ T cells, NK cells, and NKT cells to the tumor microenvironment. Activation of T cells can lead to increased tumor cell killing by the activated T cells.

[00130] In some embodiments, administration of IL-12 therapy by IT-EP enhances T cell infiltration of the tumor. Subsequent expression of CD3 half-BiTE in the tumor can activate the T cells to enhance the population of antigen specific T cells.

[00131] In some embodiments, IT-EP CXCL9 therapy enhances an IL-12 effect resulting in increased effective trafficking of tumor specific lymphocytes.

[00132] In some embodiments, IT-EP CXCL9 therapy inhibits angiogenesis in a tumor or tumor microenvironment. In some embodiments, combining IT-EP CXCL9 with IL-12 therapy increases trafficking of tumor-specific lymphocytes to tumors.

[00133] In some embodiments, intratumoral electroporation of an expression cassette encoding a CXCL9 can be administered with other therapeutic entities. In some embodiments, IT-EP CXCL9 therapy is combined IL-12 therapy. IL-12 therapy may occur before, concurrent with, and/or after IT-EP CXCL9 therapy. IL-12 therapy can occur before and concurrent with IT-EP CXCL9 therapy. IL-12 therapy can occur before and after IT-EP CXCL9 therapy. IL-12 therapy can occur concurrent with and after IT-EP CXCL9 therapy. IL-12 therapy may occur before, concurrent with, and after IT-EP CXCL9 therapy. IT-EP CXCL9 therapy may occur before, concurrent with, and/or after IL-12 therapy. IT-EP CXCL9 therapy may occur before and concurrent with IL-12 therapy. IT-EP CXCL9 therapy may occur before and after IL-12 therapy. IT-EP CXCL9 therapy may occur concurrent with and after IL-12 therapy. IT-EP CXCL9 therapy may occur before, concurrent with, and after IL-12 therapy. In some embodiments, the IL-12 therapy is administered by IT-EP of an expression cassette encoding IL-12. The CXCL9 and IL-12 can be expressed from a single expression cassette or plasmid or from multiple expression cassettes or plasmids. In some embodiments, for concurrent therapy, IT-EP CXCL9-IL12 therapy, CXCL9 and IL-12 are expressed from a single expression cassette or plasmid.

[00134] In some embodiments, intratumoral electroporation of an expression cassette encoding a CD3 half-BiTE can be administered with other therapeutic entities. In some embodiments, IT-EP CD3 half-BiTE therapy is combined IL-12 therapy. IL-12 therapy may occur before, concurrent with, and/or after IT-EP CD3 half-BiTE therapy. IL-12 therapy can occur before and concurrent with IT-EP CD3 half-BiTE therapy. IL-12 therapy can occur before and after IT-EP CD3 half-BiTE therapy. IL-12 therapy can occur concurrent with and after IT-EP CD3 half-BiTE therapy. IL-12 therapy may occur before, concurrent with, and after IT-EP CD3 half-BiTE therapy. IT-EP CD3 half-BiTE therapy may occur before, concurrent with, and/or after IL-12 therapy. IT-EP CD3 half-BiTE therapy may occur before and concurrent with IL-12 therapy. IT-EP CD3 half-BiTE therapy may occur before and after IL-12 therapy. IT-EP CD3 half-BiTE therapy may occur concurrent with and after IL-12 therapy. IT-EP CD3 half-BiTE therapy may occur before, concurrent with, and after IL-12 therapy. In some embodiments, IL-12 therapy is administered by IT-EP of an expression cassette encoding IL-12. The CD half-BiTE and IL-12 can be expressed from a single expression cassette or plasmid or from multiple expression cassettes or plasmids. In some embodiments, for concurrent therapy, IT-EP CD3 half-BiTE-IL12 therapy, CD3 half-BiTE and IL-12 are expressed from a single expression cassette or plasmid

[00135] In some embodiments, IT-EP CXCL9 therapy is combined with IT-EP CD3 half-BiTE therapy. In some embodiments, IT-EP CXCL9 and/or IT-EP CD3 half-BiTE therapy is combined with IL-12 therapy. IT-EP CD3 half-BiTE therapy may occur before, concurrent with, and/or after IT-EP CXCL9 therapy. IT-EP CD3 half-BiTE therapy can occur before and concurrent with IT-EP CXCL9 therapy. IT-EP CD3 half-BiTE therapy can occur before and after IT-EP CXCL9 therapy. IT-EP CD3 half-BiTE therapy can occur concurrent with and after IT-EP CXCL9 therapy. IT-EP CD3 half-BiTE therapy may occur before, concurrent with, and after IT-EP CXCL9 therapy. IT-EP CXCL9 therapy may occur before, concurrent with, and/or after IT-EP CD3 half-BiTE therapy. IT- EP CXCL9 therapy may occur before and concurrent with IT-EP CD3 half-BiTE therapy. IT-EP CXCL9 therapy may occur before and after IT-EP CD3 half-BiTE therapy. IT-EP CXCL9 therapy may occur concurrent with and after IT-EP CD3 half-BiTE therapy. IT- EP CXCL9 therapy may occur before, concurrent with, and after IT-EP CD3 half-BiTE therapy. Either CXCL3 or CD half-BiTE therapy can be combined with IL-12 therapy, such as by IT-EP of an expression cassette or plasmid encoding both CXCL9 and IL-12 or CD3-hallf-BiTe and IL-12, respectively (/.<?., IT-EP IL l 2-CXCL9 and IT-EP CD3 half- BiTE~IL12 therapies).

[00136] In some embodiments, IT-EP CD3 half-BiTE therapy or IT-EP CD3 half- BiTE~IL-12 therapy can be co-administered with one or more of IT-EP IL12 therapy, IT- EP CXCL9 therapy, and IT-EP IL l 2-CXCL9 therapy.

[00137] In some embodiments, a described expression cassette is combined with one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than an active pharmaceutical ingredient (API, therapeutic product) that are intentionally included with the API (molecule). Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the API during manufacture, b) protect, support or enhance stability, bioavailability or subject acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance. Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water- repelling agents, and wetting agents.

VII. Treatment Regimens/Cycles

[00138] The described IT-EP therapies can be administered at various intervals, depending upon such factors, for example, as the nature of the tumor, the condition of the subject, the size and chemical characteristics of the molecule and half-life of the molecule.

[00139] In some embodiments, methods for treating a tumor are described comprising, administering IT-EP IL12 therapy, followed by IT-EP CXCL9 and/or IT-EP

IL12~CXCL9 therapy. IT-EP CXCL or IT-EP IL12~CXCL9 therapy can increase recruitment of tumor-specific T cells to the tumor or tumor microenvironment and/or increase activation of T cells. In some embodiments, IT-EP IL12 therapy is given to a tumor on day 0 (±1 day) and IT-EP CXCL9 therapy is given to the tumor on day 4 (±2 days) and day 7 (±2 days). In some embodiments, IT-EP IL12 therapy is given to a tumor on day 0 and IT-EP IL 12-CXCL9 therapy is given to the tumor on day 4 (±2 days) and day 7 (±2 days).

