Methods for enhancing immune effector cell activity leveraging biophysical modulation in adoptive cellular therapies

Abstract

The present disclosure relates to methods for manufacturing immune effector cells (e.g., T cells, NK cells, iPSCs) with improved efficacy, comprising mechanically priming the immune effector cells (e.g., T cells, NK cells, iPSCs) in the presence of a viscoelastic hydrogel substrate matrix as described herein. Further, the present disclosure relates to such biomaterials-based hydrogel platforms for manufacturing immune effector cells (e.g., T cells, NK cells, iPSCs). The present disclosure also relates to immune effector cells (e.g., T cells, NK cells, iPSCs) whose function is enhanced by decreasing the activity of certain endogenous transcription factors and / or increasing the activity of others. The present disclosure also relates to methods of treatment and uses thereof.

Description

Attorney Docket No: 243734.000233METHODS FOR ENHANCING IMMUNE EFFECTOR CELL ACTIVITY LEVERAGING BIOPHYSICAL MODULATION IN ADOPTIVE CELLULAR THERAPIESCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U. S. Provisional Patent Application No. 63 / 752,518, filed January 31, 2025, the disclosure of which is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01NS122859 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] The application relates to methods for manufacturing immune effector cells (e.g., T cells, NK cells, iPSCs) with improved efficacy, comprising mechanically priming the immune effector cells (e g., T cells, NK cells, iPSCs) in the presence of a viscoelastic hydrogel substrate matrix as described herein. Further, the present disclosure relates to such biomaterials-based hydrogel platforms for manufacturing immune effector cells (e.g., T cells, NK cells, iPSCs). The application also relates to immune effector cells (e.g., T cells, NK cells, iPSCs) whose function is enhanced by decreasing the activity of certain endogenous transcription factors and / or increasing the activity of others. The application also relates to methods of treatment and uses thereof.BACKGROUND

[0004] Diffuse midline glioma (DMG) (including diffuse intrinsic pontine gliomas (DIPGs)) is a universally fatal pediatric brain tumor, with a five-year survival rate of less than 1% and a median survival of only 11 months (Monje, et al., 2025). Therapeutic options for DMG are severely limited to radiation therapy, which merely extends the overall survival by an average of 3 months (Johung, et al., 2017). Children with DMG desperately need new treatment interventions.

[0005] Recently, chimeric antigen receptor (CAR) T cell therapies have demonstrated promising clinical results (e.g., radiographical reduction in tumor mass after CAR T cell infusion 1324953654vlAttorney Docket No: 243734.000233and clinical improvement) in a phase 1 trial for patients with DMGs (Monje, et al., 2025;Majzner, et al., 2022). While these results are highly encouraging, patients inevitably succumb to this devastating disease - highlighting the need for continuous efforts to improve CAR T cell strategies against DMG (e.g., CAR T cell efficacy). Within solid tumors like DMGs, CAR T cells also experience significant mechanical resistances (Figure 1), commonly in the form of stiffness and viscoelasticity (Mittelheisser, et al., 2024). Stiffness accounts for a tissue’s resistance to instantaneous deformation, while viscoelasticity refers to a time-dependent response to deformation and is characterized by stress relaxation. Both stiffness and viscoelasticity have been shown to independently modulate cellular behavior (Mittelheisser, et al., 2024). However, there is an unmet need in the art to establish an improved immunotherapy for treating DMGs utilizing immune effector cells (e.g., CAR T cells).SUMMARY OF THE INVENTION

[0006] As specified in the Background section above, there is a need in the art for determining an effective treatment of a disease (e.g., tumors, a cancer, an infectious disease, an inflammatory disorder, or an autoimmune disease) including DMGs and Medulloblastomas, with immune effector cell therapy. The present application addresses these and other needs.

[0007] In one aspect, provided herein is an immune effector cell, wherein the transcriptional activity of transcription factor(s) ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof is inhibited in the immune effector cell as compared to a control cell in which the transcriptional activity of transcription factor(s) is not inhibited.

[0008] In some embodiments, the transcription factor(s) are DNMT1, FLI1, LEF1, or a combination thereof.

[0009] In some embodiments, a transcription factor gene is deleted or defective so that no detectable functional transcription factor is expressed.

[0010] In some embodiments, the enzymatic activity of the transcription factor(s) is inhibited in the immune effector cell.

[0011] In some embodiments, the enzymatic activity of the transcription factor(s) is inhibited by exposing the cell to a transcription factor inhibitor.2324953654vlAttorney Docket No: 243734.000233

[0012] In some embodiments, the transcription factor gene is mutated so that the enzymatic activity of the transcription factor is inhibited.

[0013] In some embodiments, the catalytic domain of the transcription factor is mutated.

[0014] In some embodiments, the level of enzymatic activity of the transcription factor in the immune effector cell is decreased by 50% or more.

[0015] In another aspect, provided herein is an immune effector cell, wherein the transcriptional activity of transcription factor(s) ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof is activated in the immune effector cell as compared to a control cell in which the transcriptional activity of transcription factor(s) is not activated.

[0016] In some embodiments, the enzymatic activity of the transcription factor(s) is activated in the immune effector cell.

[0017] In some embodiments, the enzymatic activity of the transcription factor(s) is activated by exposing the cell to a transcription factor activator.

[0018] In some embodiments, the transcription factor gene is mutated so that the enzymatic activity of the transcription factor is increased.

[0019] In some embodiments, the level of enzymatic activity of the transcription factor in the immune effector cell is increased by 50% or more.

[0020] In some embodiments, the immune effector cell is a T cell.

[0021] In some embodiments, the T cell is selected from a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell, an a T cell receptor (TCR) T cell, a natural killer T (NKT) cell, a y8 T cell, a memory T cell, a T-helper cell, and a regulatory T cell (Treg).

[0022] In some embodiments, the immune effector cell is a stem cell that is capable of differentiating into an immune cell.

[0023] In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC).

[0024] In some embodiments, the immune effector cell is a natural killer (NK) cell.

[0025] In some embodiments, the immune effector cell further comprises at least one surface molecule capable of binding specifically to an antigen.

[0026] In some embodiments, the antigen is selected from a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, a prion antigen, and an antigen associated with an inflammation or an autoimmune disease.3324953654vlAttorney Docket No: 243734.000233

[0027] In some embodiments, the tumor antigen is B7 Homolog 3 (B7-H3), disialoganglioside variant 2 (GD2), IL13Ra2, erythropoietin-producing human hepatocellular receptor A2 (EphA2), or human epidermal growth factor receptor 2 (HER2).

[0028] In some embodiments, the cell further comprises a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof.

[0029] In some embodiments, the cell further comprises a chimeric antigen receptor (CAR).

[0030] In some embodiments, the CAR comprises (i) an extracellular antigen-binding domain and (ii) a transmembrane domain.

