CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application 63/142,486, filed January 27, 2021, the disclosure of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The electronic copy of the Sequence Listing, created on January 5, 2022, is named 026225_WO014_SL.txt and is 23,049 bytes in size.

BACKGROUND OF THE INVENTION

[0003] In the past two decades, fundamental advances in immunology and tumor biology, combined with the identification of a large number of tumor antigens, have led to significant progress in the field of cell-based immunotherapy. T cell therapy, which aims to treat cancer by transferring autologous and ex vivo expanded T cells to patients, has resulted in some notable antitumor responses (Blattman et al., Science. (2004) 305(5681):200-5). For example, the administration of naturally occurring tumor infiltrating lymphocytes (TILs) expanded ex vivo mediated an objective response rate ranging from 50-70% in melanoma patients, including patients with bulky invasive tumors at multiple sites involving liver, lung, soft tissue and brain (Rosenberg et al., Nat Rev Cancer. (2008) 8(4):299-308; Dudley et al., J Clin Oncol. (2005) 23(10):2346-57).

[0004] A major limitation to the widespread application of TIL therapy is the difficulty in generating human T cells with antitumor potential. As an alternative approach, exogenous high- affinity TCRs can be introduced into normal autologous T cells of the patients through T cell engineering. The adoptive transfer of these cells into lympho-depleted patients has been shown to mediate cancer regression in cancers such as melanoma, colorectal carcinoma, and synovial sarcoma (Kunert et al., Front Immunol . (2013) 4:363; Robbins et al., Clin Cancer Res. (2015) 21(5): 1019-27). One of the advantages of TCR-engineered T cell therapy is that it can target the entire array of potential intracellular tumor-specific proteins, which are processed and delivered to the cell surface through MHC presentation and can be recognized even at a lower density by antigen-specific cytotoxic T cells (Kunert, supra).

[0005] Attempts have been made to engineer TCR molecules having antibody specificity with T cell receptor effector functions. In some of these approaches, the variable and constant domains of a TCR (e.g., an aP TCR or a y6 TCR) are replaced by the variable and constant domains of an antibody against a tumor antigen, creating a chimeric antibody-TCR called “abTCR” or “caTCR.” See, e.g., WO 2017/070608 and WO 2018/200582, the disclosures of which are incorporated by reference herein in their entirety. In one of the approaches, a chimeric stimulating receptor (CSR) is employed in combination with a caTCR to enhance the tumorkilling efficacy of the engineered T cells. Like a chimeric antigen receptor (CAR), a CSR has an extracellular domain that binds a target ligand, e.g., a tumor antigen, and an intracellular costimulatory domain, but unlike a CAR, the CSR does not have an intracellular primary immune cell signaling domain (which typically is a CD3 zeta chain’s intracellular domain) or not a functional primary immune cell signaling domain. The CSR and the caTCR bind to different targets/antigens or different epitopes of the same target/antigen and work synergistically to boost the cytotoxicity of the engineered T cells. See, e.g., WO 2018/200583, the disclosure of which is incorporated by reference herein in its entirety.

[0006] One challenge facing T cell therapy is the lack of persistence of T cells in vivo due to a phenomenon known as T cell exhaustion. See, e.g., Fraietta et al., Nat Med. (2018) 24(5):563- 71; Long et al., Nat Med. (2015) 21(6):581-90; and Eyquem et al., Nature (2017) 543(7643):113- 7. T cell exhaustion is characterized by marked changes in metabolic function, transcriptional programming, loss of effector functions (e.g., reduced cytokine secretion and cytotoxicity), expression of multiple surface inhibitory receptors, and apoptosis. T cell exhaustion has been attributed to constant antigen exposure, leading to continuous TCR signaling, or to tonic antigenindependent signaling through an engineered antigen receptor on T cells (see, e.g., Long, supra). Prevention or reversal of T cell exhaustion has been sought as a means to enhance T cell effectiveness, e.g., in patients with cancer or chronic infections and in T cell therapy. See, e.g., WO 2019/118902, the disclosure of which is incorporated by reference herein in its entirety. [0007] Thus, there remains a need for improved T cell therapy in which the engineered T cells have high as well as sustained tumor-killing potency.

SUMMARY OF THE INVENTION

[0008] The present disclosure provides compositions and methods for improving immune cell therapy. In one aspect, the disclosure provides one or more expression constructs comprising one or more expression cassettes for expressing: a) a chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising i) an antigen-binding module that specifically binds to a target antigen, and ii) a T cell receptor module (TCRM; e.g., a module derived from a human y6 TCR) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR- TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) a chimeric stimulating receptor (CSR) comprising i) a ligand-binding module that is capable of binding or interacting with a target ligand, ii) a transmembrane module, and iii) a costimulatory immune cell signaling module that is capable of providing a costimulatory signal to the immune cell, wherein the ligandbinding module and the costimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide.

[0009] In one aspect, the present disclosure provides a method of reducing exhaustion of an engineered immune cell (e.g., human immune cells; T cells; and human T cells), comprising introducing to the engineered immune cell an exogenous nucleic acid molecule that increases expression of c-Jun in the cell, wherein the engineered immune cell comprises one or more expression constructs comprising one or more expression cassettes for expressing: a) a caTCR comprising i) an antigen-binding module that specifically binds to a target antigen and ii) a TCRM (e.g., a TCRM derived from a human y6 TCR) comprising a first TCRD comprising a first TCR-TM and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; and b) a CSR comprising i) a ligand-binding module that is capable of binding or interacting with a target ligand, ii) a transmembrane module; and iii) a costimulatory immune cell signaling module that is capable of providing a costimulatory signal to the immune cell, wherein the ligand-binding module and the costimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain.

[0010] In some embodiments, the c-Jun is a wildtype human c-Jun, optionally comprising SEQ ID NO: 1. In other embodiments, the c-Jun is a mutant human c-Jun, optionally comprising an inactivating mutation in its transactivation domain or delta domain. In particular embodiments, the c-Jun comprises (i) S63 A and S73 A mutations or (ii) a deletion between residues 2 and 102 or between residues 30 and 50 as compared to wildtype c-Jun.

[0011] In some embodiments, the costimulatory immune cell signaling module in the CSR is derived from human CD30 and optionally comprises SEQ ID NO:21.

[0012] In some embodiments, the target antigen of the caTCR is a human AFP peptide complexed with a human MHC class I molecule. In particular embodiments, the target antigen is AFP158 complexed with HLA-A2*02:01.

[0013] In some embodiments, the antigen-binding module of the caTCR comprises (i) an immunoglobulin (Ig) heavy chain variable domain (VH) comprising the HCDR1-3 in SEQ ID NO:2 and (ii) an Ig light chain variable domain (VL) comprising the LCDR1-3 in SEQ ID NO:3. In further embodiments, the HCDR1-3 comprise SEQ ID NOs:5-7, respectively, and the LCDR1-3 comprise SEQ ID NOs:9-l 1, respectively. In particular embodiments, the VH and VL comprise SEQ ID NOs:8 and 12, respectively. In certain embodiments, the caTCR is a heterodimer comprising SEQ ID NOs:2 and 3, respectively.

[0014] In some embodiments, the target ligand of the CSR is human glypican 3 (GPC3). In further embodiments, the CSR comprises (i) an Ig VH comprising the HCDR1-3 in SEQ ID NO:4 and (ii) an Ig VL comprising the LCDR1-3 in SEQ ID NO:4. In particular embodiments, the HCDR1-3 in the CSR comprise SEQ ID NOs: 13-15, respectively, and the LCDR1-3 in the CSR comprise SEQ ID NOs: 17-19, respectively. In certain embodiments, the VH and VL in the CSR comprise SEQ ID NOs: 16 and 20, respectively. In certain embodiments, the CSR comprises SEQ ID NO:4.

[0015] In some embodiments, the expression constructs herein are viral vectors, e.g., lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors.

[0016] In some embodiments, the expression constructs comprise a quad-cistronic expression cassette for expressing a heterodimeric caTCR, the CSR, and the c-Jun. A quad-cistronic expression construct may comprise an expression cassette for expressing: a) a caTCR comprising i) an antigen-binding module that specifically binds to a human AFP peptide complex with a human MHC class I molecule, optionally AFP158 complexed with HLA-A2*02:01, and ii) a TCRM derived from human y6 TCR; b) a CSR comprising i) a ligand-binding module that is capable of binding or interacting with GPC3, ii) a transmembrane module, and iii) a costimulatory immune cell signaling module derived from an intracellular domain of human CD30; and c) a human c-Jun polypeptide. In some embodiments, the cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs:2 and 3, and a coding sequence for SEQ ID NO:4, optionally wherein the coding sequences are separated in frame by a 2A-coding sequence or by an internal ribosomal entry site (IRES).