[00140] In some embodiments, methods for treating a tumor are described comprising, administering IT-EP IL12 therapy, followed by IT-EP CD3 half-BiTE and/or CD3 half- BiTE~IL12 therapy. In some embodiments, IT-EP IL12 therapy is given to a tumor on day 0 (±1 day) and IT-EP CD3 half-BiTE therapy is given to the tumor on day 4 (±2 days) and day 7 (±2 days). In some embodiments, IT-EP IL12 therapy is given to a tumor on day 0 and IT-EP CD3 half-BiTE~IL12 therapy is given to the tumor on day 4 (±2 days) and day 7 (±2 days).

[00141] In some embodiments, methods for treating a tumor are described comprising, IT-EP IL12 therapy, following by IT-EP CXCL or IT-EP IL12~CXCL9 therapy, and/or IT-EP CD3 half-BiTE or IT-EP CD3 half-BiTE~IL-12 therapy.

[00142] In some embodiments, IT-EP IL12 therapy is administered first to increase tumor infiltrating lymphocytes. The tumor is subsequently treated with IT-EP CXCL9 or IL12~CXCL9 therapy and/or IT-EP CD3 half-BiTE or IT-EP CD3 half-BiTE~IL-12 therapy.

[00143] A treatment cycle can comprise 1-6 IT-EP treatments. In some embodiments, a treatment cycle comprises 1, 2, or 3 IT-EP treatments. A cycle can be from about 1 week to about 6 weeks, or from about 2 weeks to about 5 weeks. In some embodiments, a cycle is about 3 weeks.

[00144] In some embodiments, a cycle comprises 1-3 IT-EP treatments. The treatments can occur on days 1 (± 2 days), 5 (± 2 days) and/or day 8 (± 2 days) (i.e., days 0 (± 2 days), 4 (± 2 days) and/or day 7 (± 2 days)). Each treatment can comprise one or more of IT-EP IL2, IT-EP CXCL9, IT-EP IL l 2-CXCL9, IT-EP CD3 half-BiTE, IT-EP CD3 half- BiTE~IL12, and IT-EP anti-CTLA4 scFv.

[00145] In some embodiments, methods for treating a tumor are described comprising: administering IT-EP ILl 2 therapy on day 1 of a cycle and administering IT-EP CXCL9 or IT-EP IL12~CXCL9 on days 5 (± 2 days) and day 8 (± 2 days) of the cycle. In some embodiments, methods for treating a tumor are described comprising: administering IT- EP IL12 therapy on day 1 of a cycle and administering IT-EP CD3 half-BiTE, IT-EP CD3 half-BiTE~IL12 on days 5 (± 2 days) and day 8 (± 2 days) of the cycle. In some embodiments, methods for treating a tumor are described comprising: administering IT-EP IL12 therapy on day 1 of a cycle and administering one or more of IT-EP CXCL9, IT-EP IL12~CXCL9, IT-EP CD3 half-BiTE, and IT-EP CD3 half-BiTE~IL12 on days 5 (± 2 days) and day 8 (± 2 days) of the cycle.

[00146] In some embodiments, methods for treating a tumor are described comprising: a) administering IT-EP IL12 therapy in a first cycle, b) administering IT-EP CXCL9 or IT- EP IL12~CXCL9 therapy in a second cycle, and c) administering IT-EP CD3 half-BiTE or IT-EP CD3 half-BiTE~IL-12 therapy in a third cycle. Each cycle can comprise 1-3 administrations of the corresponding IT-EP therapy.

[00147] Described are dosing regimens encompassing administering IT-EP IL12 therapy in combination IT-EP CXCL9 therapy and/or IT-EP CD3 half-BiTE therapy. Also described are dosing regimens encompassing administering IT-EP CXCL9 or

IL12 -CXCL9 therapy with IT-EP CD3 half-BiTE or IT-EP CD3 half-BiTE- lL 12 therapy. The therapies may be administered concurrently, sequentially, or separately. In some embodiments, IT-EP IL12 therapy is administered in a first cycle and IT-EP CXCL9 therapy or IT-EP IL12~CXCL9 therapy is administered in a second cycle. In some embodiments, IT-EP D_12 therapy is administered in a first cycle and IT-EP CD3 half- BiTE therapy or IT-EP CD3 half-BiTE- ILl 2 therapy is administered in a second cycle. In some embodiments, IT-EP IL12 therapy is administered in a first cycle, IT-EP CXCL9 therapy or IT-EP CXCL9-TL12 therapy is administered in a second cycle, and IT-EP CD3 half-BiTE therapy or IT-EP CD3 half-BiTE IL12 therapy is administered in a third cycle. The IT-EP therapy may be delivered on day 1 of each cycle. One or more of the cycles may be repeated as necessary. Within a cycle, the IT-EP therapy may be administered on a least one, two, or three days of the cycle. For example, a given expression cassette may be administered on day 1, day 5 (± 2 days) and/or day 8 (± 2 days). [00148] In some embodiments, a CXCL9 or IL12~CXCL9 plus IL-12 expression cassette is administered on days 1, 5±2, and 8±2 of a cycle. In some embodiments, a CTLA-4 scFv or anti-CTLA-4 scFv plus IL-12expression cassette is administered on days 1, 5±2, and 8±2 of a cycle. In some embodiments, a CD3 half-BiTE or CD3 half-BiTE plus IL-12 expression cassette is administered on days 1, 5±2, and 8±2 of a cycle.

[00149] In some embodiments, a CXCL9 or CXCL9 plus IL-12 expression cassette ( e.g ., IL12~CXCL9) is administered on days 1 and 5±2, and a CD3 half-BiTE or CD3 half-BiTE plus IL-12 expression cassette (e.g., CD3 half-BiTE~IL12) is administered on day 8±2 of a cycle. In some embodiments, a CXCL9 or CXCL9 plus IL-12 expression cassette is administered on day 1, and a CD3 half-BiTE or CD3 half-BiTE plus IL-12 expression cassette is administered on days 5±2 and 8±2 of a cycle. In some embodiments, a CXCL9 or CXCL9 plus IL-12 expression cassette is administered on days 1 and 8±2, and a CD3 half-BiTE or CD3 half-BiTE plus IL-12 expression cassette is administered on day 5±2 of a cycle.

[00150] In some embodiments, a CD3 half-BiTE or CD3 half-BiTE plus IL-12 expression cassette is administered on days 1 and 5±2, and a CXCL9 or CXCL9 plus IL- 12 expression cassette is administered on day 8±2 of a cycle. In some embodiments, a CD3 half-BiTE or CD3 half-BiTE plus IL-12 expression cassette is administered on days 1, and a CXCL9 or CXCL9 plus IL-12 expression cassette is administered on days 5±2 and 8±2 of a cycle. In some embodiments, a CD3 half-BiTE or CD3 half-BiTE plus IL-12 expression cassette is administered on days 1 and 8±2, and a CXCL9 or CXCL9 plus IL- 12 expression cassette is administered on day 5±2 of a cycle.

[00151] In some embodiments, an IL-12-2A expression cassette is administered on day 1 and, and a CXCL9 or IL 12-CXCL9 expression cassette is administered on days 5±2 and 8±2 of a cycle. In some embodiments, an IL-12-2A expression cassette is administered on days 1 and 5±2, and a CXCL9 or IL12~CXCL9 expression cassette is administered on day 8±2 of a cycle.