[0031] In some embodiments, the CAR further comprises a cytoplasmic domain.

[0032] In some embodiments, the extracellular antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

[0033] In some embodiments, the extracellular antigen-binding domain comprises an scFv capable of binding to B7 Homolog 3 (B7-H3).

[0034] In some embodiments, the scFv capable of binding to B7-H3 comprises the amino acid sequence of SEQ ID NO: 2.

[0035] In some embodiments, the extracellular antigen-binding domain further comprises a leader sequence.

[0036] In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 3.

[0037] In some embodiments, the transmembrane domain is derived from CD3ζ, CD28, CD4, or CD8α.

[0038] In some embodiments, the transmembrane domain is derived from CD3ζ and comprises the amino acid sequence SEQ ID NO: 4.

[0039] In some embodiments, the transmembrane domain is derived from CD28 and comprises the amino acid sequence SEQ ID NO: 5.

[0040] In some embodiments, the transmembrane domain is derived from CD8a and comprises the amino acid sequence SEQ ID NO: 6 or SEQ ID NO: 17.

[0041] In some embodiments, the transmembrane domain is derived from CD4 and comprises the amino acid sequence SEQ ID NO: 7.

[0042] In some embodiments, the CAR further comprises a linker domain between the extracellular antigen-binding domain and the transmembrane domain.4324953654vlAttorney Docket No: 243734.000233

[0043] In some embodiments, the linker domain comprises a hinge region.

[0044] In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 8. In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 9. In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 16.

[0045] In some embodiments, the linker domain comprises the amino acid sequence SEQ ID NO: 10.

[0046] In some embodiments, the CAR cytoplasmic domain comprises one or more lymphocyte activation domains.

[0047] In some embodiments, the lymphocyte activation domain is derived from DAP10, DAP12, Fc epsilon receptor I γ chain (FCER1G), CD3δ, CD3ε, CD3γ, CD3ζ, CD27, CD28, CD40, CD134, CD137, CD226, CD79A, ICOS, or MyD88.

[0048] In some embodiments, the lymphocyte activation domain is derived from CD3ζ and comprises the amino acid sequence SEQ ID NO: 11.

[0049] In some embodiments, the CAR cytoplasmic domain comprises one or more costimulatory domains.

[0050] In some embodiments, the co-stimulatory domain is derived from CD28 and comprises the amino acid sequence SEQ ID NO: 12.

[0051] In some embodiments, the immune effector cell is an allogeneic cell.

[0052] In some embodiments, the immune effector cell is an autologous cell.

[0053] In some embodiments, the immune effector cell is isolated from a subject having a disease.

[0054] In some embodiments, the disease is a cancer, an infectious disease, an inflammatory disorder, or an autoimmune disease.

[0055] In some embodiments, the cancer is a cancer expressing B7-H3, GD2, IL13Rα2, or EphA2, or HER2.

[0056] In some embodiments, the immune effector cell is derived from a blood, marrow, tissue, or a tumor sample.

[0057] In another aspect, provided herein is a pharmaceutical composition comprising the immune effector cell as described herein and a pharmaceutically acceptable carrier and / or excipient.5324953654vlAttorney Docket No: 243734.000233

[0058] In a further aspect, provided herein is a method for generating the immune effector cell as described herein, said method comprising (a) deleting or modifying the gene or gene product of ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell so that the transcription factor activity is decreased and / or (b) overexpressing or modifying the gene or gene product of ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell so that the transcription factor activity is increased.

[0059] In some embodiments, the transcription factor gene in the immune effector cell is deleted, overexpressed, or modified as a result of an activity of a site-specific nuclease.

[0060] In some embodiments, the site-specific nuclease is an RNA-guided endonuclease.

[0061] In some embodiments, the RNA-guided endonuclease is a Cas9 protein, Cpfl (Casl2a) protein, C2cl protein, C2c3 protein, or C2c2 protein.

[0062] In some embodiments, the RNA-guided endonuclease is a Cas9 protein.

[0063] In some embodiments, the Cas9 protein is programmed with a guide RNA (gRNA) that comprises a nucleotide sequence encoded by SEQ ID NOs: 18-24.

[0064] In some embodiments, the site-specific nuclease is a zinc finger nuclease, a TALEN nuclease, or mega-TALEN nuclease.

[0065] In some embodiments, the transcription gene product in the immune effector cell is deleted or modified as a result of an activity of an RNA interference (RNAi) molecule or an antisense oligonucleotide.

[0066] In some embodiments, the RNAi molecule is a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

[0067] In some embodiments, the site-specific nuclease or the RNAi molecule or the antisense oligonucleotide is introduced into the immune effector cell via a viral vector, a non-viral vector or a physical means.

[0068] In some embodiments, the method further comprises genetically modifying the immune effector cell to express a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof that is capable of binding specifically to an antigen.6324953654vlAttorney Docket No: 243734.000233

[0069] In some embodiments, the CAR, antigen specific T-cell receptor, or antibody or antigen-binding fragment thereof is expressed from a transgene introduced into the immune effector cell.

[0070] In some embodiments, the CAR-, antigen specific T-cell receptor-, or antibody- or antigen-binding fragment-expressing transgene is introduced into the immune effector cell using a viral vector, a non-viral vector or a physical means.

[0071] In some embodiments, the viral vector is a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, or a baculoviral vector.

[0072] In some embodiments, the retroviral vector is a lentiviral vector.

[0073] In some embodiments, the non-viral vector is a transposon.

[0074] In some embodiments, the transposon is a sleeping beauty transposon or PiggyBac transposon.

[0075] In some embodiments, the physical means is electroporation, microinjection, magnetofection, ultrasound, a ballistic or hydrodynamic method, or a combination thereof.

[0076] In some embodiments, the immune effector cell is activated and / or expanded ex vivo.

[0077] In another aspect, provided herein is a method of treating a disease in a subject in need thereof comprising administering to the subject an effective amount of the immune effector cells as described herein or the pharmaceutical composition as described herein.

[0078] In some embodiments, the immune effector cell is an autologous cell.

[0079] In some embodiments, the immune effector cell is an allogeneic cell.

[0080] In some embodiments, the disease is a cancer, an infectious disease, an inflammatory disorder, or an autoimmune disease.

[0081] In some embodiments, the cancer is a solid tumor.

[0082] In some embodiments, the cancer is breast, prostate, urinary bladder, skin, lung, ovary, sarcoma, or brain cancer.

[0083] In some embodiments, the cancer is a central nervous system (CNS) tumor.

[0084] In some embodiments, the CNS tumor is a pediatric CNS malignancy.

[0085] In some embodiments, the pediatric CNS malignancy is Diffuse Midline Glioma (DMG).

[0086] In some embodiments, the CNS tumor is medulloblastoma.