[0017] The expression constructs (e.g., viral vectors such as lentiviral vectors) herein may comprise a constitutive or inducible promoter, optionally an EF-la promoter, optionally wherein the expression construct(s) are lentiviral vector(s).

[0018] In one aspect, the present disclosure provides a recombinant virus comprising the quad-cistronic expression construct herein, optionally wherein the expression construct is a lentiviral vector.

[0019] In another aspect, the present disclosure provides a method of engineering immune cells, comprising: (a) providing a starting cell population (e.g., a human cell population), (b) introducing the expression constructs or the recombinant virus herein into the starting cell population, (c) optionally selecting cells that express the caTCR, the CSR, and the c-Jun, and (d) deriving engineered immune cells from the cells of step (b) or (c). In some embodiments, the starting cell population comprises immune cells (e.g., autologous or allogeneic T cells). In other embodiments, the starting cell population comprises pluripotent or multipotent cells and step (d) comprises differentiating the cells of step (b) or (c) into immune cells, optionally T cells.

[0020] The present disclosure also provides human and/or immune cells (e.g., T cells) comprising the expression constructs or virus herein, as well as cells obtained by the present engineering methods. In some embodiments, the engineered T cells comprise CD8 ⁺ T cells. In some embodiments, the engineered cells express a lower level (e.g., at least 10, 20, 30, 40, 50, 60, or 70% lower) of an exhaustion marker (e.g., is CD39, PD-1, TIM-3, or LAG-3) compared to corresponding cells that do not overexpress c-Jun, and optionally wherein the exhaustion marker. [0021] The present disclosure also provides pharmaceutical compositions comprising the expression constructs, viruses, or engineered cells herein, and a pharmaceutically acceptable carrier.

[0022] In another aspect, the present disclosure provides a method of killing target cells, comprising contacting the target cells with the engineered immune cells herein under conditions that allow killing of the target cells by the immune cells, wherein the target cells express the target antigen and the target ligand, optionally wherein the immune cells express a lower level (e.g., at least 10, 20, 30, 40, 50, 60, or 70% lower) of an exhaustion marker (e.g., CD39, PD-1, TIM-3, or LAG-3) when in contact with the target cells, as compared to corresponding immune cells that do not comprise an exogenous nucleic acid molecule that causes c-Jun overexpression. In some embodiments, the immune cells are T cells and/or the target cells are cancer cells. In some embodiments, the costimulatory immune cell signaling module in the CSR is derived from human CD30 and the immune cells express a lower level (e.g., at least 10, 20, 30, 40, 50, 60, or 70% lower) of an exhaustion marker optionally selected from CD39, PD-1, TIM-3, and LAG-3, as compared to corresponding immune cells engineered to express a CSR whose costimulatory immune cell signaling module is derived from human CD28.

[0023] In one aspect, the present disclosure provides a method of treating a patient (e.g., a human patient) in need thereof, comprising administering the human cells or pharmaceutical composition herein to the patient. The patient may have, e.g., cancer or tumor (e.g., a solid tumor), such as hepatocellular carcinoma or gastric cancer.

[0024] Also provided in the present disclosure are the use of the expression constructs, viruses, or engineered cells herein for the manufacture of a medicament for treating a patient in need thereof. Further provided are expression constructs, viruses, cells, or pharmaceutical compositions for use in treating a patient in need thereof in a treatment method as described herein.

[0025] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESRIPTION OF THE DRAWINGS

[0026] FIG. 1A is a schematic representation of a caTCR and CSR combination.

[0027] FIG. IB is a schematic representation of (i) a lentiviral vector containing a tri- cistronic expression cassette for an anti-AFP chimeric antibody-TCR (caTCR) with a y6 TCR module and an anti-GPC3 chimeric stimulating receptor (CSR) containing CD30 transmembrane and intracellular domains (termed “Construct 1”); and (ii) a lentiviral vector containing a quad- cistronic expression cassette encoding the two caTCR chains and the CSR, as well as a human c- Jun (termed “Construct 2”). EFla(s): an EFla promoter.

[0028] FIG. 1C is a schematic representation showing the plasmid comprising [or containing] the lentiviral construct.

[0029] FIG. 2 shows the results of a long-term killing assay comparing primary T cells expressing AFP-caTCR, GPC3-CD30-CSR, and c-Jun (+cJun) with primary T cells expressing only AFP-caTCR and GPC3-CD30-CSR but no c-Jun (-eJun). In this assay, HepG2 cells were used as target cells at an effector-to-target ratio of 1 : 1. T cells were counted by FACS staining, and HepG2 cells were counted by using crystal violet dye staining. El to E5: the first to the fifth T cell engagement with HepG2 cells (addition of fresh HepG2 cells), sequentially. The four data points for each engagement represent the data obtained on day 0, and days 3, 5, and 7 after each engagement, respectively.

[0030] FIG. 3 shows the results of a short-term (overnight) killing assay comparing primary effector T cells expressing AFP-caTCR, GPC3-CD30-CSR, and c-Jun (+cJun) with primary effector T cells expressing only AFP-caTCR and GPC3-CD30-CSR but no c-Jun (-eJun). HepG2 cells were used as target cells, while SK-Hep-1 cells were used as the negative control. The effector-to-target ratio was 2: 1.

[0031] FIG. 4 shows the secretion of cytokines IL-2, IFN-y, TNF-a, and GM-CSF by the T cells in the short-term killing study shown in FIG. 3. “Unstimulated” control: only T cells in the wells (no target cells).

[0032] FIG. 5A is a graph showing that c-Jun overexpression enhanced the potency of the “AFP-caTCR + GPC3-CD30-CSR” T cells in vivo as measured by a decrease in tumor volume. Mock: animals injected with un-transduced human primary T cells. The T cells were isolated from healthy human Donor 1. [0033] FIG. 5B is a graph showing that c-Jun-expressing T cells did not cause weight loss in the studies of FIG. 5A as compared to T cells not expressing c-Jun. The T cells were isolated from Donor 1.

[0034] FIG. 6 is a graph showing the in vivo effects of c-Jun overexpression in mice injected with “AFP-caTCR + GPC3-CD30-CSR” T cells derived from an additional human donor. The T cells were isolated from healthy human Donor 2.

[0035] FIGs. 7A and 7B are graphs showing that the c-Jun effect was also observed in vivo when the T cells were engineered to express AFP-caTCR + GPC3-CD28-CSR construct. In FIG. 7A, the animals were injected with 5 million T cells, whereas in FIG. 7B, the animals were injected with 2.5 million T cells. The T cells were isolated from Donor 1.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present application provides engineered human cells (e.g., immune cells such as T cells) comprising constructs for expressing a chimeric antibody-T cell receptor (caTCR), a chimeric stimulating receptor (CSR; also termed “chimeric signaling receptor” herein), and a c- Jun protein. The caTCR comprises an antigen-binding module that specifically binds to a target antigen and a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling molecule. The CSR comprises a ligand-binding domain that specifically binds to a target ligand and a costimulatory immune cell signaling domain capable of providing a stimulatory signal to the immune cell, and does not comprise a functional primary T cell signaling sequence. The target antigen and target ligand are different or the same proteins or protein complexes expressed on the cell surface of a target cell (e.g., a diseased cell). In some embodiment, the target antigen and target ligand are the same protein or protein complex but the caTCR and the CSR bind to different regions on the same protein or protein complex. In some embodiments, the target antigen may be a protein complex comprising a peptide and a major histocompatibility complex (MHC) protein, such as a disease-associated antigen peptide presented by an MHC on the surface of a diseased cell. The disease may be, for example, gastric or liver cancer. The caTCR is regulated by the naturally occurring machinery that controls TCR activation, while the CSR potentiates the immune response mediated by the caTCR. c-Jun expression helps sustain the active state of the T cells by, e.g., alleviating or preventing T cell exhaustion. [0037] The present engineered immune cells such as T cells exhibit sustained, potent cytotoxicity against target-bearing tumor cells. As compared to T cells that do not overexpress c-Jun (e.g., through an exogenously introduced c-Jun gene sequence), the present engineered T cells display fewer signs of T cell exhaustion. The engineered cells may have one or more of the following characteristics: (i) they do not have increased expression of exhaustion markers PD-1, TIM-3, LAG-3, and/or CD39 over time, (ii) have reduced rates of apoptosis, (iii) they maintain an active biological state including secretion of cytokines including IL-2, TNF-a, INF-y, and GM-CSF, (iv) they have increased memory cell formation and/or maintenance of memory markers (e.g., CD62L); (v) they have enhanced cytotoxicity; (vi) they display increased recognition of tumor targets with low surface antigen; (vii) they have enhanced proliferation in response to antigen; (viii) maintain survival and functionality after repeated antigen stimulation; and (ix) they display increased tumor-infiltrating abilities.