[00152] In some embodiments, an IL-12-2A expression cassette is administered on day 1 and, and a CD3 half-BiTE or CD3 half-BiTE~IL-12 expression cassette is administered on days 5±2 and 8±2 of a cycle. In some embodiments, an IL-12-2A expression cassette is administered on days 1 and 5±2, and a CD3 half-BiTE or CD3 half-BiTE~IL-12 expression cassette is administered on day 8±2 of a cycle. [00153] In some embodiments, an IL12-2A expression cassette is administered on day 1, a CD3 half-BiTE or CD3 half-BiTE~IL-12 expression cassette is administered on day 5±2, and a CXCL9 or IL12~CXCL9 expression cassette is administered on day 8±2 of a cycle. In some embodiments, an IL-12-2A expression cassette is administered on day 1, a CXCL9 or IL12~CXCL9 expression cassette is administered on day 5±2, and a CD3 half- BiTE or CD3 half-BiTE~IL-12 expression cassette is administered on day 8±2 of a cycle.

[00154] In some embodiments, a subject is administered either IT-EP IL-12~CXCL9 therapy or IT-EP CD3 half-BiTE~IL12 therapy on days 0, 4 (±2 days), and 7(±2 days) provided the subject receives at least one IT-EP treatment with IL- 12-CXCL9 and one IT- EP treatment with CD3 half-BiTE~IL12.

[00155] In some embodiments, a treatment can be administered every cycle or every other cycle. A cycle may be repeated such that 2 or more cycles are administered to a subject. Repeated cycles may be administered consecutively, alternated with one or more different cycles of treatment, or run concurrently with one or more difference cycles of treatment. Any of the above described treatments can be combined with other cancer therapies. For example, an IT-EP cycle can be combined with checkpoint inhibitor therapy.

VIII. Combination Therapy

[00156] In some embodiments, a therapeutic method includes a combination therapy. A combination therapy comprises a combination of therapeutic molecules or treatments. Therapeutic treatments include, but are not limited to, electric pulse (i.e., electroporation), radiation, antibody therapy, checkpoint inhibitor therapy, and chemotherapy. In some embodiments, administration of a combination therapy is achieved by electroporation alone. In some embodiments, administration of a combination therapy is achieved by a combination of electroporation and systemic delivery. In some embodiments,

administration of a combination therapy is achieved by a combination of electroporation and radiation. In some embodiments, administration of a combination therapy is achieved by a combination of electroporation and oral medication. Therapeutic electroporation can be combined with, or administered with, one or more additional therapeutic treatments. The one or more additional therapeutics can be delivered by systemic delivery, intratumoral injection, intratumoral injection with electroporation, and/or radiation. The one or more additional therapeutics can be administered prior to, concurrent with, or subsequent to the CXCL9 and/or CD3 half-BiTE electroporation therapy.

[00157] In some embodiments, methods of treating cancer as described comprising: administering IT-EP therapy on day 1, days 1 and 5 (±2 days), days 1 and 8 (±2 days), or days 1, 5 (±2 days), and 8 (±2 days) and administering an additional therapeutic treatment on day 1 of a 3-6 week cycle. In some embodiments, methods of treating cancer as described comprising: administering IT-EP therapy on day 1, days 1 and 5 (±2 days), days 1 and 8 (±2 days), or days 1, 5 (±2 days), and 8 (±2 days) of every other cycle {i.e., every 6 weeks) and administering an additional therapeutic treatment on day 1 of each 3 week cycle (i.e., every 3 weeks). In some embodiments, the additional therapeutic treatment comprises a checkpoint inhibitor.

IX. Electroporation (EP) therapy

[00158] Electroporation therapy comprises administering at least one electroporative pulse to a cell, tissue, or tumor. Electroporation therapy can be performed using any known electroporation device suitable for use in a mammalian subject. The described expression cassettes can be administered to a subject before, during, or after

administration of the electric pulse. The expression cassette can be administered at or near the tumor in a subject. The described expression cassettes can be injected into a tumor using a hypodermic needle.

[00159] In some embodiments, electroporation therapy comprises the administration of one or more voltage pulses. The nature of the electric field to be generated is determined by the nature of the tissue, the size of the selected tissue and its location. The voltage pulse that can be delivered to the tumor may be about 100 V/cm to about 1500V/cm. In some embodiments, the voltage pulse is about 700 V/cm to 1500 V/cm. In some embodiments, the voltage pulse may be about 600 V/cm, 650 V/cm, 700 V/cm, 750 V/cm, 800 V/cm,

850 V/cm, 900 V/cm, 950 V/cm, 1000 V/cm, 1050 V/cm, 1100 V/cm, 1150 V/cm, 1200 V/cm, 1250 V/cm, 1300 V/cm, 1350 V/cm, 1400 V/cm, 1450 V/cm, or 1500 V/cm. In some embodiments, the voltage pulse is about 10 V/cm to 700 V/cm. In some

embodiments, the electric is about 100 V/cm, 150 V/cm, 200 V/cm, 250 V/cm, 300 V/cm, 350 V/cm, or 400 V/cm, 450 V/cm, 500 V/cm, 550 V/cm, 600 V/cm 650 V/cm. or 700 V/cm.

[00160] The pulse duration of the electroporative pulse of the may be from 10 psec to 1 second. In some embodiments, the pulse duration is from about 10 psec to about 100 milliseconds (ms). In some embodiments, the pulse duration is 100 psec, 1 ms, 10 ms, or 100 ms. The interval between pulses sets can be any desired time, such as one second. The waveform, electric field strength and pulse duration may also depend upon the type of cells and the type of molecul es that are to enter the cells via electroporation.

[00161] The waveform of the electrical signal provided by the pulse generator can be an exponentially decaying pulse, a square pulse, a unipolar oscillating pulse train, a bipolar oscill ating pulse train, or a combination of any of these forms. Square wave

electroporation systems deliver controlled electric pulses that rise quickly to a set voltage, stay at that level for a set length of time (pulse length), and then quickly drop to zero.

[00162] 1 to 100 pulses may be administered. In some embodiments, 1, 2, 3, 4, 5, 6, 7,

8, 9, or 10 pulses are administered. In some embodiments, 6 pulses are administered. In some embodiments, 6x0.1 msec pulses are administered. In some embodiments, 6 pulses are administered. In some embodiments, 6x0.1 msec pulses are administered at 1300-1500 V/cm. In some embodiments 8 pulses are administered. In some embodiments 8x 10 msec pulses are administered. In some embodiments 8x 10 msec pulses are administered at 300- 500 V/cm.

[00163] The electroporation device can compri se a single needle electrode, a pair of needle electrode, or a plurality or array of needle electrodes. In some embodiments, the electroporation device an comprise a hypodermic needle or equivalent. In some embodiments, the electroporation device can comprise an electro-kinetic device ("EKD device") able to produce a series of programmable constant-current pulse patterns between electrodes in an array based on user control and input of the pulse parameters.

[00164] Electroporation devices suitable for use with the described compounds, compositions, and methods include, but are not limited to, those described in U. S. Patent Nos. 7245963, 5439440, 6055453, 6009347, 9020605, and 9037230, and U.S. Patent Publication Nos. 2005/0052630, 2019/0117964, and patent applications

PCT/US2019/030437 and US. Patent Applications Serial No. 16/269,022.