[0087] In some embodiments, the CNS tumor is an ependymoma.7324953654vlAttorney Docket No: 243734.000233

[0088] In some embodiments, the cancer is a cancer expressing B7-H3, GD2, IL13Ra2, EphA2, or HER2.

[0089] In some embodiments, the method comprises:a) isolating an immune effector cell from the subject or a donor;b) modifying a transcription factor gene or gene product in the immune effector cell; and c) introducing the immune effector cell into the subject.

[0090] In some embodiments, step b) comprises (i) deleting or modifying the gene or gene product of ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell so that the transcription factor activity is decreased and / or (b) overexpressing or modifying the gene or gene product of ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell so that the transcription factor activity is increased.

[0091] In some embodiments, the method further comprises genetically modifying the immune effector cell to express a chimeric antigen receptor (CAR), antigen specific T-cell receptor, or antibody or antigen-binding fragment thereof that is capable of binding specifically to an antigen.

[0092] In some embodiments, the subject is a human.

[0093] In a further aspect, provided herein is a method of enhancing an antitumor activity of an immune effector cell, comprising:

[0094] a) inhibiting the transcriptional activity of transcription factor(s) ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell and / or

[0095] b) activating the transcriptional activity of transcription factor(s) ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell.

[0096] In another aspect, provided herein is a method of inhibiting mechanical suppression of an immune effector cell, comprising:

[0097] a) inhibiting the transcriptional activity of transcription factor(s) ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell and / or8324953654vlAttorney Docket No: 243734.000233

[0098] b) activating the transcriptional activity of transcription factor(s) ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell.

[0099] In some embodiments, the method further comprises genetically modifying the immune effector cell to express a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof that is capable of binding to an antigen.

[0100] In some embodiments, the method comprises activation and / or expansion of the immune effector cell ex vivo.

[0101] In some embodiments, the immune effector cell is a T cell, NK cell, or an induced pluripotent stem cell (iPSC).

[0102] In another aspect, provided herein is a method for manufacturing an immune effector cell with improved efficacy, comprising mechanically priming the immune effector cell in the presence of a viscoelastic hydrogel substrate matrix comprising crosslinked hydrogels.

[0103] In a further aspect, provided herein is a method for manufacturing an immune effector cell resistant to mechanical suppression, comprising mechanically priming the immune effector cell in the presence of a viscoelastic hydrogel substrate matrix comprising crosslinked hydrogels.

[0104] In some embodiments, the viscoelastic hydrogel substrate matrix comprises poly(ethylene-glycol)-based hydrogels, alginate-based hydrogels, collagen-I based platforms, hyaluronic acid and collagen-I hydrogels, and / or elastin-like protein (ELP) based hydrogel systems.

[0105] In some embodiments, the crosslinked hydrogel is a poly(ethylene-glycol)-based hydrogel, optionally an 8-arm poly(ethylene-glycol) macromer functionalized with reactive end groups.

[0106] In some embodiments, the viscoelastic hydrogel substrate matrix comprises dynamic crosslinks (e.g., alkyl-hydrazone crosslinks) and / or stable crosslinks (e.g., benzyl-hydrazone crosslinks).

[0107] In some embodiments, the viscoelastic hydrogel substrate matrix comprises a poly(ethylene-glycol)-based hydrogel comprising benzyl-hydrazone crosslinks and the immune effector cell experiences enhanced antitumor efficacy or resistance to mechanical suppression as compared to an immune effector cell with no mechanical priming or mechanical priming with a poly(ethylene-glycol)-based hydrogel comprising alkyl-hydrazone crosslinks.9324953654vlAttorney Docket No: 243734.000233

[0108] In some embodiments, the poly(ethylene-glycol)-based hydrogel comprising benzylhydrazone crosslinks is prepared by:

[0109] mixing poly(ethylene-glycol)-hydrazine in a buffer;

[0110] mixing the mixture of step a) with a collagen;

[0111] adding a neutralization buffer to the mixture of step b);

[0112] adding poly(ethylene-glycol)-benzyl-aldehyde to the mixture of step c);

[0113] casting the mixture of step d) in a cell culture dish, a mold, or coverslip;

[0114] incubating the cell culture dish, a mold, or coverslip containing the mixture of step d) until the mixture gels;

[0115] adding buffer or culture medium to fully cover the gel of step f); and

[0116] seeding the cell culture dish, a mold, or coverslip with the immune effector cell.

[0117] In some embodiments, a molar ratio of hydrazine: aldehyde in the mixture of step d) is about 2: 1 to about 1:2.

[0118] In some embodiments, a molar ratio of hydrazine: aldehyde in the mixture of step d) is about 1:1.

[0119] In some embodiments, the buffer comprises an osmolarity of about 270-300 mOsm.

[0120] In some embodiments, the buffer is phosphate buffered saline.

[0121] In some embodiments, the collagen is present in the mixture of step b) at a concentration of about 2.0 mg / mL to about 5 mg / mL, or about 2.5 mg / mL.

[0122] In some embodiments, the collagen is collagen-I.

[0123] In some embodiments, the neutralization buffer is added to the mixture of step b) in an amount effective to adjust the pH of the mixture to about pH 7.0 to about pH 7.4.

[0124] In some embodiments, the neutralization buffer comprises sodium hydroxide.

[0125] In some embodiments, the mixture of step d) comprises a total polyethylene glycol concentration of about 2.0 % w / v to about 5.0 % w / v, or about 2.5% w / v.

[0126] In some embodiments, the mixture of step d) is mixed by pipetting.

[0127] In some embodiments, the mixture of step d) is not mixed for more than 30 seconds.

[0128] In some embodiments, the incubation step of step f) is conducted at 37°C.

[0129] In some embodiments, the hydrogel comprises 100% benzyl-hydrazone crosslinks.

[0130] In some embodiments, the viscoelastic hydrogel substrate matrix comprises a poly(ethylene-glycol)-based hydrogel comprising alkyl-hydrazone crosslinks and wherein the10324953654vlAttorney Docket No: 243734.000233immune effector cell experiences enhanced antitumor efficacy or resistance to mechanical suppression as compared to an immune effector cell with no mechanical priming or mechanical priming with a poly(ethylene-glycol)-based hydrogel comprising benzyl-hydrazone crosslinks.

[0131] In some embodiments, the poly(ethylene-glycol)-based hydrogel comprising alkylhydrazone crosslinks is prepared by:

[0132] mixing poly(ethylene-glycol)-hydrazine in a buffer;

[0133] mixing the mixture of step a) with a collagen;

[0134] adding a neutralization buffer to the mixture of step b);

[0135] adding poly(ethylene-glycol)-alkyl-aldehyde to the mixture of step c);

[0136] casting the mixture of step d) in a cell culture dish, a mold, or coverslip;

[0137] incubating the cell culture dish, a mold, or coverslip containing the mixture of step d) until the mixture gels;

[0138] adding buffer or culture medium to fully cover the gel of step f); and

[0139] seeding the cell culture dish, a mold, or coverslip with the immune effector cell.