[0038] In some embodiments, the CSR comprises a costimulatory immune cell signaling domain derived from the intracellular domain of CD30 (e.g., human CD30). The present inventors have unexpectedly discovered that c-Jun overexpression significantly reduces exhaustion in T cells engineered to express caTCR (e.g., caTCR against AFP) and a CD30-based CSR (e.g., one that targets GPC3). This significant reduction was not observed when the CSR had an intracellular costimulatory domain derived from another immune cell signaling domain, such as one derived from CD28.

I. Immune cell Sources

[0039] The source of the engineered immune cells of the present disclosure may be a patient to be treated (i.e., autologous cells) or from a donor who is not the patient to be treated (e.g., allogeneic cells). In some embodiments, the engineered immune cells are engineered T cells. The engineered T cells herein may be CD4 ⁺ CD8‘ (i.e., CD4 single positive) T cells, CD4'CD8 ⁺ (i.e., CD8 single positive) T cells, or CD4 ⁺ CD8 ⁺ (double positive) T cells. Functionally, the T cells may be cytotoxic T cells, helper T cells, natural killer T cells, suppressor T cells, or a mixture thereof. The T cells to be engineered may be autologous or allogeneic.

[0040] Primary immune cells, including primary T cells, can be obtained from a number of tissue sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and/or tumor tissue. Leukocytes, including PBMCs, may be isolated from other blood cells by well-known techniques, e.g., FICOLL™ separation and leukapheresis. Leukapheresis products typically contain lymphocytes (including T and B cells), monocytes, granulocytes, and other nucleated white blood cells. T cells are further isolated from other leukocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3 ⁺ , CD25 ⁺ , CD28 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ , GITR ⁺ , and CD45RO ⁺ T cells, can be further isolated by positive or negative selection techniques (e.g., using fluorescence-based or magnetic-based cell sorting). For example, T cells may be isolated by incubation with any of a variety of commercially available antibody-conjugated beads, such as Dynabeads®, CELLection™, DETACHaBEAD™ (Thermo Fisher) or MACS® cell separation products (Miltenyi Biotec), for a time period sufficient for positive selection of the desired T cells.

[0041] In some instances, autologous T cells are obtained from a cancer patient directly following cancer treatment. It has been observed that following certain cancer treatments, in particular those that impair the immune system, the quality of T cells collected shortly after treatment may have an improved ability to expand ex vivo and/or to engraft after being engineered ex vivo.

[0042] Whether prior to or after genetic modification, T cells can be activated and expanded generally using methods as described, for example, in U.S. Pats. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; 10,786,533; and U.S. Patent Publication 20060121005. Generally, T cells may be expanded in vitro or ex vivo by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD3 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatins) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule may be used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4 ⁺ T cells or CD8 ⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody may be employed.

[0043] The cell culture conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the culture conditions include addition of IL-2, IL-7 and/or IL-15. [0044] In some embodiments, the cells to be engineered may be pluripotent or multipotent cells that are differentiated into mature T cells after engineering. These non-T cells may be allogeneic and may be, for example, human embryonic stem cells, human induced pluripotent stem cells, or hematopoietic stem or progenitor cells. For ease of description, pluripotent and multipotent cells are collectively called “progenitor cells” herein.

[0045] Where allogeneic cells are used, they are preferably engineered to reduce graft-versus- host rejection (e.g., by knocking out the endogenous B2M and/or TRAC genes).

II. Engineering of Immune or Progenitor cells

[0046] As used herein, the term “cell engineering” or “cell modification” (including derivatives thereof) refers to the targeted modification of a cell, e.g., an immune cell disclosed herein. In some aspects, the cell engineering comprises viral genetic engineering, non-viral genetic engineering, introduction of receptors to allow for tumor specific targeting (e.g., an anti- AFP caTCR and anti-GPC3 CSR), introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve immune cell, e.g., T cell, function (e.g., a polynucleotide encoding a c-Jun polypeptide, such that the immune cell exhibits increased c-Jun expression compared to a corresponding cell that has not been modified), or any combination thereof. As further described elsewhere in the present disclosure, in some aspects, a cell can be engineered or modified with a transcription activator (e.g., CRISPR/Cas systembased transcription activator), wherein the transcription activator is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun).

[0047] In some aspects, a cell described herein has been modified with a transcriptional activator, which is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun) in the cell. As used herein, the term “transcriptional activator” refers to a protein that increases the transcription of a gene or set of genes (e.g., by binding to enhancers or promoter-proximal elements of a nucleic acid sequence and thereby, inducing its transcription). Non-limiting examples of such transcriptional activators that can be used with the present disclosure include: Transcription Activator-like Effector (TALE)-based transcriptional activator, zinc finger protein (ZFP)-based transcriptional activator, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system-based transcriptional activator, or a combination thereof. See, e.g., Kabadi et al., Methods (2014) 69(2): 188-197, which is incorporated herein by reference in its entirety.

[0048] In some aspects, a cell described herein has been modified with a CRISPR/Cas- system-based transcriptional activator, such as CRISPR activation (CRISPRa). See, e.g., Nissim et al., Molecular Cell (2014) 54(4):698-710; Perez-Pinera et al., Nat. Methods (2013) 10(10):973-976; Maeder et al., Nat. Methods (2013) 10(10):977-979; Cheng et al., Cell Res. (2013) 23(10): 1163-71; Farzadfard et al., ACS Synth. Biol. (2013) 2(10):604-13; all of which are incorporated herein by reference in their entirety. CRISPRa is a type of CRISPR tool that comprises the use of modified Cas proteins that lacks endonuclease activity but retains the ability to bind to its guide RNA and the target DNA nucleic acid sequence. Non-limiting examples of such modified Cas proteins which can be used with the present disclosure are known in the art. See, e.g., Pandelakis et al., Cell Systems (2020) 10(1): 1 - 14, which is incorporated herein by reference in its entirety. In some aspects, the modified Cas protein comprises a modified Cas9 protein (also referred to in the art as “dCas9”). In some aspects, the modified Cas protein comprises a modified Casl2a protein. In some aspects, a modified Cas protein that is useful for the present disclosure is bound to a guide polynucleotide (e.g., small guide RNA) (“modified Cas-guide complex”), wherein the guide polynucleotide comprises a recognition sequence that is complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In certain aspects, the guide polynucleotide comprises a recognition sequence that is complementary to the promoter region of an endogenous nucleic acid sequence encoding a protein of interest. In some aspects, one or more transcriptional activators are attached to the modified Cas-guide complex (e.g., the N- and/or C-terminus of the modified Cas protein), such that when the modified Cas-guide complex is introduced into a cell, the one or more transcription activators can bind to a regulatory element (e.g., a promoter region) of an endogenous gene and thereby induce and/or increase the expression of the encoded protein (e.g., c-Jun). Illustrative examples of common general activators that can be used include the omega subunit of RNAP, VP16, VP64 and p65 (see, e.g., Kabadi and Gersbach, Methods (2014) 69(2): 188-97).

[0049] In some aspects, one or more transcriptional repressors (e.g., Kruppel-associated box domain (KRAB)) can be attached to the modified Cas-guide complex (e.g., the N- and/or C- terminus of the modified Cas protein), such that when introduced into a cell, the one or more transcriptional repressors can repress or reduce the transcription of a gene, e.g., such as those that can interfere with the expression of c-Jun (e.g., Bach2). See, e.g., US20200030379A1 and Yang et al., J Transl Med. (2021) 19:459, each of which is incorporated herein by reference in its entirety. In some aspects, a modified Cas protein useful for the present disclosure can be attached to both one or more transcriptional activators and one or more transcriptional repressors. [0050] Not to be bound by any one theory, in some aspects, the use of such modified Cas proteins can allow for the conditional transcription and expression of a gene of interest. For example, in some aspects, a cell (e.g., T cells) is modified to comprise a recombinant antigen receptor (e.g., an anti-AFP caTCR and an anti-GPC3 CSR), which is linked to a protease (e.g., tobacco etch virus (TEV)) and a single guide RNA (sgRNA) targeting the promoter region of c- Jun. In some aspects, the cell is modified to further comprise a linker for activation of T cells (LAT), complexed to the modified Cas protein attached to a transcriptional activator (e.g., dCas9-VP64-p65-Rta transcriptional activator (VPR)) via a linker (e.g., TEV-cleavable linker). Upon activation of the antigen receptor, the modified Cas protein is released for nuclear localization and conditionally and reversibly induces the expression of c-Jun. See, e.g., Yang et al., J Immunother Cancer (2021) 9(Suppl2):A164, which is herein incorporated by reference in its entirety.