List of embodiments:

[00165] E An expression cassette comprising: a first nucleotide sequence encoding a CD3 half-BiTE, wherein the CD3 half-BiTE comprises an anti-CD3 scFv and a transmembrane domain wherein the transmembrane domain is linked to the C-terminal end of the anti-CD3 scFv. [00166] 2. The expression cassette of embodiment 1, wherein the first nucleotide sequence is operatively linked to a promoter.

[00167] 3. The expression cassette of embodiment 2, wherein the promoter is selected from the group consisting of: CMY promoter, mPGK, SY40 promoter, b-actin promoter, SRa promoter, herpes thymidine kinase promoter, herpes simplex virus (HSV) promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), rous sarcoma virus (RSV) promoter, and EF la promoter.

[00168] 4. The expression cassette any one of embodiments 1-3, wherein the anti-CD3 scFv comprises CDR regions of the VH and VL domains of OKT3 (Muromonab-CD3), 145-2C11, 17A2, SP7, or UCHTl antibodies.

[00169] 5. The expression cassette of embodiment 4, wherein the anti-CD3 scFv comprises the VF and VL domains of OKT3 (Muromonab-CD3), 145-2C11, 17A2, SP7, or UCHT1 or a humanized version thereof.

[00170] 6. The expression cassette of any one of embodiments 1-5, wherein the transmembrane domain is selected from the group consisting of: PDGFRa transmembrane domain, and PDGFRfS transmembrane domain.

[00171] 7. The expression cassette of any one of embodiments 1-6, wherein the first nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 60, 62, 74, or 76 or a polypeptide having at least 70%, 72%, 75%, 78%, 80%,

82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identify to SEQ ID NO: 60, 62, 74, or 76.

[00172] 8. The expression cassette any one of embodiments 1-7, wherein the first nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 59, 61, 73, or 75 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identify to SEQ ID NO: 59, 61, 73, or 75.

[00173] 9. The expression cassette of any one of embodiments 2-8, further comprising a second nucleotide sequence encoding IL-12.

[00174] 10. The expression cassette of embodiment 9, wherein the second nucleotide sequence encoding IL-12 comprises a first coding sequence encoding IL-12 p35 and a second coding sequence encoding IL-12 p40. [00175] 11. The expression cassette of embodiment 10, wherein the expression cassette comprises the formula represented by:

P - A - T - B - T - B'

wherein P is the promoter, A encodes the CD3 half-BiTE, T is a translation modification element, B encodes IL-12 p35, and B' encodes IL-12 p40.

[00176] 12. The expression cassette of embodiment 11, wherein T encodes a 2A peptide selected from the group consisting of: a P2A peptide, a T2A peptide, a E2A peptide, and a F2A peptide.

[00177] 13. The expression cassette of embodiment 12, wherein A encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 60, 62, 74, or 76 or a polypeptide having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%,

90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identify to SEQ ID NO: 60, 62, 74, or 76; B encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 or 53, or a polypeptide having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 31 or 53; and B' encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 33 or 56 or a polypeptide having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 33 or 56.

[00178] 14. The expression cassette of embodiment 12, wherein the A comprises the nucleotide sequence of SEQ ID NO: 59, 61, 73, or 75 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% to the nucleotide sequence of SEQ ID NO: 59, 61, 73, or 75; B comprises the nucleotide sequence of SEQ ID NO: 30, 51, or 52 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 30, 51, or 52; and B' comprises the nucleotide sequence of SEQ ID NO: 32, 54, or 55 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 32, 54, or 55.

[00179] 15. The expression cassette of any one of embodiments 1-14, wherein the first nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 64, 66, 78, or 80 or a polypeptide having at least 70%, 72%, 75%, 78%, 80%,

82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identify to SEQ ID NO: 64, 66, 78, or 80.

[00180] 16. The expression cassette of any one of embodiments 1-15 wherein the expression cassette comprises the sequence of SEQ ID NO: 63, 65, 77, or 79 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% to the nucleotide sequence of SEQ ID NO: 63, 65, 77, or 79.

[00181] 17. The expression cassette of any one of embodiments 1-14, wherein the expression cassette further encodes a tag sequence linked to either the N-terminal or C- terminal end of the anti-CD3 scFv.

[00182] 18. The expression cassette of embodiment 17, wherein the tag sequence comprises at least one tag sequence selected from the group consisting of: an HA tag and a Myc tag.

[00183] 19. A plasmid for expressing a CD3 half-BiTE comprising the expression cassette of any one of embodiments 1-18.

[00184] 20. A CD3 half-BiTE comprising: anti-CD3 single-chain variable fragment

(scFv) fused to a transmembrane domain.

[00185] 21. The expression cassette of any one of embodiments 1-18 or the plasmid of embodiment 19, for use in treating cancer.

[00186] 22. An expression cassette comprising the formula represented by:

P - B - T - B' - T - A

wherein P is a promoter, A encodes a CXCL9, T is a translation modification element, B encodes IL-12 p35, and B' encodes IL-12 p40.

[00187] 23. The expression cassette of embodiment 22, wherein T comprises a 2A peptide selected from the group consisting of: a P2A peptide, a T2A peptide, a E2A peptide, and a F2A peptide.

[00188] 24. The expression cassette of embodiment 22 or 23, wherein A encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or 58 or a polypeptide having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identify to SEQ ID NO: 35 or 57; B encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 or 53 or a polypeptide having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%,

90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 31 or 53; and B' encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 33 or 56 or a polypeptide having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 33 or 56.

[00189] 25. The expression cassette of any one of embodiments 22-24, wherein A comprises the nucleotide sequence of SEQ ID NO: 34 or 57 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% to the nucleotide sequence of SEQ ID NO: 34 or 57; B comprises the nucleotide sequence of SEQ ID NO: 30, 51, or 52 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 30, 51, or 52; and B' comprises the nucleotide sequence of SEQ ID NO: 32, 54, or 55 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 32, 54, or 55.

[00190] 26. The expression cassette of any one of embodiments 22-25, wherein the expression cassette encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 68 or 82 or a polypeptide having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%,

85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identify to SEQ ID NO: 68 or 82.

[00191] 27. The expression cassette of any one of embodiments 22-26, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 67 or 81 or a nucleotide sequence having at least 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% to the nucleotide sequence of SEQ ID NO: 67 or 81.

[00192] 28. A plasmid for expressing CXCL9 and IL-12, comprising the expression cassette of any one of embodiments 22-27.

[00193] 29. The expression cassette of any one of embodiments 22-27 or the plasmid of embodiment 28, for use in treating cancer.

[00194] 30. An expression cassette encoding a CD3 half-BiTE and an expression cassette encoding CXCL9 for use in treating cancer, wherein the expression cassettes are formulated for intratumoral electroporation therapy.

[00195] 31. A method of treating a subject having a tumor comprising injecting the tumor with an effective dose of at least one plasmid according to embodiment 19 or 28 and administering electroporation therapy to the tumor.

[00196] 32. The method of embodiment 31, wherein the electroporation therapy comprises administration of at least one voltage pulse over a duration of about 100 microseconds to about 1 millisecond.