[0140] In some embodiments, a molar ratio of hydrazine: aldehyde in the mixture of step d) is about 2: 1 to about 1:2.

[0141] In some embodiments, a molar ratio of hydrazine: aldehyde in the mixture of step d) is about 1:1.

[0142] In some embodiments, the buffer comprises an osmolarity of about 270-300 mOsm.

[0143] In some embodiments, the buffer is phosphate buffered saline.

[0144] In some embodiments, the collagen is present in the mixture of step b) at a concentration of about 2.0 mg / mL to about 5 mg / mL, or about 2.5 mg / mL.

[0145] In some embodiments, the collagen is collagen-I.

[0146] In some embodiments, the neutralization buffer is added to the mixture of step b) in an amount effective to adjust the pH of the mixture to about pH 7.0 to about pH 7.4.

[0147] In some embodiments, the neutralization buffer comprises sodium hydroxide.

[0148] In some embodiments, the mixture of step d) comprises a total polyethylene glycol concentration of about 2.0 % w / v to about 5.0 % w / v or about 2.5% w / v.

[0149] In some embodiments, the mixture of step d) is mixed by pipetting.

[0150] In some embodiments, the mixture of step d) is not mixed for more than 30 seconds.

[0151] In some embodiments, the incubation step of step f) is conducted at 37°C.11324953654vlAttorney Docket No: 243734.000233

[0152] In some embodiments, the hydrogel comprises 100% alkyl-hydrazone crosslinks.

[0153] In some embodiments, the stiffness of the gel is adjusted by changing the wt / vol% of the PEG components.

[0154] In some embodiments, the method further comprises pre-incubating the cell culture dish of step e) with poly-L-lysine.

[0155] In some embodiments, steps a)-e) are performed at 0°C to 5°C.

[0156] In some embodiments, the immune effector cell is a T cell, NK cell, or an induced pluripotent stem cell (iPSC).

[0157] In some embodiments, the immune effector cell expresses a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof that binds to an antigen.

[0158] In some embodiments, the antigen is selected from a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, a prion antigen, and an antigen associated with an inflammation or an autoimmune disease.

[0159] In some embodiments, the tumor is a solid tumor.

[0160] In some embodiments, the tumor is breast, prostate, urinary bladder, skin, lung, ovary, sarcoma, or brain cancer.

[0161] In some embodiments, the tumor is a central nervous system (CNS) tumor.

[0162] In some embodiments, the CNS tumor is a pediatric CNS malignancy.

[0163] In some embodiments, the pediatric CNS malignancy is Diffuse Midline Glioma (DMG).

[0164] In some embodiments, the CNS tumor is medulloblastoma.

[0165] In some embodiments, the CNS tumor is an ependymoma.

[0166] In some embodiments, the tumor is a tumor expressing B7-H3, GD2, IL13Ra2, EphA2, or HER2.

[0167] In another aspect, provided herein is abiomaterials-based hydrogel platform for manufacturing immune effector cells, wherein the platform comprises a hydrogel substrate (e.g., crosslinked hydrogels) comprising tunable viscoelastic and stress relaxation properties.

[0168] In some embodiments, the biomaterials-based hydrogel platform comprises poly(ethylene-glycol)-based hydrogels, alginate-based hydrogels, collagen-I based platforms,12324953654vlAttorney Docket No: 243734.000233hyaluronic acid and collagen-I hydrogels, and / or elastin-like protein (ELP) based hydrogel systems.

[0169] In some embodiments, the biomaterials-based hydrogel platform comprises hydrazonebased crosslinking chemistry (e.g., PEG macromers functionalized with reactive end groups (e.g., alkyl-aldehyde, benzaldehyde) to form either dynamic (alkyl-hydrazone) or stable (benzylhydrazone) crosslinks).

[0170] In some aspects, the transcriptional activity of one or more transcription factors of the immune effector cell is reduced (e.g., knocked out), the transcriptional activity of one or more transcription factors of the immune effector cell is activated (e.g., overexpressed), and / or the immune effector cell is mechanically primed in the presence of a viscoelastic hydrogel substrate matrix comprising slow-relaxing crosslinked hydrogels. These methods, including individual steps within the methods, may be applied simultaneously or sequentially, in any order, and with any length of time between them. In some embodiments, the knockout of one or more transcription factors of the immune effector cell, the overexpression of one or more transcription factors of the immune effector cell, and the mechanical priming of the immune effector cell on the slow-relaxing hydrogel substrate matrix are performed in any combination. In some embodiments, the knockout of one or more transcription factors of the immune effector cell, the overexpression of one or more transcription factors of the immune effector cell, and the mechanical priming of the immune effector cell on the slow-relaxing hydrogel substrate matrix are performed individually (i.e., separately from one another).BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1 shows schematic of CAR T cells as exemplary immune effector cells expressing a CAR functioning within a complex tumor microenvironment. CAR T cells are exposed to various suppressive cues, involved in various cell-cell interactions, and exposed to various soluble factors, and exposed to mechanical resistances that can suppress the CAR T effector function.

[0172] Figures 2A-2I show rheological characterization of viscoelastic and stiffness mechanical properties of DMG (DIPG007, DIPG7c), Medulloblastoma (HDMB03, D341), healthy brain, lung tumor, and healthy lung samples using rheology and atomic force microscopy13324953654vlAttorney Docket No: 243734.000233(AFM). Figure 2A shows a schematic of harvested tumor samples or healthy controls measured under a rheometer to determine mechanical properties and a schematic of the timeline from implantation in mice to collection of tumors. Figure 2B shows stress relaxation profdes and Figure 2C shows the quantification using the time at which half of the modulus of the sample has relaxed (T’Y). Figure 2D shows stiffness (Young’s modulus) measurements of the different samples. Figure 2E shows loss tangent parameters of the different samples. Figure 2F shows a schematic of harvested tumor samples measured under atomic force microscopy (AFM) to determine mechanical properties at the micron-scale length scale. Figure 2G shows a representative workflow of a DMG tumor sample indented by AFM and sample heatmaps of stiffness or dissipation that are generated from AFM measurements. Figures 2H-2I show weighted average calculations of dissipation, as a metric for viscoelasticity (Figure 2H), and stiffness (Figure 21) across DMG, Medulloblastoma, or lung tumor samples, n > 3 animals / tumor type. *: p< 05, **: p< 01, ***: p<0.001, ****: p<0.0001.