[0051] As will be apparent to those skilled in the art, in some aspects, a cell described herein has been modified using a combination of multiple approaches. For instance, in some aspects, a cell has been modified to comprise (i) an exogenous nucleotide sequence encoding one or more proteins (e.g., an anti-AFP caTCR, an anti-GPC3 CSR, and a truncated EGFR (EGFRt)) and (ii) an exogenous transcriptional activator (e.g., CRISPRa) that increases expression of an endogenous protein (e.g., c-Jun). In some aspects, a cell has been modified to comprise (i) an exogenous nucleotide sequence encoding a first protein (e.g., an anti-AFP caTCR), (ii) an exogenous nucleotide sequence encoding a second protein (e.g., an anti-GPC3 CSR), and (iii) an exogenous nucleotide sequence encoding a protein protein (e.g., a c-Jun protein). In some aspects, the modified cell can further comprise an exogenous nucleotide sequence encoding a third protein (e.g., EGFRt). As described herein, in some aspects, the exogenous nucleotide sequences encoding the first, second, and third proteins can be part of a single polycistronic vector.

[0052] Unless indicated otherwise, the one or more exogenous nucleotide sequences and/or transcriptional activators can be introduced into a cell using any suitable methods known in the art. Non-limiting examples of suitable methods for delivering one or more exogenous nucleotide sequences to a cell include: transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof.

[0053] In some aspects, a cell has been modified with a transcriptional activator (e.g., CRISPR/Cas-sy stem -based transcription activator, e.g., CRISPRa), such that the expression of the endogenous c-Jun protein is increased compared to a corresponding cell that has not been modified with the transcriptional activator.

[0054] While certain disclosures provided herein generally relate to modifying an immune cell to comprise an exogenous nucleotide sequence encoding a c-Jun protein (wild-type c-Jun or a variant thereof), it will be apparent to those skilled in the art that other suitable methods can be used to induce and/or increase c-Jun protein expression (either wild-type or a variant thereof) in a cell. For instance, as described herein, in some aspects, the endogenous c-Jun protein expression can be increased with a transcriptional activator (e.g., CRISPRa). Unless indicated otherwise, disclosures provided herein using exogenous nucleotide sequences equally apply to other approaches of inducing and/or increasing c-Jun protein expression in a cell provided herein (e.g., transcriptional activator, e.g., CRISPRa).

[0055] The immune cells (e.g., T cells) or progenitor cells herein may be engineered to express a caTCR and a CSR, and overexpress c-Jun (e.g., a human c-Jun). The caTCR may bind specifically to a ligand on a tumor cell (e.g., a tumor antigen), and the CSR may bind specifically to the same or a different ligand on the tumor cell. As used herein, a receptor (e.g., a caTCR or a CSR) is said to specifically bind to a ligand (e.g., an antigen) when the KD for the binding is < 100 nM, e.g., < 10 nM or < 1 nM. A KD binding affinity constant can be measured, e.g., by surface plasmon resonance (using, e.g., a Biacore™ or Octet™ system). A. caTCR

[0056] The caTCR is an engineered construct comprising an antigen-binding module that specifically binds to a target antigen and a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling molecule. The antigen-binding module may be derived from the variable domains of an immunoglobulin or antibody. The TCRM may comprise a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule. A TCR-associated signaling molecule refers to a molecule having a cytoplasmic immunoreceptor tyrosine-based activation motif (IT AM) that is part of the TCR-CD3 complex. TCR-associated signaling molecules include CD3ys, CD36s, and ( (also known as CD3(^ or CD3(£). The caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, and wherein the stabilization module may be an immunoglobulin constant domain, e.g., selected from the group consisting of a CHI-CL module, a CH2-CH2 module, a CH3-CH3 module, a CH4-CH4 module, and a TCR constant region module such as a pair of gamma and delta TCR constant regions. The Ig constant domains may be from any of the IgG heavy chain isotype (e.g., IgGi, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD) or light chain type (kappa or lambda light chain).

[0057] In some embodiments, the caTCR is an engineered construct comprising a TCRM derived from a y6 TCR, where the variable and constant domains of the gamma TCR chain are replaced with an Ig variable domain (VH) and an Ig CH domain (e.g., CHI), while the variable and constant domains of the delta TCR chain are replaced with an Ig variable domain (VL) and an Ig CL domain. In some embodiments, the caTCR comprises a TCRM derived from a y6 TCR, where the variable and constant domains of the delta TCR chain are replaced with an Ig VH and an Ig CH domain (e.g., CHI), while the variable and constant domains of the gamma TCR chain are replaced with an Ig VL and an Ig CL domain. The caTCR chains dimerize to form an antigenbinding domain formed by the VH and VL, supported by the respective constant domain. The TCR’s intracellular portions remain capable of recruiting one or more CD3 chains to form a functional TCR complex (e.g., CD36s, CD3ys, and CD3( ). The caTCRs described herein lack a functional primary immune cell signaling sequence, such as a functional signaling sequence comprising an ITAM (e.g., the intracellular domain of CD3Q. In some embodiments, the caTCRs lack any primary immune cell signaling sequence. A “functional” primary immune cell signaling sequence is a sequence that is capable of transducing an immune cell activation signal when operably coupled to an appropriate receptor, e.g., the intracellular domain of CD3(^. “Nonfunctional” primary immune cell signaling sequences, which may comprise fragments or variants of primary immune cell signaling sequences, are unable to transduce an immune cell activation signal.

[0058] The antigen-binding module of the caTCR may bind to a cell surface antigen, selected, for example, from a protein, a carbohydrate, and a lipid. In some embodiments, the cell surface antigen is a disease-associated antigen expressed in a diseased cell. In some embodiments, the disease is cancer and the disease-associated antigen is a cancer-associated antigen expressed in a cancer cell, e.g., an oncoprotein. In some embodiments, the disease is viral infection and the disease-associated antigen is a virus-associated antigen expressed in an infected cell.

[0059] In some embodiments, the caTCR specifically binds to a cell surface antigen selected from CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL5, including variants or mutants thereof.

[0060] In some embodiments, the antigen-binding module specifically binds a complex comprising a peptide and an MHC protein. Peptide/MHC complexes include, for example, a surface-presented complex comprising a peptide derived from a disease-associated antigen expressed in a diseased cell and an MHC protein. The peptide may be derived from, for example, WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, Histone H3.3, and PSA, including variants or mutants thereof. Specific binding to a complex comprising a peptide and an MHC protein is sometimes referred to as “MHC -restricted binding.” An MHC protein may be an MHC class I protein such as HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some embodiments, the MHC class I protein is HLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-A11, HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74, or HLA-A80. In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is any one of HLA-A*02:01-555, such as HLA-A* 02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:08, HLA-A*02:09, HLA-A*02: 10, HLA-A*02: l l, HLA-A*02: 12, HLA-A*02: 13, HLA-A*02: 14, HLA-A*02: 15, HLA-A*02: 16, HLA-A*02: 17, HLA-A*02: 18, HLA-A*02: 19, HLA-A*02:20, HLA-A*02:21, HLA-A*02:22, or HLA-A*02:24. In some embodiments, the MHC class I protein is HLA-A*02:01.

[0061] In some embodiments, the caTCR comprises an antigen-binding module that specifically binds to a complex comprising a peptide derived from a disease-associated antigen (such as a tumor-associated or virally-encoded antigen) and an MHC class II protein, wherein the MHC class II protein is HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, the MHC class II protein is HLA-DP. In some embodiments, the MHC class II protein is HLA-DQ. In some embodiments, the MHC class II protein is HLA-DR.

[0062] In particular embodiments, the caTCR binds specifically to a complex comprising an alpha-fetoprotein (AFP) peptide and a major histocompatibility (MHC) class I protein. Such a caTCR is called anti-AFP caTCR or anti-AFP/MHC caTCR. In certain embodiments, the caTCR binds specifically to a peptide fragment of human AFP, the AFP158 peptide (FMNKFIYEI; SEQ ID NO:22), complexed with (also called “presented by”) the HLA-A*02 serotype of MHC I. In certain embodiments, the HLA-A*02 is of the subtype HLA-A*02:01, A*02:02, A*02:03, A*02:05, A*02:06, A*02:07, or A*02: 11. For example, the anti-AFP caTCR has a structure shown in FIG. 1A, where the AFP -binding domain is formed by the heavy and light chain variable domains (VH and VL) of a human antibody that binds to the AFP158 peptide presented by HLA-A*02:01. See also WO 2016/161390. In the caTCR, the Ig VH and VL domains replace the variable domains of the delta and gamma chains, respectively, of human y6 TCR; and a human IgGi CHi constant domain and a human Ig kappa constant domain replace the constant domains of the delta and gamma chains, respectively, of human y6 TCR.