[00197] 33. The method of embodiment 32, wherein the electroporation therapy comprises administration of 1-6 voltage pulses.

[00198] 34. The method of embodiment 32 or 33, wherein the 1-10 voltage pulses have a field strength of about 200 V/cm to about 1500 V/cm.

[00199] 35. The method of any one of embodiments 31-34, wherein the at least one plasmid is injected into the tumor and the electroporation therapy is administered on day 1, day 5±2 days, and day 8±2 days.

[00200] 36. The method of any one of embodiments 31-34, wherein

(a) the plasmid according to embodiment 19 is injected into the tumor and the

electroporation therapy is administered on day 1 and day 5±2 days, and the plasmid according to embodiment 28 is injected into the tumor and the electroporation therapy is administered on day 8±2 days;

(b) the plasmid according to embodiment 19 is injected into the tumor and the

electroporation therapy is administered on day 1 and day 8±2 days, and the plasmid according to embodiment 28 is injected into the tumor and the electroporation therapy is administered on day 5±2 days;

(c) the plasmid according to embodiment 19 is injected into the tumor and the

electroporation therapy is administered on day 5±2 days and day 8±2 days, and the plasmid according to embodiment 28 is injected into the tumor and the electroporation therapy is administered on day 1;

(d) the plasmid according to embodiment 28 is injected into the tumor and the

electroporation therapy is administered on day 1 and day 5±2 days, and the plasmid according to embodiment 19 is injected into the tumor and the electroporation therapy is administered on day 8±2 days;

(e) the plasmid according to embodiment 28 is injected into the tumor and the

electroporation therapy is administered on day 1 and day 8±2 days, and the plasmid according to embodiment 19 is injected into the tumor and the electroporation therapy is administered on day 5±2 days; and

(f) the plasmid according to embodiment 28 is injected into the tumor and the

electroporation therapy is administered on days 5±2 days and 8±2 days, and the plasmid according to embodiment 19 is injected into the tumor and the electroporation therapy is administered on day 1.

[00201] 37. The method of any one of embodiments 31-36, further comprising administering to the subject at least one additional therapeutic.

[00202] 38. The method of any one of embodiments 31-37, wherein the method results in one or more or: increased tumor infiltrating lymphocytes, increased activation and/or proliferation of tumor-specific T cells, regression of the treated tumor, and regression of one or more untreated tumors.

[00203] 39. The method of claim 37, wherein the at least one additional therapy comprises administering IT-EP anti-CLTA-4 scFv therapy.

[00204] 40. A method for treating of treating a subject having a tumor comprising injecting the tumor with an effective dose of at least one plasmid encoding an anti-CTLA- 4 scFv and administering electroporation therapy to the tumor.

[00205] 41. The method of claim 40, wherein the method further comprises one or more of: IT-EP IL12 therapy, IT-EP CXCL9 therapy, IT-EP IL12-CXCL9 therapy, IT-EP CD3 half-BiTE therapy, and IT-EP CD3 half-BiTE~IL12 therapy.

Table 1. Sequences (nucleotides or amino acids in parenthesis may or may not be present)

EQKLISEEDL

ggctctggcacagactttaccttgaaaatctccagagttgaggctgaggaccttggtgta tactactgct tccaaggctctcatgttccctacactttcggaggcggaacaaaactggagataaaacgag ccgacgcagc ccccactgtg y q

gaggcaaagcttcaggaatctggtccagtgttggtgaaaccaggtgcatccgtgaaaatg tcctgcaaag caagcggttacacttttactgactattatatgaactgggtaaagcaatcccacggcaaat ccctggaatg gattggtgtcatcaacccttacaacggtgatacaagttacaaccaaaagttcaaaggtaa ggctacattg accgtagataagagtagcagtactgcatacatggaacttaactctcttacatccgaggac tccgctgttt actattgtgcacgctactacgggagctggttcgcttactggggtcaaggcaccctgataa cagtgtccac agccaaaaccacacctccctccgtctatcctctcgctcca

Mouse anti-CTLA-4 9D9 variable heavy chain amino acid sequence (SEQ ID NO: 39)

EAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTSY NQKFKGKATL

TVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFAYWGQGTLITVSTAKTTPPSVYP LAP

Mouse anti-CTLA-4 9H10 variable light chain nucleic acid sequence (SEQ ID NO: 40) gacattgtgatgacacagagtccttcatcccttgcagtcagtgtcggcgaaaaagtaaca atttcatgca agtctagtcaatctctgttgtacggctcctctcattacctcgcatggtatcaacaaaaag tgggtcaatc tcccaaattgttgatatactgggcttcaactagacacactggaatccctgacaggttcat tggtagcgga tcagggactgactttacactgtccctcagcagcgtacaagcagaagacatggccgactat ttctgccaac

caagtgcagctgcttcaatccgaatcagaactcgtgaagccaggcgcttcagtgaaattg tcttgtaaga cttcaggatacactttcactgattactatatacactgggttaagcagaagcctggtcagg gtcttgaatg gattggcctcatcaatcccaataacgatggcacaaactacaaccagaaatttcaaggaaa agccacactt accgcagacaaatccagttctaccgcatacatggaacttaatagtctcacttttgatgac tcagtaatat atttctgtgccagggccagtagccgacttagaatggctaggactacctctgactactatg ccatggacta ttggggacagggcattcaagtgaccgtgagctct

Mouse anti-CTLA-4 9H10 variable heavy chain amino acid sequence (SEQ ID NO: 43)

QVQLLQSESELVKPGASVKLSCKTSGYTFTDYYIHWVKQKPGQGLEWIGLINPNNDG TNYNQKFQGKATL

TADKSSSTAYMELNSLTFDDSVIYFCARASSRLRMARTTSDYYAMDYWGQGIQVTVS S

mlgGl Fc domain nucleic acid sequence (SEQ ID NO: 44)

ggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttcccccca aagcccaagg atgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaagg atgatcccga ggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccg ggaggagcag ttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaat ggcaaggagt tcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaa ccaaaggcag accgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataa agtcagtctg acctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatggg cagccagcgg agaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagca agctcaatgt gcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcct gcacaaccac catact a aa a cctctcccactctcct taaat a

ar a e eavy c a n nuc e c ac sequence :

caggtgcagctgcagcaatctggggctgaactggcaagacctggggcctcagtgaagatg tcctgcaagg cttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagg gtctggaatg gattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaa ggccacattg actacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggac tctgcagtct attactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcacca cactcaccgt ctcctca

OKT3 Variable heavy chain amino acid sequence (SEQ ID NO: 47)

[00206] Although the invention has been described in detail for purposes of clarity of understanding, certain modifications may be practiced within the scope of the appended claims. All publications, accession numbers, web sites, patent documents and the like cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so individually denoted. To the extent different information is associated with a citation at different times, the information present as of the effective filing date of this application is meant. Unless otherwise apparent from the context any element, embodiment, step, feature or aspect of the invention can be performed in combination with any other.