[0173] Figures 3A-3E show hydrazone-based crosslinked hydrogels demonstrated the ability to mimic DMG and Medulloblastoma viscoelasticity and stiffness. Figure 3A shows a schematic representing 8-arm PEG macromers functionalized with reactive end groups (alkyl-aldehyde or benzaldehyde) which facilitate dynamic alkyl-hydrazone (AH) or stable benzyl-hydrazone (BH) crosslinks. Adapted from Sinha et al., Adv Healthc Mater. 2022 Nov 16;12(l):e2202147 (incorporated herein by reference for all intended purposes). Figure 3B shows a schematic illustrating how viscoelasticity can be tuned by using either dynamic AH or stable BH crosslinks.Figure 3C shows stress relaxation profdes of DMGs (including DMG implanted in either the cortex or brainstem), Medulloblastomas, and hydrogels with either fast-relaxing or slow-relaxing viscoelastic properties. Figure 3D shows quantification using the time at which half of the modulus of the sample has relaxed (T’ / ). Figure 3E shows stiffness (Young’s modulus) measurements of the different samples. ****: p<.0001.

[0174] Figures 4A-4G show CAR T cells tasked to kill and persist on DMG-mimicking, fastrelaxing viscoelastic substrates have dampened functionality. Figure 4A shows a schematic of CAR T cells tasked to kill or persist against DMGs on fast-relaxing matrix, slow-relaxing matrix, or 2D tissue culture plastic (TCP). Figure 4B shows the relative tumor viability of three cell lines (DIPG007, DIPG7c, and HDMB03) when treated with CAR T cells at various EffectorTarget (E: T) ratios at 24 hrs. n = 3 different healthy T cell donors. Figures 4C-4D show14324953654vlAttorney Docket No: 243734.000233percent tumor killing of CAR T cells against DMGs (DIPG007, DIPG7c) (Figure 4C) or Medulloblastomas (HDMB03) (Figure 4D) on either fast- or slow-relaxing hydrogels at various EffectorTarget (E: T) ratios at 24 hrs. Figure 4E shows cytokine production of TNFa, IFNy, and Granzyme B after 24 hrs of CAR T cell co-culture with DMGs or Medulloblastomas (MBs) on either fast- or slow-relaxing hydrogels. Figure 4F shows CAR T cell cytokine production of TNFa, IFNY, IL2, and Granzyme B with DMGs (DIPG007, DIPG7c) or Medulloblastomas (MB) on either fast-relaxing matrix, slow-relaxing matrix, or 2D TCP. Figure 4G shows CAR T cell expansion measured after each stimulation with DMG cells every 3.5 days at 2: 1 E: T ratio across 3 different healthy T cell donors. *: p< 05, **: p< 01, ***: p< 001, ****: p< 0001.

[0175] Figures 5A-5T show CAR T cells mechanically primed or pre-exposed to DMG-viscoelasticity exhibit poor in vitro and in vivo functionality. Figure 5A shows a schematic of CAR T cells mechanically primed on fast-relaxing (DMG-tumor mimicking), slow-relaxing, or 2D TCP. After 3 days of priming, CAR T cells are assessed for their cytotoxicity and persistence functionality against DMGs on either fast-relaxing substrates or 2D TCP. Figures 5B-5C show relative cell viability of DMG cells on (Figure 5B) fast-relaxing substrates or (Figure 5C) 2D TCP when treated with CAR T cells at various Effector: Target (E: T) ratios at 24 hrs. n = 3 different healthy T cell donors. Figures 5D-5E show CAR T cell expansion measured after each stimulation with DMG cells on either (Figure 5D) fast-relaxing substrates or (Figure 5E) 2D TCP every 3.5 days at 2: 1 E: T ratio across 4 different healthy T cell donors. Figures 5F-5G show CAR T cell expansion measured after each stimulation with DMG cells on either fastrelaxing substrate or 2D TCP in two different DMG cell lines. Figure 5H shows a schematic for in vivo experiments. Tumor cells are implanted intracranially into the cortex, and mechanically primed CAR T cells are delivered intracranially 7 days later. Tumor growth is assessed by bioluminescence imaging of DMG luciferase+ cells. Figure 51 shows total flux of DMG tumor growth in vivo after infused with CAR T cells that were mechanically primed on fast-relaxing substrates, slow-relaxing substrates, or 2D Tissue Culture Plastic (TCP). Dashed vertical line indicate time of T cell treatment at day 7. n = 4-5 animals / group. Figure 5J shows representative bioluminescent images of the DMG tumor growth in vivo from Figure 51. Figure 5K shows flow cytometry analysis of CD4+ / CD8+ CAR T cells at different time points of priming and stimulation with DMG cells, n = 3 different healthy T cell donors. Figure 5L shows the timeline for DMG in vivo experiment set up. Tumor cells were implanted intracranially into the cortex,15324953654vlAttorney Docket No: 243734.000233and mechanically primed CAR T cells were delivered intracranially 7 days later. Tumor growth was assessed by BLI imaging of DMG luciferase+ cells. Figure 5M shows quantitative bioluminescence imaging shown as total flux in photons per second (p / s). The vertical dashed line indicates time of CAR T cell injection. Figure 5N shows Kaplan-Meier overall survival curves of mechanically primed CAR T cells infused in DMG xenograft in vivo models using 2 healthy T cell donors. Figure 50 shows the timeline for Medulloblastoma in vivo experiment set up. Tumor cells were implanted intracranially into the cortex, and mechanically primed CAR T cells were delivered intracranially 14 days later. Tumor growth was assessed by BLI imaging of tumor luciferase+ cells. Figure 5P shows quantitative bioluminescence imaging shown as total flux in photons per second (p / s). The vertical dashed line indicates time of CAR T cell injection.Figure 5Q shows Kaplan-Meier overall survival curves of mechanically primed CAR T cells infused in Medulloblastoma xenograft in vivo models using 2 healthy T cell donors. Figure 5R shows the timeline for DMG in vivo experiment set up. Tumor cells were implanted intracranially into the cortex, and mechanically primed CAR T cells generated from Loc3CAR patient T cells were delivered intracranially 7 days later. Tumor growth was assessed by BLI imaging of DMG luciferase+ cells. Figure 5S shows quantitative bioluminescence imaging shown as total flux in photons per second (p / s). The vertical dashed line indicates time of CAR T cell injection. Figure 5T shows Kaplan-Meier overall survival curves of mechanically primed CAR T cells infused in DMG xenograft in vivo models using 2 Loc3CAR patient T cell donors. * p< 05, **: p< 01, ***: p<0.001, ****: pO. OOOl.