[0063] In certain embodiments, the delta chain of the caTCR is anti-AFP158/HLA-A*02:01- caTCR-1-0 delta and has the following amino acid sequence, wherein the Ig VH domain is underlined and in boldface, exemplary HCDRs are italicized, the IgGi CHi domain is double underlined, the TCR delta intracellular tail region is dotted underlined, and the TCR delta transmembrane domain is boxed:

EVQLVQSGAE VKKPGESLTI SCKASGYSFP NYWITWVRQM SGGGLEWMGR IDPGDSYTT NPSFQGHVTI SIDES TNTAY LHWNSLKASD TAMYYCARYY VSLVDI GQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSWTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC EVKTDSTDHV KPKETENTKg PSKSCHKPKA IVHTEKVNMM SLTjVLGLRML FAKTVAVNFL ILTAKLFFLI ( SEQ ID NO : 2 ) [0064] In these certain embodiments, the gamma chain of the caTCR is anti-AFP158/HLA- A*02:01 -caTCR- 1-0 gamma and has the following amino acid sequence, wherein the IgVL domain is underlined and in boldface, exemplary LCDRs are italicized, the Ig kappa CL domain is double underlined, the TCR gamma extracellular stem region is dotted underlined, the TCR gamma transmembrane domain is boxed, and the TCR gamma intracellular domain is single underline:

QSVLTQPASV SGSPGQSITI SCTGTSSDVG GY YVSWYQQ HPGKAPKLMI

YDVNNRPSEV SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTTGSRAV

FGGGTKLTVL GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDEYPGAVTV

AWKADGSPVK AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT

HEGSTVEKTV APTECSPIKT .P iTMDPKDN CSKDANDTLL LQLTNTSAJYY

MYLLLLLKSV

[0065] The precursors of the caTCR chains may contain a signal peptide, such as an immunoglobulin kappa light chain signal peptide derived from, e.g., murine or human sources. [0066] caTCR chains that are variants of the above polypeptide sequences may also be used, so long as they can bind the AFP158/HLA-A2 complex and function as a chimeric TCR, e.g., it can recruit at least one TCR-associated signaling molecule such as CD36, CD3y, CD3s, and CD3(^ chains, which may be endogenous to the cell or exogenously introduced into the cell.

[0067] In some embodiments, the caTCR of the present disclosure comprises the complementarity-determining regions (CDRs; e.g., all six CDRs) in SEQ ID NOs: 2 and 3. The delineation of CDRs may be made in accordance with one of the well-known systems. See, e.g., Kabat et al., J Biol Chem. (1977) 252:6609-16 (1977); Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J Mol Biol. (1987) 196:901-17; Al-Lazikani et al., J Mol Biol. (1997) 273:927-48; MacCallum et al, J Mol Biol. (1996) 262:732-45; Abhinandan and Martin, Mol Immunol. (2008) 45:3832-9 (2008); Lefranc et al., Dev Comp Immunol. (2003) 27:55-77 (“IMGT”); and Honegger and Pliickthun, J Mol Biol. (2001) (“AHo”). The amino acid residues that encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, 2008, supra, Ehrenmann et al., Nucleic Acids Res. (2010) 38:D301-D307; and Adolf-Bryfogle et al., Nucleic Acids Res. (2015) 43:D432-D438. In particular embodiments, the caTCR comprises HCDR1-3 comprising SEQ ID NOs:5-7, respectively, and LCDR1-3 comprising SEQ ID NOs:9-l 1, respectively.

Table 1

[0068] In some embodiments, the caTCR of the present disclosure comprises the VH and VL sequences (SEQ ID NOs:8 and 12) in SEQ ID NOs:2 and 3, respectively.

B. CSR

[0069] The CSR specifically binds to a target ligand (such as a cell surface antigen or a peptide/MHC complex) and is capable of stimulating an immune cell on the surface of which it is functionally expressed upon target ligand binding. The CSR comprises a ligand-binding module that provides the ligand-binding specificity, a transmembrane module, and a costimulatory immune cell signaling module that allows for stimulating the immune cell. The CSR lacks a functional primary immune cell signaling sequence.

[0070] In some embodiments, the target ligand is a disease-associated ligand such as a cancer-associated ligand or a pathogen-associated ligand (e.g., a virus-associated ligand). In some embodiments, the target ligand is an immunomodulatory molecule, e.g., an immunosuppressive receptor, in which case the CSR can comprise a fragment of an antagonist or agonist of the immunosuppressive receptor. In some embodiments, the target ligand is an immune checkpoint molecule, an inhibitory cytokine, or an apoptotic molecule.

[0071] In some embodiments, the ligand-binding module in the CSR is an antibody moiety. In some embodiments, the ligand-binding module is derived from the extracellular domain of a receptor.

[0072] The target ligand may be a cell surface antigen or a peptide/MHC complex. In some embodiments, the target ligand is the same as or different from the target antigen of a caTCR expressed in the same immune cell. For example, the target antigen of the caTCR is a cancer- associated antigen presented on a cancer cell, and the target ligand is a ubiquitous molecule expressed on the surface of the cancer cell, such as an integrin. In some embodiments, the target ligand is a disease-associated ligand, e.g., a cancer-associated ligand such as CD 19, CD20, CD22, CD47, IL4, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, the cancer-associated ligand is a peptide/MHC complex comprising a peptide derived from a protein including WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the target ligand is a virus-associated ligand. In some embodiments, the target ligand is an immune checkpoint molecule such as PD-L1, PD-L2, CD80, CD86, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL9. In some embodiments, the target ligand is an apoptotic molecule such as FasL, FasR, TNFR1, and TNFR2. In some embodiments, the ligand-binding module is (or is derived from) all or a portion of the extracellular domain of a receptor for the target ligand. In some embodiments, the receptor includes, for example, FasR, TNFR1, TNFR2, PD-1, CD28, CTLA-4, ICOS, BTLA, KIR, LAG- 3, 4-1BB, 0X40, CD27, and TIM-3.

[0073] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a transmembrane protein including, for example, CD28, CD3. epsilon., CD3.zeta., CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the CSR comprises a fragment of a transmembrane protein (fTMP).

[0074] In some embodiments, the costimulatory signaling domain comprises, consists essentially of, or consists of all or a portion of the intracellular domain of an immune cell costimulatory molecule including, for example, CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.

[0075] Examples of costimulatory immune cell signaling domains for use in the CSRs of the invention include the cytoplasmic sequences of co-receptors of the T cell receptor (TCR), which can act in concert with a caTCR to initiate signal transduction following caTCR engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Under some circumstances, signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or costimulatory signal is also required. Thus, in some embodiments, T cell activation is mediated by two distinct classes of intracellular signaling sequence: those that initiate antigen-dependent primary activation through the TCR (referred to herein as “primary T cell signaling sequences”) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (referred to herein as “costimulatory T cell signaling sequences”).

[0076] Primary immune cell signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing primary immune cell signaling sequences include those derived from CD3< CD3y, CD38, CD3s, FcRy, FcRp, CD5, CD22, CD79a, CD79b, and CD66d. A “functional” primary immune cell signaling sequence is a sequence that is capable of transducing an immune cell activation signal when operably coupled to an appropriate receptor. “Non-functional” primary immune cell signaling sequences, which may comprise fragments or variants of primary immune cell signaling sequences, are unable to transduce an immune cell activation signal. The CSRs described herein lack a functional primary immune cell signaling sequence, such as a functional signaling sequence comprising an ITAM, e.g., the intracellular domain of CD3(^. In some embodiments, the CSRs lack any primary immune cell signaling sequence.

[0077] In some embodiments, the CSR may contain a costimulatory domain that is derived from an intracellular domain from CD30 such as human CD30. In some embodiments, the CD30 costimulatory domain comprises SEQ ID NO:21 or a functional equivalent thereof (e.g., a sequence having at least 90, 95, 98, or 99% homology to SEQ ID NO:21). The CSR may have a transmembrane domain derived from the transmembrane domain of CD30, CD8, CD28, 41-BB, CD27, 0X40, or another cell surface protein.

[0078] In further embodiments, the CSR binds specifically to human glypican-3 (GPC3) and contains a human CD30 costimulatory domain. For example, the anti-GPC3 CSR having a human CD30 costimulatory domain is anti-GPC3 scFv CSR7 and has the following sequence, where the Ig VL and VH domains of a human anti-GPC3 scFv are italicized and in boldface, respectively, exemplary LCDRs and HCDRs are dotted underlined, peptide linkers are boxed, the CD30 sequence (representing amino acid residues of 215 to 484 of human CD30) is underlined, and the myc tag is double underlined:

QSVLTQPPSV SAAPGQRVTI SCSGTRSNIG^ SDYVSWYQHL PGTAPKLLVY GDNLRPSGIP DRFSASKSGT SATLGITGLQ TGDEADYYCG^ TWDYTLNGVV FGGGTKLTVL dSRGGGGSGGl iGGSGGGGSLEl BAIQVQLVESG GGLVQPGGSL RLSCAASGFT FSSYAMSWVR QAPGKGLEWV SVIYSGGSST YYADSVKGRF TISRDNSKNT LYLQMNSLRA EDTAVYYCAR TSYLNHGDYW GQGTLVTVSS

EQKLISEEDL AAATG|APPLG TQPDCNPTPE NGEAPASTSP TQSLLVDSQA SKTLPIPTSA PVALSSTGKP VLDAGPVLFW VILVLWWG SSAFLLCHRR ACRKRIRQKL HLCYPVQTSQ PKLELVDSRP RRSSTQLRSG ASVTEPVAEE RGLMSQPLME TCHSVGAAYL ESLPLQDASP AGGPSSPRDL PEPRVSTEHT NNKIEKIYIM KADTVIVGTV KAELPEGRGL AGPAEPELEE ELEADHTPHY PEQETEPPLG SCSDVMLSVE EEGKEDPLPT AASGK ( SEQ ID NO : 4 )

[0079] Variants of the above polypeptide sequence may also be used, so long as they can bind the GPC3 and provide costimulation as CD30 does.