EXAMPLES

CXCL9

[00207] Example 1. CXCL9 plasmid construction. Mouse CXCL9 (mCXCL9) or human CXCL9 (hCXCL9) nucleic acid sequence was cloned into an expression vector using standard molecular biology techniques to. Alternatively, mCXCL9 or hCXCL9 was cloned downstream of mouse (mIL12-2A) or human (hIL12-2A) IL12 p35-P2A-IL12 p40 to yield mIL12~mCXCL9 and hIL12~hCXCL9 (FIG. 1 A-B_. IL12 p35-P2A-IL12 p40 constructs were made essentially as described in WO2017/106795 or

WO2018/229696.

[00208] The resulting plasmids contained IL-12 p35, IL-12 p40 and CXCL9, all expressed from the same promoter, with intervening exon skipping (P2A) motifs to allow all three proteins to be expressed from a single polycistronic message. Similar methods were sued to make mCXCL9~mCherry

[00209] Example 2. Protein expression. The mIL12-2A, mCXCL9, and

mIL12~mCXCL9 expression vectors were transfected into HEK293 cells in vitro. 96 h after transfection, supernatants were collected and IL12 and CXCL9 protein expression were assayed by ELISA. The results, shown in FIG. 2 show that, while expression was decreased in the cells transfected with the mIL12~mCXCL9 expression vector, detectable levels of both IL12 and CXCL9 were produced. FIG. 27 shows high levels of secreted hTEI 2 and hCXCL9 in cells transfected with hIL-12~hCXCL9 expression vector.

[00210] Similarly, hIL12-2A, hCXCL9, and hIL12~hCXCL9 expression vectors were transfected into HEK293 cells in vitro. 96 h after transfection, supernatants were collected and IL12 and CXCL9 protein expression were assayed by ELISA. hIL12 was expressed nearly equally from both the hIL12-2A (1.59 pg/mL) and hIL12~hCXCL9 (1.37 pg/mL) expression vectors (FIG. 10A). Decreased, but still substantial levels of hCXCL9 was expressed in cells transfected with the hIL12~hCXCL9 expression vector (1.75 pg/mL) compared to cells transfected with the hCXCL9 expression vector (5.19 pg/mL) (FIG. 10B).

[00211] mIL12 protein produced from the mIL12~mCXCL9 expression vector was further tested for activity. mIL12 produced from cells transfected with the mIL12-2A or mIL12~mCXCL9 expression vectors was incubated with HEK-Blue IL-12 cells. HEK- Blue IL-12 cells are used to detect bioactive human and mouse IL-12. HEK-Blue IL-12 cells are used to validate the functionality of recombinant native or engineered human or mouse IL-12. Functional IL-12 binds to IL-12 receptor in HEK-Blue IL-12 cells and activates a STAT-4 pathway and a STAT4-inducible SEAP reporter gene. SEAP expression is then assayed. The response ratio was calculated by dividing the OD at 630 nm for treated cells by the OD at 630 nm for untreated cells. The results, shown in FIG. 3, demonstrate that IL-12, produced from either the mIL12-2A or the mIL12~mCXCL9 expression vectors is functional.

[00212] Similarly, hIL12 protein produced from the hIL12~hCXCL9 expression vector was also tested for activity. hIL12 produced from cells transfected with the

hIL12~hCXCL9 expression vector was incubated with HEK-Blue IL-12 cells. The results, shown in FIG. 11, demonstrate that IL-12, produced from the hIL12-2A expression vector, is functional.

[00213] Example 3. CXCL9-induced migration of T cells in vitro. Mammalian

(HEK293) cells were transfected with CXCL9 expression vectors (CXCL9 or

IL12-CXCL9 ). OT-I mouse splenocytes were pulsed with 1 pg/mL SIINFEKL peptide for 24 h, then allowed to recover for 72 h. The CXCL9 transfected cells were then assayed for the induction of chemotaxis of the SIINFEKL-pulsed OT-I splenocytes through polycarbonate membranes with 5.0-micron pores. Migration index was defined as the number of observed chemotactic cells, normalized to the number of cells that passively migrated through the membrane in the OptiMEM negative control. Results are shown in FIG. 4A, 4B, and 4C. mCXCL9, produced from mCXCL9 and mIL12~mCXCL9 expression vectors caused about 7-fold and about 3-fold increases in chemotactic cells, respectively. The increase in chemotaxis was inhibited by the addition of CXCL9 neutralizing antibodies, indicating the effect was dependent on mCXCL9.

[00214] Example 4. In vivo expression of mCXCL9. CT-26 (colon carcinoma) tumors were implanted in mice. Tumors were subsequently treated with IT-EP pUMCV3 control vector or IT-EP mCXCL9 expression vector. 48 h after IT-EP, tumors were homogenized and assay for CXCL9 expression by ELISA (DuoSet ELISA DY392; n=3; * P<0.05; T test with Welch correction). The results in FIG. 5 show that the IT-EP treated tumors expressed CXCL9.

[00215] Example 5. Tumor regression in mice treated with mIL12-2A and mCXCL9. Mice were implanted with tumor cells. Anesthetized mice were subcutaneously injected with cells into the right and/or left flank. Tumor growth was monitored by digital caliper measurements until average tumor volume reached -100 mm ³ .

[00216] Tumors were treated on day 0 with IT-EP control vector or IT-EP IL12-2A expression vector and on days 4 and 7 with IT-EP control vector or IT-EP CXCL9

(optionally with mcherry reported protein). Tumor volumes and survival were monitored. Mice were euthanized when the total tumor burden of the primary and contralateral reached 2000 mm ³ .

[00217] The data, shown in FIG. 6, show that mice treated with IT-EP mIL12-2A plus mCXCL9 therapy showed increased survival compared to untreated mice, mice treated with control vehicle, or mice treated with IT-EP mIL12-2A alone. Tumor bearing mice treated with IT-EP mIL12-2A plus mCXCL9~mCherry therapy also showed decreased primary (treated) and contralateral (untreated) tumor progression (FIG. 7A-B).

[00218] Example 6. IT-EP IL12-2A + IT-EP CXCL9 drives systemic expansion of antigen specific CD8 and short-lived effector cells (SLECs). On day -8, mice were implanted with tumor as described above. On day 0, tumors were treated with IT-EP mIL12-2A. On days 4 and 7, mice were treated with control plasmid or mCXCL9

(n=3/group) using IT-EP as described above. On day 9, spleens were harvested and CD3 ⁺ CD8 ⁺ cells analyzed by FACS. FIG. 8 shows that CD3 ⁺ T cell populations were significantly increased in mice treated with IL12-2A + CXCL9. Fold increase in the number of AH1+ CD8+ T cells is shown in FIG. 9.

[00219] Example 7. Intratumoral CXCL9 synergizes with IL-12 to modulate the tumor microenvironment, expand antigen-specific T cells, and control contralateral tumor growth. A mouse model was used to evaluate intratumoral expression post electroporation.

[00220] CT26 tumors were implanted in mice on day -7. For NanoString analysis and flow based assays single, tumor model was used. Mice were treated on day 1 with IT-EP with a suboptimal dose of IL12-2A followed by treatment on days 4 and 7 with IT-EP using 100 pg of either mCXCL9 or pUMVC3. Tumor and immune response were then monitored. Tumor and splenocytes were harvested 2 days after last EP (z.e., Day 9) for NanoString and flow based analysis. Alternatively, tumor volumes were measured three times a week for regression/survival studies. Gene expression changes in electroporated CT26 lesions were assessed by NanoString nCounter® technology. Intratumoral expression of mCXCL9 was confirmed using ELISA for mCXCL9 48hrs post

electroporation in tumor lysates from mice bearing CT26 tumors (n=3; * P<0.05; T test with Welch correction).