[0176] Figures 6A-6G show slow primed CAR T cells demonstrated durable in vivo tumor control after rechallenging. Figure 6A shows a schematic of in vivo experiments in DMG cell line DIPG007. Tumor cells are implanted intracranially into the cortex, and primed CAR T cells are delivered intracranially 7 days later. Animals were rechallenged (Haydar, et al., 2021) day 97 (indicated by arrow in Figure 6B). Tumor growth was assessed by bioluminescence imaging of DMG luciferase+ cells. Figure 6B shows total flux of DMG tumor growth in vivo after infused with CAR T cells that were mechanically primed on fast-relaxing substrates, slow-relaxing substrates, or 2D Tissue Culture Plastic (TCP). Dashed vertical line indicate time of T cell treatment at day 7. Figure 6C shows luminescent images of the DMG tumor growth in vivo from Figure 6B. Figure 6D shows the CD4+ / CD8+ ratio between CAR T cells that were mechanically primed on fast-relaxing substrates, slow-relaxing substrates, or 2D Tissue Culture16324953654vlAttorney Docket No: 243734.000233Plastic (TCP). Figure 6E shows a schematic of an additional rechallenge in vivo experiment with DMG cell line DIPG7c. Figure 6F shows quantitative bioluminescence imaging shown as total flux in photons per second (p / s) within a DMG DIPG7c model. The vertical dashed line indicates time of CAR T cell injection. Figure 6G shows Kaplan-Meier overall survival curves of mechanically primed CAR T cells infused in DMG xenograft (DIPG7c) in vivo models using two T cell donors.

[0177] Figures 7A-7D shows fast primed CAR T cells exhibit decreased calcium signaling and decreased. Figure 7A shows fold change of calcium flux quantifications of mechanically primed CAR T cells with DMG or Medulloblastoma tumor cells. For DMGs, n = 40 cells (fast) or 56 cells (slow) across two T cell donors. For Medulloblastomas, n = 60 cells (fast), 56 cells (slow), or 49 cells (2D) across two T cell donors. Figure 7B shows cell avidity between CAR T cells that were mechanically primed on fast-relaxing substrates, slow-relaxing substrates, or 2D Tissue Culture Plastic (TCP), n = 3 donors. Figure 7C shows single-cell testing of mechanically primed CAR T cell avidity to DMG tumor cells. At least 100 cells were analyzed across three donors. Two DMG cell lines were used - DIPG007 and DIPG7c. Figure 7D shows area under the curve analysis for avidity measurements across fast, slow, and 2D primed groups. *p: <.05, **: p< 01, ***: pO. OOl, ****: p<0.0001.

[0178] Figure 8 shows antigen differences in B7-H3 molecules per cell.

[0179] Figures 9A-9B show activation markers in CD4+ / G4S linker+ and CD8+ / G4S linker+ populations, n = 3 donors.

[0180] Figures 10A-10B show exhaustion markers in CD4+ / G4S linker+ and CD8+ / G4S linker+ populations, n = 3 donors.

[0181] Figure 11 shows compiled activation and exhaustion markers, n = 3 donors.

[0182] Figures 12A-12F show scRNA-seq analysis of CAR T cells pre-exposed or primed on fast-relaxing vs. slow-relaxing viscoelastic hydrogels. Figure 12A shows a schematic of CAR T cells (generated from either patient or healthy donors) pre-exposed or primed on fast-relaxing (tumor-mimicking) or slow-relaxing (control) hydrogels. After 3 days, CAR T cells were collected for scRNA sequencing. Patient donors were from Loc3CAR: NCT05835687. Figure 12B shows scRNA-seq UMAP plot of CAR T cells primed on fast- vs. slow-relaxing hydrogels.Figure 12C shows gene set enrichment analysis of top immune related pathways between CAR17324953654vlAttorney Docket No: 243734.000233T cells primed on fast- vs. slow-relaxing hydrogels. Figures 12D-12F show scRNA-seq cluster annotations.

[0183] Figures 13A-13G show scRNA-seq analysis revealed potential biophysical regulators driving CAR T cell response to viscoelasticity. Figure 13A shows transcription factor inference analysis revealed regulators differentially active in fast vs. slow primed CAR T cells. Figure 13B shows Western blot to confirm knockout (KO) of FLU and LEF1 with quantification on the bottom. Figure 13C shows next generation sequencing (NGS) analysis to confirm KO efficiency. Figure 13D shows a timeline for the DMG in vivo experiment set up. Tumor cells were implanted intracranially into the cortex, and CRISPR KO CAR T cells were delivered intracranially 7 days later. Tumor growth was assessed by BLI imaging of DMG luciferase+ cells. Figure 13E shows early in vivo DMG tumor clearance at day 6 post CAR T cell treatment. CAR T cells had individual knockouts of leading regulators active after fast priming. Figure 13F shows Kaplan-Meier overall survival curves of KO CAR T cells infused in DMG xenograft in vivo models (n=5 / group) using one healthy donor. Mix = mixed KOs, equal parts AAVS1: DNMT1: LEF1: FLI1. Figure 13G shows quantitative bioluminescence imaging as total flux in photons per second (p / s). The vertical dashed line indicates time of CAR T cell injection. Modified CAR T cells (either AAVS1 KO, FLU KO, or FLU overexpression (OE)) were tested and n=5 / group.

[0184] Figure 14 shows a picture of different size droplets of the hydrogel solutions.DETAILED DESCRIPTION

[0185] To identify the defining mechanical features of DMG tumors and to investigate how DMG-specific mechanical cues impact immune effector cell (e.g., CAR T cell) function, a biomaterials-based hydrogel platform is utilized herein. The biomaterials-based hydrogel platform herein mimics the viscoelastic and stiffness mechanical properties of diffuse midline gliomas (DMGs) via independent tuning of stiffness and viscoelasticity.

[0186] It was identified herein that DMG tumors are viscoelastic and demonstrated that DMG-specific viscoelasticity significantly impaired CAR T cell efficacy (e.g., cytotoxicity, cytokine production, activation, avidity, and antitumor efficacy) in vitro and in vivo. Interestingly, the hydrogel group that exhibited slow viscoelastic properties, which was used initially as a control, has yielded surprising and unexpected enhancement of immune effector cell efficacy. For18324953654vlAttorney Docket No: 243734.000233example, when CAR T cells were exposed to the slow viscoelastic hydrogel substrate, the CAR T cells exhibited enhanced antitumor activity both in vitro and in vivo. This demonstrated that pre-exposing or mechanically priming immune effector cells to a substrate with specific mechanical properties may elicit improved immune effector cell efficacy (e.g., cytotoxicity, cytokine production, activation, avidity, and antitumor efficacy). The biomaterials-based hydrogel platform with unique mechanical properties can be used in the immune effector cell manufacturing process to mechanically prime immune effector cells, resulting in enhanced immune effector cell efficacy (e.g., antitumor activity, e.g., against DMGs and other pediatric brain tumors).

[0187] In certain aspects, dasatinib, a tyrosine kinase inhibitor, is used in the CAR T cell manufacturing process to improve CAR T cell expansion and quality. The use of dasatinib has also been found to help against CAR T cell exhaustion by temporarily suppressing T cell activity (Weber, et al., 2021).