[0080] In some embodiments, the CSR of the present disclosure comprises the CDRs, e.g., all six CDRs, in SEQ ID NO: 4. The delineation of CDRs may be made in accordance with one of the well-known systems as disclosed above. In some embodiments, the CSR comprises HCDR1- 3 comprising SEQ ID NOs: 13-15, respectively, and LCDR1-3 comprising SEQ ID NOs: 17-19, respectively.

[0081] In some embodiments, the CSR comprises VH and VL sequences (SEQ ID NOs: 16 and

20) in SEQ ID NON.

C. c-Jun

[0082] In some embodiments, the c-Jun is a human c-Jun, such as wildtype human c-Jun having the following sequence (available at GenBank under accession number AAA59197.1 or at UniProtKB (under accession number P05412.2):

MTAKMETTFY DDALNASFLP SESGPYGYSN PKILKQSMTL NLADPVGSLK PHLRAKNSDL LT|S]PDVGLLK LA(S]PELERLI IQSSNGHITT TPTPTQFLCP KNVTDEQEGF AEGFVRALAE LHSQNTLPSV TSAAQPVNGA GMVAPAVASV AGGSGSGGFS ASLHSEPPVY ANLSNFNPGA LSSGGGAPSY GAAGLAFPAQ PQQQQQPPHH LPQQMPVQHP RLQALKEEPQ TVPEMPGETP PLSPIDMESQ ERIKAERKRM RNRIAASKCR KRKLERIARL EEKVKTLKAQ NSELASTANM LREQVAQLKQ KVMNHVNSGC QLMLTQQLQT F ( SEQ ID 1 0 : 1 )

See also Hattori et al., PNAS (1988) 85:9148-52. Alternatively, the c-Jun is a mutant human c- Jun so long as the mutant c-Jun does not impact the mutant’s ability to rescue dysfunctional (exhausted) T cells. In some embodiments, a mutant c-Jun comprises at least 70% (e.g., at least 75, 80, 85, 90, 95, or 99%) sequence identity with the C-terminal amino acid residues (e.g., C- terminal 50, 75, 100, 150, 200, or 250 or more residues), the C-terminal portion (e.g., quarter, third, or half) or C-terminal domains (e.g., epsilon, bZIP, and amino acids C-terminal thereof) of a wildtype c-Jun. In some embodiments, the N-terminal amino acid residues (e.g., N-terminal 50, 75, 100, or 150 or more), the N-terminal portion (e.g., quarter, third, or half) or N-terminal domains (e.g., delta, transactivation domain, and amino acids N-terminal thereof) of a wildtype c-Jun are deleted, mutated, or otherwise inactivated.

[0083] In some embodiments, the c-Jun comprises an inactivating mutation (e.g., substitutions, deletions, or insertions) in its transactivation domain and/or its delta domain. In some embodiments, the c-Jun comprises one or both of S63 A and S73 A mutations (the positions are boxed above). In some embodiments, the c-Jun has a deletion between residues 2 and 102 or between residues 30 and 50 as compared to wildtype human c-Jun.

[0084] Due to introduction of an exogenously introduced c-Jun coding sequence, the engineered T cells overexpress, i.e., express a higher level (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% more, or at least 2-, 3-, 4-, 5-, or 10-fold more) of, c-Jun than T cells without such a sequence.

[0085] In certain embodiments, the caTCR and/or the CSR and/or the c-Jun polypeptides further comprise an affinity or purification tag available for various use purposes; for example, it may be used to enhance the purification efficiency of the target polypeptide. In one embodiment, the tag is a myc, HIS or HA tag.

[0086] In some embodiments, the immune cells herein are engineered to overexpress c-Jun through activation of the endogenous c-Jun gene in the cells, as described above.

D. Nucleic Acids

[0087] The caTCR, the CSR, and the c-Jun may be introduced to the T cells or progenitor cells through one or more nucleic acid molecules (e.g., DNA or RNA such as mRNA). In some embodiments, the nucleic acid molecules may be placed on one or more DNA or RNA vectors for introduction into the host cells.

[0088] The nucleic acid molecules (e.g., DNA or RNA vectors containing them) may be introduced into the cells by well-known techniques, including without limitation, electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, colloidal dispersion systems (e.g., as macromolecule complexes, nanocapsules, microspheres, and beads), and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, and liposomes). Alternatively, the nucleic acid molecules may be introduced into the cells by transduction of recombinant viruses whose genomes comprise the nucleic acid molecules. Examples of viral vectors include, without limitation, vectors derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, and vaccinia virus. [0089] In some embodiments, the coding sequences for the two polypeptide chains of the caTCR, the CSR, and the c-Jun may be placed on a single expression construct. The four coding sequences may be placed into one or more expression cassettes on the construct, each cassette being its own transcription unit (e.g., with its own promoter and polyadenylation site and other transcription control elements). In particular embodiments, the four coding sequences may be placed into a single expression cassette (e.g., a quad-cistronic expression cassette), with the four coding sequences being transcribed under a common promoter. In a polycistronic arrangement, the coding sequences are in-frame and separated from each other by the coding sequence of a self-cleaving peptide (e.g., a 2A self-cleaving peptide such as a T2A, P2A, E2A, or F2A peptide). Alternatively, the coding sequences may be separated from each other by a ribosomal internal entry site (IRES). Thus, the polycistronic (e.g., quad-cistronic) expression cassette is transcribed into a single RNA but ultimately the single RNA is processed and translated into separate polypeptides.

[0090] The expression cassettes (polycistronic or monoci stronic) may contain a promoter that is constitutively active in mammalian (e.g., human or human T) cells. Such promoters include, without limitation, an immediate early cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an elongation factor- la (EF-la) promoter, an MND promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Core or minimal promoters derived from the aforementioned promoters also are contemplated. Alternatively, the expression cassettes may comprise an inducible promoter system. Exemplary inducible promoter systems include, without limitation, hormone-regulated elements, synthetic ligand-regulated elements, ionizing radiation-regulated elements, tetracycline (Tet) systems (e.g., “Tet-Off” and “Tet-On” systems), and NF AT systems (see, e.g., Kallunki et al., Cells (2019) 8(8):796; Uchibori et al., Mol Ther Oncolytics. (2018) 12: 16-25).

[0091] In some embodiments, the expression cassettes also include Kozak sequences, polyadenylation sites, and other elements that facilitate transcription and/or translation of the coding sequences. For example, a woodchuck hepatitis virus post-transcriptional response element (WPRE) or variants thereof may be included at the 3 ’ untranslated region of the expression cassette. [0092] In the expression cassettes, the transcription/translation regulatory elements such as the promoters, any enhancers, and the like are operably linked to the coding sequences so as to allow efficient expression of the coding sequences and efficient translation of the RNA transcripts.

[0093] In certain embodiments, the present disclosure provides a single-vector construct (e.g., a lentiviral vector) comprising a quad-cistronic expression cassette, comprising a mammalian promoter, a c-Jun coding sequence, coding sequences for the two caTCR chains, a coding sequence for the CSR, and a polyadenylation signal sequence. The coding sequences are linked by a nucleotide linker that may be an IRES or a coding sequence for a self-cleaving peptide (e.g., P2A, T2A, E2A, F2A, or functional equivalents thereof). By way of example, FIG. IB illustrates such an expression cassette, where the promoter is an EF-la promoter. In further embodiments, the caTCR binds to an AFP peptide presented by an HLA-A2, the CSR binds to GPC3 and contains a CD30 costimulatory domain, and the c-Jun is a human c-Jun.

[0094] In particular embodiments, the expression cassette encodes a c-Jun comprising SEQ ID NO: 1, a caTCR comprising two polypeptide chains comprising SEQ ID NOs:2 and 3, respectively, and a CSR comprising SEQ ID NO:4. The construct may be a recombinant lentiviral vector and may further comprises a central polypurine tract (cPPT) upstream of the EFla promoter, and a WPRE sequence between the CSR coding sequence and an SV40 EEL polyadenylation signal (see, e.g., FIG. 1C), or other sequences for efficient transduction and expression in mammalian cells.

[0095] The coding sequences in the expression cassettes may be codon-optimized for optimal expression levels in a host cell of interest (e.g., human cells).