[00221] Volcano plots displaying p-values and log2 fold change for each gene were generated in mice treated with CXCL9 alone or CXCL9 in combination with IL12-2A (FIG. 28A). Analysis of cell type scores showed in increase in Cytotoxic immune cells in response to treatment with either CXCL9 or IL12-2A. A synergistic increase in Cytotoxic immune cell scores was further seen when CXCL9 was administered in combination with IL12-2A.‘Cytotoxic immune cells’ cell type scores are shown in FIG. 28B.

[00222] Flow cytometric analysis of was used to analyze splenocytes in treated mice. Antigen specific AH1+ CD8+ T cells were measured via tetramer analysis (Immudex). Cells are gated on Singlets<Live<CD3+CD4- splenocytes (FIG. 8). The fold increase in the number of AH1+ CD8+ T cells compared to empty vector control (N=2 independent experiments with 3-5 animals/group; * P<0.05, ** P<0.005; One way ANOVA). In mice treated with control plasmid only, 0.79% of AH1 tetramer were CD8+. In mice treated with IT-EP IL12-2A, 1.43% of AH1 tetramer were CD8+. In mice treated with IT-EP IL12-2A and CXCL9, 3.22% of AH1 tetramer were CD8+. Fold increase in the number of AH1+ CD8+ T cells is shown in FIG. 9.

[00223] The results show that IT-EP CXCL9 can substantially enhance anti-tumor immune response in animal previously treated with a suboptimal dose of IT-EP IL12-2A.

CD3 half-BiTE

[00224] Example 8. The Half-BiTE expression cassettes were made in a manner similar to that described above for the generation of CXCL9 plasmids (FIG. 12A and 12B).

[00225] Example 9. Protein expression. The OKT3 scFv and 2C11 scFv, expression vectors were transfected into HEK293 cells in vitro. HA-2C11 scFv and HA-2C11 scFv~mIL12 were transfected into B16-F10 tumor cells. 24 h after transfection, supernatants were collected, and proteins were separated by gel electrophoresis. CD3 scFv, Cadherin (membrane protein) and Hsp90 were detected by Western blot analysis. The results, shown in FIG. 13 show that the expression vectors expressed the CD3 scFv protein. The CD3 scFv protein was predominantly located in the membrane fraction expression vectors

[00226] HA-OKT3 scFv, OKT3 scFv~hIL12 expression vectors were transfected into HEK293 cells in vitro. 72 h after transfection, cells were analyzed by FACS to detect CD3 scFv (FIG. 14A-C). HA-2C11 scFv and HA-2C11 scFv~mIL12 expression vectors were transfected into B16-F10 cells. Cells were analyzed by FACS to detect surface expression of CD3 scFv (FIG. 14D). Expression of IL12 from IL12-2A and HA-2C11 scFv~mIL12 expression vectors is shown in FIG. 14E.

[00227] HA-OKT3 scFv~hIL12 and OKT3 scFv~hIL12expression vectors were transfected into HEK293 cells in vitro. 72 h after transfection cells supernatant was collected and assayed for IL12p70 by ELISA. The results confirm that cells transfected with the HA-OKT3 scFv~hIL12 and OKT3 scFv~hIL12 expression vectors express and secrete hIL12p70 (FIG. 15).

[00228] In vivo expression: Mice were inoculated with B16F10 melanoma cells or 4T1 breast cancer cells on day -7. On day 0, tumors were treated with IT-EP HA-2C11 scFv~hIL12 (FIG. 16). FIG 16A-B show the CD3 half-BiTE is expressed on the surface of melanoma and breast cancer tumors following IT-EP. FIG. 16C shows that following IT- EP of HA-OKT3 scFv~hIL12, the expression vector also expresses IL-12.

[00229] Example 10. In vitro Functional assay. B16F10 cells were transfected in vitro with control vector and 2C11 scFv expression vector with or without recombinant mouse IL12. Transfected B16F10 cells were then co-cultured with naive mouse splenocytes for 23, 48, or 72 hours. Following co-culture, supernatants were assayed for IFNy and cell proliferation was evaluated by FACS. Plate bound anti-CD3 was used as a positive control. The results, shown in FIG. 17, show that IFNy expression was substantially increased when splenocytes were co-cultured with B16F10 expressing 2C11 scFv. FACS analyses were performed to analyze proliferation of CFSE labeledCD3+CD45+ T cells following co-culture of naive mouse splenocytes with B16F10 cells transfected in vitro with control vector (Tfx control), 2C11 scFv expression vector with or without recombinant mouse IL12, or with plate bound anti-CD3 (positive control) (FIG. 18).

[00230] Example 11. In vivo functional assay. On day -9, B16-OYA cells were implanted in mice (n=8/group). On day 0, tumors were treated by IT-EP with 2C11 scFv expression vector or empty vector (negative control). On day 0, mice were also implanted, by adoptive transfer, with a 1 : 1 mix of OT-1 (GFP) CD8 ⁺ cells T cells and naive mouse lymphocytes. On day 5, adoptive transferred T cell proliferation in spleen and draining lymph node (DLN) were examined by FACS. Endogenous T cell populations and

SIINFEKL expression in tumor infiltrating lymphocytes (TILs) were also examined by FACS. An increase in polyclonal T cell proliferation in DLN was observed in mice treated with IT-EP 2C11 scFv (FIG. 19). Increases in OT-1 and polyclonal T cell populations were also observed in splenocytes in mice treated with IT-EP 2C11 scFv. An increase CD8+ T cells in CD45.1+ live cells in TILs was observed in B16-OVA tumor model mice treated with 2C11 scFv IT-EP (FIG. 20). An increase antigen specific (SIINFEKL+)

CD8+ T cells in TILs was observed in B 16-OVA tumor model mice treated with 2C11 scFv IT-EP FIG. 21. The results demonstrate that IT-EP with 2C11 results in proliferation of polyclonal T cells and enhanced tumor specific T cell response in the tumor.

[00231] Example 12. In vivo cytotoxic T cells killing assay. Lymphocytes were harvested from naive mice and labeled with CFSE. Label lymphocytes were then either pulsed with OVA peptide to activate T cells (CFSE ¹¹¹ , treated) or left untreated (CFSE ¹⁰ , unpulsed). CFSE ¹¹¹ and CFSE ¹⁰ lymphocytes were combined in an about 1 : 1 ratio for administration into the tumor bearing mice.

[00232] On Day -7, mice were implanted with B 16-OVA tumor cells (B16 melanoma cells expressing ovalbumin) into the flank of c57/bl/6 mice. On Day 1, mice were treated with IT-EP anti-2Cl 1 scFv or empty vector (pUMVC3). On day 2, mice administered pulsed target cells (cells pulsed with 2 pg/ml SIINFEKL peptide labeled with ImM CFSE (5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester)) and unpulsed cells by adoptive transfer. 18 hours after adoptive transfer, spleen and draining lymph nodes were collected and analyzed.