[0188] Further, biophysical regulators that drive immune effector cell dysfunction in response to tumor mechanics were identified. The biophysical regulators of note were transcription factors, some of which were upregulated and some of which were downregulated when immune effector cells (e.g., CAR T cells) were exposed to the fast viscoelastic hydrogel substrate compared to the slow substrate. Genetic modifications of these regulators (e.g., FLI1, LEF1, and DNMT1) enhanced in vivo efficacy, thereby demonstrating a potential therapeutic strategy to overcome viscoelasticity-induced immunosuppression. These findings establish tumor viscoelasticity as a mechanical checkpoint that suppresses immune effector cell function and demonstrate that engineering immune effector cells (e.g., CAR T cells) to overcome mechanical immunosuppression may improve therapeutic outcomes.Definitions

[0189] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present application. For purposes of the present application, the following terms are defined.

[0190] The term “immune effector cell” as used herein refers to a cell that is involved in an19324953654vlAttorney Docket No: 243734.000233immune response, e.g., in the promotion of an immune effector response. Non-limiting examples of immune effector cells include T cells (e.g., a(3 T cells and y8 T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes. Stem cells, such induced pluripotent stem cells (iPSCs), that are capable of differentiating into immune cells are also included here.

[0191] The terms “T cell” and “T lymphocyte” are interchangeable and used synonymously herein. As used herein, T cell includes thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a CD8+ T cell, a CD4+ T cell, a helper T cell or T-helper cell (HTL; CD4+ T cell), a cytotoxic T cell (CTL; CD8+ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+ T cell), CD4+CD8+ T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. Also included are “ct(3 T cell receptor (TCR) T cells”, which refer to a population of T cells that possess a TCR composed of a- and P-TCR chains. Also included are “NKT cells”, which refer to a specialized population of T cells that express a semi-invariant aP T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+ andNKl.l-, as well as CD4+, CD4-, CD8+ and CD8- cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance. Also included are “gamma-delta T cells (y5 T cells),” which refer to a specialized population that to a small subset of T cells possessing a distinct TCR on their surface, and unlike the majority of T cells in which the TCR is composed of two glycoprotein chains designated a- and P-TCR chains, the TCR in y6 T cells is made up of a y-chain and a 8-chain. y6 T cells can play a role in immunosurveillance and immunoregulation, and were found to be an important source of IL- 17 and to induce robust CD8+ cytotoxic T cell response. Also included are “regulatory T cells” or “Tregs”, which refer to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance. Tregs cells are typically transcription factor Foxp3 -positive CD4+T cells and can also include transcription factor Foxp3 -negative regulatory T cells that are IL-10-producing CD4+T cells.20324953654vlAttorney Docket No: 243734.000233

[0192] The terms “natural killer cell” and “NK cell” are interchangeable and used synonymously herein. As used herein, NK cell refers to a differentiated lymphocyte with a CD16+ CD56+ and / or CD57+ TCR- phenotype. NKs are characterized by their ability to bind to and kill cells that fail to express “self’ MHC / HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0193] The term “chimeric antigen receptor” or “CAR” as used herein is defined as a cellsurface receptor comprising an extracellular target-binding domain, a transmembrane domain and a cytoplasmic domain, comprising a lymphocyte activation domain and optionally at least one co-stimulatory signaling domain, all in a combination that is not naturally found together on a single protein. This particularly includes receptors wherein the extracellular domain and the cytoplasmic domain are not naturally found together on a single receptor protein. The chimeric antigen receptors of the present invention are intended primarily for use with lymphocyte such as T cells and natural killer (NK) cells.

[0194] As used herein, the term “antigen” refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) molecule capable of being bound by a T-cell receptor, chimeric antigen receptor (CAR), or antibody or antigen-binding fragment thereof. An antigen is also able to provoke an immune response. An example of an immune response may involve, without limitation, antibody production, or the activation of specific immunologically competent cells, or both. A skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components, organisms, subunits of proteins / antigens, killed or inactivated whole cells or lysates.

[0195] The term “antigen-binding moiety”, “antigen-binding fragments,” or “target-binding moiety” refers to a target-specific binding element that may be any ligand that binds to the antigen of interest or a polypeptide or fragment thereof, wherein the ligand is either naturally derived or synthetic. Examples of antigen-binding moi eties include, but are not limited to,21324953654vlAttorney Docket No: 243734.000233antibodies; polypeptides derived from antibodies, such as, for example, single chain variable fragments (scFv), Fab, Fab', F(ab')2, and Fv fragments; polypeptides derived from T Cell receptors, such as, for example, TCR variable domains; secreted factors (e.g., cytokines, growth factors) that can be artificially fused to signaling domains (e.g., “zytokines”); and any ligand or receptor fragment (e.g., CD27, NKG2D) that binds to the antigen of interest. Combinatorial libraries could also be used to identify peptides binding with high affinity to the therapeutic target.

[0196] The terms “antibody” and “antibodies” refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies and anti-idiotypic (anti-Id) antibodies (including, e g., anti-Id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above. The terms “antibody” and “antibodies” also refer to covalent diabodies such as those disclosed in U. S. Pat. Appl. Pub. 2007 / 0004909 and Ig-DARTS such as those disclosed in U. S. Pat. Appl. Pub. 2009 / 0060910, each of which are incorporated by reference in their entirety for all purposes. Antibodies useful as a TCR-binding molecule include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl and IgA2) or subclass. Also included are “bispecific antibodies”, which refer to antibodies that are capable of binding to two different antigens or different epitopes of the same antigen.

[0197] The term “host cell” means any cell that contains a heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, a host cell can be a cell from any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. An appropriate host may be determined. For example, the host cell may be selected based on the vector backbone and the desired result. By way of example, a plasmid or cosmid can be introduced into a prokaryote host cell for replication of several types of vectors. Bacterial cells such as, but not limited to DH5ot, JM109, and KCB, SURE® Competent Cells, and SOLOPACK Gold Cells, can be used as host cells for vector replication and / or expression. Additionally, bacterial cells such as E. coli22324953654vlAttorney Docket No: 243734.000233LE392 could be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to yeast (e.g., YPH499, YPH500 and YPH501), insects and mammals. Examples of mammalian eukaryotic host cells for replication and / or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12.

[0198] Host cells of the present invention also include T cells, natural killer cells (NK cells), and stem cell that is capable of differentiating into an immune cell (e.g., T cells and NK cells) that contain the DNA or RNA sequences the heterologous nucleic acid. In some embodiments, the stem cell that is capable of differentiating into an immune cell may be derived from induced pluripotent stem (iPS) cells (iPSCs). Non-limiting examples of iPSCs that may be used in accordance with the disclosure may be any such cells as described in, e.g., Zhu et al., 2019 and Iriguchi et al., 2021, the contents of each of which is incorporated herein by reference in its entirety for all purposes.