[0096] The nucleic acid molecules encoding the caTCR, the CSR, and the c-Jun may be integrated into the genome of the engineered cells, or remain episomal. The integration may be targeted integration occurring through gene editing (e.g., mediated by CRISPR, TALEN, zinc finger nucleases, and meganucleases).

[0097] The engineered cells can be enriched for by positive selection techniques. For example, the cells can be selected for their ability to bind to the target antigen (AFP or AFP158/HLA-A2) and/or GPC3 in, e.g., flow cytometry assays. To confirm c-Jun expression, RT-PCT may be performed on the engineered T cells. The positive selection may lead to enrichment of caTCR ⁺ CSR ⁺ c-Jun ⁺ cells in a cell population, where the triple positive T cells constitute more than 30, 35, 40, 45 ,50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the total cell population. The engineered cells may be cryopreserved until use.

III. Pharmaceutical Compositions and Uses

[0098] The present disclosure provides pharmaceutical compositions comprising the engineered T cells described herein. The pharmaceutical compositions may comprise a pharmaceutically acceptable carrier that is suitable to maintain the health of the cells before introduction into the patient.

[0099] In some embodiments, engineered cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline (e.g., phosphate buffered saline), physiological saline, water, Hanks' solution, Ringer’s solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof. It is preferred that the carrier is isotonic. In some embodiments, the carrier can be supplemented with ingredients such as human serum albumin (HSA) or other human serum components, 5% glucose or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol may also be included.

[0100] The pharmaceutical T cell compositions may be administered in a therapeutically effective amount to a cancer patient systemically (e.g., through intravenous or portal vein injection) or locally (e.g., through intratumoral injection). In some embodiments, the compositions such as those targeting AFP are used to treat a patient with hepatocellular carcinoma (HCC), stomach cancer, pancreatic cancer, or a cancer in the reproductive system (see, e.g., Wang and Wang, Canadian J Gastroent Hep. (2018) art. 9049252). As used herein, the term “treatment” or “treating” refers to an approach for obtaining beneficial or desired results in the treated subject. Such results include, but are not limited to: alleviating one or more symptoms resulting from the disease (e.g., HCC), diminishing the extent of the disease (e.g., reducing tumor volumes), stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence or relapse of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, improving the quality of life, restoring body weight, and/or extension of survival (e.g., overall survival or progression-free survival).

[0101] A therapeutically effective amount of the composition refers to the number of engineered T cells sufficient to achieve a desired clinical endpoint. In some embodiments, a therapeutically effective amount contains more than 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , or 10 ⁹ of the engineered cells.

[0100] The pharmaceutical composition in some embodiments comprises the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0101] In certain embodiments, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0102] The cells and compositions in some embodiments are administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present disclosure (e.g., a pharmaceutical composition containing a genetically modified cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0103] In one aspect, the present disclosure provides pharmaceutical compositions comprising the nucleic acid molecules for expressing the caTCR, the CSR, and c-Jun. The nucleic acid molecules may be as described above, such as the viral vectors (e.g., lentiviral vectors) described above. The pharmaceutical compositions are used ex vivo to engineer T or progenitor cells, which are then introduced to the patient. The pharmaceutical compositions comprise the nucleic acid molecules or the recombinant viruses whose genome comprise the expression cassettes for the caTCR, the CSR, and c-Jun and a pharmaceutically acceptable carrier such as a buffered solution that optionally comprises other agents such as preservatives, stabilizing agents, and the like.

[0104] The pharmaceutical compositions may be provided as articles of manufacture, such as kits, that include vials (e.g., single-dose vials) comprising the biological materials (the cells or the nucleic acid molecules or recombinant viruses) and optionally instructions for use.

[0105] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of immunology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0106] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLES

[0107] In the studies described in the Examples below, the cell lines HepG2 (ATCC HB-8065) and SK -Hep-1 (ATCC HTB-52) were obtained from the American Type Culture Collection. HepG2 is a hepatocellular carcinoma cell line that expresses HLA-A2, AFP, and GPC3. SK- HEP1 is a liver adenocarcinoma cell line that expresses HLA-A2 but does not express AFP or GPC3. Cell lines were cultured in RPMI 1640 or DMEM supplemented with 10% FBS and 2 mM glutamine at 37°C/5% CO2.

[0108] Two lentiviral vectors were constructed. The first one, named “AFP-caTCR + GPC3- CD30-CSR” or “Construct 1” herein, contained a tri-cistronic expression cassette encoding the AFP158/HLA-A*02:01-specific caTCR (SEQ ID NOs:2 and 3) and the GPC3-CD30-CSR (SEQ ID NO:4). The second vector, named “c-Jun + AFP-caTCR + GPC3-CD30-CSR” or “Construct 2” herein, contained a quad-ci str onic expression cassette encoding the caTCR, the CSR, and human c-Jun (SEQ ID NO: 1; placed upstream of the caTCR and CSR coding sequences). For both vectors, the expression cassette was under the transcriptional control of an EF- la promoter and the coding sequences for the different polypeptides were linked in frame by a coding sequence for a 2A peptide (F2A or P2A). The recombinant viruses were produced by transfection of 293T cells with vectors encoding the constructs using known lentiviral production protocols and packaging systems. [0109] For in vitro studies, primary human T cells were used for lentiviral transduction after one-day stimulation with CD3/CD28 beads (Dynabeads®, Invitrogen) in the presence of interleukin-2 (IL-2) at 100 U/ml. Concentrated lentiviruses were applied to the T cells in 6-well plates coated with Retronectin® (Takara) and incubated for 96 hours. Transduction efficiencies were assessed by flow cytometry, using biotinylated AFP 158/HLA-A* 02:01 tetramer (“AFP158 tetramer”) with PE-conjugated streptavidin. For anti-GPC3 CSR, an anti-myc antibody was used. Repeat flow cytometry analyses were done on day 5 and every 3 or 4 days thereafter.

[0110] Tumor cytotoxicity was assayed by Cytox 96® Non-Radioactive Cytotoxicity Assay (Promega), which quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. CD3 ⁺ T cells were prepared from PBMC-enriched whole blood using EasySep™ Human T Cell Isolation Kit (StemCell Technologies), which negatively depletes cells expressing CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, and glycophorin A. Human T cells were activated and expanded with CD3/CD28 Dynabeads® (Invitrogen) according to manufacturer’s protocol. Activated T cells (ATCs) were cultured and maintained in RPMI1640 medium with 10% FBS plus 100 U/ml IL-2, and used at day 7-14. The activated T cells and target cells were co-cultured at various effector-to-target ratios (e.g., 2.5: 1 or 5: 1) for 16 hours and then assayed for cytotoxicity.

[OHl] Monoclonal antibody against human HLA-A02 (clone BB7.2) conjugated to FITC or APC and its isotype control - mouse IgG2b conjugated to FITC or APC; and antibodies against human or mouse CD3, various subunits of human TCR, 3xFLAG tag, HA tag, goat F(ab)2 antihuman IgG conjugated with PE or FITC, and fluorescence-conjugated goat F(ab’)2 anti-mouse immunoglobulins were purchased (Invitrogen). The anti-idiotypic antibody against an AFP158/HLA-A*02:01-specific antibody was developed and produced in house. Flow cytometry data were collected using BD FACSCanto II™ and analyzed using the FlowJo software package.

[0112] For in vivo studies, the anti-tumor activity of primary human T cells expressing Construct 1 or Construct 2 were tested in an established HepG2 liver cancer xenograft model. HepG2 cells were implanted subcutaneously (s.c.) in the right flank of NSG mice. When tumors reached about 200 mm ³ in size, the mice were intratumorally (i.t.) injected with either (1) 5 x 10 ⁶ un-transduced donor-matched (Mock) primary human T cells, (2) 5* 10 ⁶ primary human T cells expressing Construct 1, or (3) 5 * 10 ⁶ primary human T cells expressing Construct 2 (n=6 mice/group). Health effects resulting from the T cell infusions in mice were assessed by monitoring the animals’ general appearance, body weight, and other clinical signs of adverse response (including hypothermia, labored respiration, and hind-limb paralysis/weakness).

Example 1: Enhanced Long-Term Potency and Survival of Anti-AFP/MHC+GPC3 CSR T Cells Overexpressing c-Jun

[0113] This Example describes a study that evaluated the effects of c-Jun overexpression on the cytotoxicity and survival of human effector T cells that were engineered to express AFP- caTCR and GPC3-CD30-CSR.

[0114] The primary T cells isolated from healthy human donors were transduced with lentiviral Construct 1 or Construct 2 for 7-9 days. The effector cells were normalized to 30% caTCR ⁺ based on AFP158 tetramer staining (adjusting all effector cell samples to the same percentage of receptor ⁺ (caTCR ⁺ or CAR ⁺ ) cells among the total number of T cells, using un- transduced/mock-transduced T cells). A FACS-based assay for counting effector cells was used to compare the long-term killing potential of the transduced cells.