[00233] Western blot analysis indicated the tumors expressed the CD3 half-BiTE. On day 3, 18 h after adoptive transfer, DLN were isolated. DLNs were then analyzed by FACS for the presence of CFSE ¹⁰ and CFSE ¹¹¹ cells. The results, shown in FIG. 22, show a substantial decrease in the number of CFSE ¹¹¹ cells, indicated antigen-specific killing of cells displaying the OVA peptide. The decrease was quantitated using the following formula:

[pulsed / unpulsed of treated]

% lysis = 1—

[pulsed / unpulsed of naive] [pulsed (CFSE ^ht )EP anti-CD3 ÷ unpulsed (CFSE ⁱ⁰ )EP anti-CD3]

% lysis = 1—

[pulsed (CFSE ^ht )EP contol ÷ unpulsed (CFSE ⁱ⁰ )EP control]

Results are shown in FIG. 23, showing an increase in lysis of CFSE ^M cells in both splenocytes (SP) and DLN. FACS analysis of CFSE cells is shown in FIG. 24. In control mice, percent lysis of CFSE ^M cells was 54.63 ± 12.79%. In mice receiving IT-EP CD3 half-BiTE therapy, percent lysis of CFSE ^M cells was 82.44 ± 11.35%. OVA expressing cells were specifically killed in mice treated with IT-EP CD3 half-BiTE, indicating the enhancement of an antigen-specific cytotoxic T cell response. Activated T lymphocytes were preferentially retained in tumors expressing a CD3 half-BiTE. Thus, electroporation of nucleic acid encoding a CD3 half-BiTE provides an effective tumor therapy.

[00234] IT-EP of CD3 half-BiTE resulted in increased targeting of tumor cells by T cells. Flow cytometric analysis of cells from spleen and draining lymph node

demonstrating significant antigen specific killing in the IT-EP anti-CD3(2Cl 1) group (FIG. 23 and 26).

[00235] Example 13. Tumor Regression.

[00236] A. Melanoma : On Day -7, mice were implanted with B16 melanoma cells. On Day 0, mice were treated with IT-EP with control empty vector, expression vector encoding IL12-2A. On days 4 and 7, mice were treated with IT-EP control vector or IT-EP 2C11 (CD3 half-BiTE) expression vector. Tumor progression was monitored every three days. The results show improved contralateral (untreated) tumor regression in mice treated with IL12-2A plus CD3 half-BiTE compared to treatment with IL12-2A alone (FIGs. 25A and 25B).

[00237] B. Breast Cancer : On Day -7, mice were implanted with 4T1 breast cancer cells. On Day 0, mice were treated with IT-EP with control vector, or IT-EP IL12-2A. On days 4 and 7, mice were treated with IT-EP with control vector or IT-EP 2C11 (CD3 half- BiTE) expression vector. Tumor progression was monitored every three days. The results show that combining IT-EP IL12-2A with CD3 half-BiTE therapy improves breast cancer tumor regression (FIG. 26A). IL12-2A plus CD3 half-BiTE therapy was also effective in treating lung metastases nodules in 4T1 breast cancer model mice (FIG. 26B). The absolute number of effector T cells (CD127-CD62L-CD3+) per pL peripheral blood in 4T1 breast cancer model mice is shown in FIG. 26C. CXCL9/CD3 half-BiTE combination therapy

[00238] Example 14. CXCL9 plus CD3 half-BiTE combination therapy. B16.F10 tumor bearing mice were treated with IT-EP (days 1, 5, and 8) with 10 pg IL-12 expression plasmid, 100 pg IL-12 expression plasmid, or 100 pg IL-12-CXCL9/CD3 half- BiTE~IL12. For IL-12-CXCL9/CD3 half-BiTE~IL12, either IL-12-CXCL9 or CD3 half- BiTE~IL12 is administered on each of days 1, 5, and 8 provided the subject receives at least one IT-EP treatment with IL-12~CXCL9 and one IT-EP treatment with CD3 half- BiTE~IL12. Intratumoral expression of IL-12 was confirmed (ELISA) 48 hr post IT-EP in tumor lysates (n=8 animals). IL12 70 expression is shown in FIG. 29A. Growth of primary (electroporated lesion) and contralateral (non-electroporated lesion) B16.F10 lesions was measured 12 days after IT-EP therapy (FIG. 29B-C). With respect to IL12 p70 expression, animal treated with IT-EP with 10 pg IL12-2A expressed the same amount of IL12 as animals treated with 100 pg IL-12-CXCL9/CD3 half-BiTE~IL12 (FIG. 29A).

Contralateral tumors were significantly smaller in IL-12-CXCL9/CD3 half-BiTE~IL12 treated animals compare to 10 pg IL12-2A treated mice (8-10 animals/group; statistical significance determined using two way ANOYA * p<0.05), illustrating enhancement of tumor regression using IT-EP IL-12~CXCL9/CD3 half-BiTE~IL12 therapy.

CLTA-4 scFv

[00239] Example 15. Intratumoral expression of anti-CTLA4 scFv. Mouse IgGl ELISA (ab 133045) was performed on RENCA tumor lysates to quantify intratumoral expression of anti-CTLA4 scFv. Expression of anti-CTLA4 scFv was detected only in the tumor and not in the serum highlighting local expression of the antibody upon intratumoral electroporation.

[00240] Plasmid encoded anti-CTLA4 scFv bound to recombinant CTLA4 protein. Transfection-derived secreted anti-CTLA4 (scFv) were evaluated for their binding capacity to CTLA-4. Recombinant mouse CTLA-4/human IgGl chimera (R&D Systems) were immobilized in 96-well plates (1 or 5 pg/mL, or 50 pg/well or 250 pg/well) for 18 hr at room temperature. Wells were washed three times with 0.1% Tween in PBS and blocked with 1% BSA in PBS. Conditioned medium from HEK293 cells transfected with 9H10-scFv (168 ng/mL) or 9D9-scFv (130 ng/mL) was added to the wells, and incubated for 2 h at room temperature. Wells were washed three times, and anti-mouse IgG- horseradish peroxidase (Jackson ImmunoResearch, 0.2 pg/mL) were added and incubated for 1.5 hours at room temperature. Wells were again washed three times, developed with HRP Substrate Reagent (R&D Systems) and stopped with Stop Solution, 2N sulfuric acid (R&D Systems). Optical density of each well was measured at 450nm. Graphical representation of average OD values for each condition group are displayed demonstrating binding of plasmid derived anti CTLA4scFv to recombinant CTLA4 protein (FIG. 30 A).

[00241] Mouse IgGl ELISA (abl33045) was performed on RENCA tumor lysates to quantify intratumoral expression of anti-CTLA4 scFv. Expression of anti-CTLA4 scFv was detected in the tumor (FIG. 30B). Statistically significant levels of anti-CTLA4 scFv was not observed in serum, indicating local expression of the antibody upon intratumoral electroporation.

[00242] It will be understood that the present invention has been described above by way of example only. The examples are not intended to limit the scope of the invention. Various modifications and embodiments can be made without departing from the scope and spirit of the invention, which is defined by the following claims only.