[0115] The target cells used in the cytotoxicity assay were HepG2 cells (HLA- A2 ⁺ AFP ⁺ GPC3 ⁺ ). The effector-to-target ratio (E:T ratio) was 1 : 1. Specifically, 50,000 caTCR ⁺ T cells and 50,000 HepG2 cells were incubated together in each well in RPMI+10% FBS with no cytokine. The cells were re-challenged with 100,000 HepG2 cells per well every 7 days. The cells were split 1:3 before re-challenging on day 14 and day 21, and split 1 :6 on day 28 to reduce E:T ratio and stress the effector cells. The numbers of remaining target cells and caTCR ⁺ T cells were quantified on selected days after each target cell engagement. The results of the long-term killing (represented by remaining target cells’ percentage relative to target cells incubated with mock-transduced T cells) and T cell survival are shown in FIG. 2. The data in the left panel of the figure show that both T cells expressing Construct 1 and T cells expressing Construct 2 effectively mediated the killing of almost all of the initially engaged target cells as well as the rechallenged target cells. But starting in the fifth round of engagement (E5), T cells expressing Construct 2 killed many more target cells than T cell expressing Construct 1. The data in the right panel of the figure shows that, in spite of multiple rounds of target cell addition and nonreplenishment of T cells, T cells expressing Construct 2 survived and expanded better than T cells expressing Construct 1 after the third and fourth rounds of engagement of target cells. Even after the fifth round of target cell engagement/addition, T cells expressing Construct 2 survived better than T cells expressing Construct 1. Together, these results show that c-Jun promotes the prolonged survival of T cells co-expressing the AFP-caTCR and GPC3-CD30-CSR and maintained their long-term target-cell-killing capabilities.

Example 2: Enhanced Short-Term Potency of Anti-AFP/MHC+GPC3 CSR T Cells Overexpressing c-Jun

[0116] This Example describes a study that the short-term killing efficiency of T cells expressing Construct 2 as compared to T cells expressing Construct 1. CD3 ⁺ T cells were prepared from PBMC-enriched whole blood using EasySep® Human T Cell Isolation Kit (StemCell Technologies) and activated with CD3/CD28 Dynabeads®. The activated and expanded cell population was >99% CD3 ⁺ as determined by flow cytometry. The primary T cells were mock-transduced (no DNA added) or transduced with lentiviral Construct 1 or Construct 2 for 7-9 days. The transduction efficiency was determined by staining with PE- labeled AFP 158/HLA-A* 02:01 tetramers (“AFP158 tetramers”). The T cells were normalized to 30% caTCR ⁺ and tested for their abilities to kill cancer cells in an LDH-based assay as described above. Activated T cells and target cells HepG2 were co-cultured at an effector-to-target ratio of 2: 1. Antigen-negative target cells SK-Hep-1 were used as a negative control, to confirm that there was no antigen-independent killing. As shown in FIG. 3, T cells transduced with Construct 2 (+cJun) had higher in vitro tumor cell killing efficacies than corresponding T cells transduced by Construct 1 (-eJun).

Example 3: Characterization of Anti-AFP/MHC+GPC3 CSR T Cells Overexpressing c- Jun After Co-Culture with Target Cells

[0117] This Example describes the characterization of T cells expressing Construct 2 after coculture with target cells. The concentration of cytokines released into the supernatant of the in vitro killing experiments was measured with a Bio-Plex® 200 Systems (Bio-Rad) using the Bio- Plex Pro™ Human Cytokine 8-Plex Assay kit (Bio-Rad). The data show that T cells expressing Construct 2 (+cJun) released more cytokines, including IL-2, IFN-y, TNF-a, GM-CSF, than T- cells expressing Construct 1 (-eJun), when co-cultured with HepG2 target cells (FIG. 4). [0118] To examine the levels of exhaustion markers expressed on T cells upon antigen stimulation, CD3 ⁺ T cells were prepared and activated with CD3/CD28 Dynabeads® as describe above. The activated and expanded cell population was >99% CD3 ⁺ by flow cytometry. The T cells were transduced with lentiviral Construct 1 or Construct 2 for 7-9 days. The effector cells were normalized to 30% caTCR ⁺ based on AFP158 tetramer staining. The T cells were incubated and re-challenged with HepG2 cells as described in Example 1. The expression levels of exhaustion markers CD39, PD-1, LAG-3, and TIM-3 on the caTCR ⁺ CD8 ⁺ T cells were analyzed by flow cytometry on selected days after each target cell engagement (BioLegend®). CD39, PD-1, LAG-3, and TIM-3 are inhibitory receptors that accumulate on T cells as T cells lose function; thus, these molecules are used as markers for T cell exhaustion. The fold of difference in the expression level (mean fluorescence intensity) of an exhaustion marker on T cells expressing c-Jun versus T cells not expressing c-Jun is shown in Table 2 below (E: engagement with target cells; D: days post engagement; n=2 (Donor 1 and Donor 2)).

Table 2 Exhaustion Marker Expression Levels in T cells with CD28-CSR or CD30-CSR

[0119] As shown above, before engagement, the two groups of T cells displayed similar levels of the exhaustion markers (with a ratio of close to 1). Seven days after the fifth engagements with target cells, T cells expressing Construct 2 displayed exhaustion markers CD39, PD-1, and TIM-3 at levels that were half or less than half of those on T cells expressing Construct 1. These results demonstrate that overexpression of c-Jun results in T cells that are significantly more functional and less exhausted in the long term.

[0120] However, this effect was not observed with T cells engineered to express the same caTCR and anti-GPC3 CSR, except that the CSR contained a CD28, rather than CD30, costimulatory domain. As shown in Table 2 above, at E5D7, T cells engineered with the CD28- CSR showed a markedly increased exhaustion markers CD39, LAG-3, and TIM-3 in T cells expressing c-Jun as compared to T cells not expressing c-Jun. The data show that in T cells expressing an anti-AFP/MHC caTCR and an anti-GPC3-CD28-CSR, c-Jun overexpression did not alleviate T cell exhaustion. The results thus suggest that c-Jun expression made a particularly positive impact in alleviating T cell exhaustion in T cells engineered to expressed a caTCR and a CD30-based CSR.

[0121] In addition, subpopulations of CD4 or CD8 single positive and CD4/CD8 double positive caTCR ⁺ T cells were analyzed by flow cytometry on selected days after each target cell engagement. The data in Table 3 show that c-Jun overexpressing resulted in higher percentages of CD4'CD8 ⁺ T cells (E: engagement with target cells; D: days post engagement).

Table 3 Percentages of CD4 ⁺ and/or CD8 ⁺ Cells Among receptor ⁺ T cells

Following HepG2 Target Cell Engagement [0122] As shown in the above table, the CD8 single positive population expanded from 60.3% of total T cell population to 79.9% of total T cell population at E5D7. These results demonstrate that c-Jun expression promoted preferential expansion of CD8 single positive T cells.

Example 4: In vivo Efficacy Study of Anti-AFP/MHC+GPC3 CSR T Cells Overexpressing c-Jun

[0123] Primary human T cells from Donor 1 expressing Construct 1 or 2 were assessed for their in vivo cytotoxic potency as described above. The data show that c-Jun overexpression significantly enhanced the tumor-killing potential of the “AFP-caTCR + GPC3-CD30-CSR” T cells. Around day 10 post injection of T cells expressing Construct 2 (+cJun), tumors started to regress; complete tumor regression was achieved around 60-70 days (FIG. 5A). By contrast, in animals injected with T cells expressing Construct 2 (-eJun), the tumor relapsed (after an initial drop) around day 40 and continued to grow, reaching a peak around day 60 (FIG. 5A). The data also show that animals injected with T cells expressing Construct 1 and T cells expressing Construct 2 did not have significant difference in body weight change, indicating that T cells overexpressing c-Jun did not cause more adverse effects in the animal (FIG. 5B).

[0124] Similarly, T cells obtained from another donor (Donor 2) prevented tumor relapse and maintained tumor suppression throughout the study period when they were transduced with Construct 2 and dosed at day 17 (FIG. 6). But T cells transduced with Construct 1 did not suppress tumor growth over the long run; it only delayed tumor growth (FIG. 6).

[0125] Interestingly, an enhancement effect of c-Jun was also observed, though less markedly, on T cells expressing AFP-caTCR with a GPC3-CD28-CSR (FIGs. 7A and 7B). c-Jun provided increased benefit with the GPC3-CD30 CSR as compared to the GPC3-CD28 CSR. As such, it appears that c-Jun overexpression acts synergistically with CD30 trans-signaling in the AFP- caTCR setting.

[0126] Exemplary sequences of the present disclosure are provided in Table 4 below (SEQ: SEQ ID NO).

Table 4