[0199] The terms “activation” or “stimulation” means to induce a change in their biologic state by which the cells (e.g., T cells and NK cells) express activation markers, produce cytokines, proliferate and / or become cytotoxic to target cells. All these changes can be produced by primary stimulatory signals. Co-stimulatory signals can amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity. A “co-stimulatory signal” refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell and / or NK cell proliferation and / or upregulation or downregulation of key molecules.

[0200] The term “proliferation” refers to an increase in cell division, either symmetric or asymmetric division of cells. The terms “expand” or “expansion” when used in relation to an immune cell refer to the ability of the immune cell to undergo cellular proliferation (i.e., to increase the number of cells). The terms used herein encompass both in vivo and in vitro immune cell expansion.

[0201] The term “differentiation” refers to a method of decreasing the potency or proliferation of a cell or moving the cell to a more developmentally restricted state.

[0202] The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become produced, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as23324953654vlAttorney Docket No: 243734.000233a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular or transmembrane.

[0203] The term “transfection” means the introduction of a “foreign” (i.e., extrinsic or extracellular) nucleic acid into a cell using recombinant DNA technology. The term “genetic modification” means the introduction of a “foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences operably linked to polynucleotide encoding the chimeric antigen receptor, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “genetically engineered.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0204] The term “transduction” means the introduction of a foreign nucleic acid into a cell using a viral vector.

[0205] The terms “genetically modified” or “genetically engineered” refers to the addition of extra genetic material in the form of DNA or RNA into a cell.

[0206] Percent sequence identity can be determined using any method known to one of skill in the art. In a specific embodiment, the percent identity is determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wisconsin). Information regarding hybridization conditions (e.g., high, moderate, and typical stringency conditions) have been described, see, e.g., U. S. Patent Application Publication No. US 2005 / 0048549 (e.g., paragraphs 72-73).

[0207] The term “variant” as used herein refers to a modified polypeptide, protein, or polynucleotide that has substantial or significant sequence identity or similarity to a wild type polypeptide, protein, or polynucleotide. The variant may retain the same, or have altered (e.g., improved, reduced or abolished) biological activity relative to the wild type polypeptide, protein,24324953654vlAttorney Docket No: 243734.000233or polynucleotide of which it is a variant. The variant may contain an insertion, a deletion, a substitution of at least one amino acid residue or nucleotide.

[0208] The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, synthesized RNA and DNA molecules, phages, viruses, etc. In some embodiments, the vector is a viral vector such as, but not limited to, viral vector is an adenoviral, adeno-associated, alphaviral, herpes, lentiviral, retroviral, baculoviral, or vaccinia vector.

[0209] The term “regulatory element” refers to any cis-acting genetic element that controls some aspect of the expression of nucleic acid sequences. In some embodiments, the term “promoter” comprises essentially the minimal sequences required to initiate transcription. In some embodiments, the term “promoter” includes the sequences to start transcription, and in addition, also include sequences that can upregulate or downregulate transcription, commonly termed “enhancer elements” and “repressor elements”, respectively.

[0210] As used herein, the term “operatively linked,” and similar phrases, when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.

[0211] By “enhance” or “promote,” or “increase” or “expand” or “improve” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. A measurable physiological response may include an increase in T cell expansion, activation, effector function, persistence, and / or an increase in antitumor activity (e.g., cancer cell death killing ability), among others apparent from the25324953654vlAttorney Docket No: 243734.000233understanding in the art and the description herein. In some embodiments, an “increased” or “enhanced” amount can be a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.

[0212] By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refers generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. In some embodiments, a “decrease” or “reduced” amount can be a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell lineage.

[0213] The terms “inhibit” or “inhibition” as used herein refer to reducing a function or activity to an extent sufficient to achieve a desired biological or physiological effect. Inhibition may be complete or partial.

[0214] The terms “activate” or “activation” as used herein refer to increasing a function or activity to an extent sufficient to achieve a desired biological or physiological effect. Activation may be complete or partial.

[0215] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0216] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to26324953654vlAttorney Docket No: 243734.000233a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0217] The term “in need thereof’ refers to a judgment made by a physician or another caregiver that a subject requires and / or will benefit from treatment. The judgment is made based on a variety of factors in the realm of the expertise of the physician or caregiver.

[0218] The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0219] The term “protein” is used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0220] The terms “nucleic acid”, “nucleotide”, and “polynucleotide” encompass both DNA and RNA unless specified otherwise. By a “nucleic acid sequence” or “nucleotide sequence” is meant the nucleic acid sequence encoding an amino acid, the term may also refer to the nucleic acid sequence including the portion coding for any amino acids added as an artifact of cloning, including any amino acids coded for by linkers.

[0221] The terms “patient”, “individual”, “subject”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g, cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.27324953654vlAttorney Docket No: 243734.000233

[0222] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin.

[0223] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0224] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. The term “about X to Y” has the same meaning as “about X to about Y.”

[0225] If aspects of the disclosure are described as “comprising” a feature, or versions thereof (e.g., comprise), embodiments also are contemplated “consisting of’ or “consisting essentially of’ the feature.

[0226] The practice of the present invention employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John28324953654vlAttorney Docket No: 243734.000233Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g., in U. S. Patent No. 7,912,698 and U. S. Patent Appl. Pub. Nos. 2011 / 0202322 and 2011 / 0307437.

[0227] The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein.

[0228] The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed.Immune effector cells

[0229] Products and methods are disclosed for improved efficacy of immune effector cells. Non-limiting examples of immune effector cells include T cells (e.g., αβ T cells and γδ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes. Stem cells, such induced pluripotent stem cells (iPSCs), that are capable of differentiating into immune cells are also included herein.

[0230] In some embodiments, the immune effector cell is a T cell. T cells may include, but are not limited to, thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4+ T cell) CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+ T cell), CD4+ CD8+ T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells memory T cells, and NKT cells.

[0231] In some embodiments, the T cell is selected from a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell, an αβ T cell receptor (TCR) T cell, a natural killer T (NKT) cell, a yb T cell, a memory T cell, a T-helper cell, and a regulatory T cell (Treg).29324953654vlAttorney Docket No: 243734.000233

[0232] In some embodiments, the immune effector cell is a natural killer (NK) cell. NK cell refers to a differentiated lymphocyte with a CD3- CD16+, CD3- CD56+, CD16+ CD56+ and / or CD57+ TCR- phenotype.

[0233] In some embodiments, the immune effector cell is a stem cell that is capable of differentiating into an immune cell. In some embodiments, the stem cell that is capable of differentiating into an immune cell may be derived from induced pluripotent stem (iPS) cells (iPSCs). Non-limiting examples of iPSCs that may be used in accordance with the disclosure may be any such cells as described in, e.g., Zhu et al., 2019 and Iriguchi et al., 2021, the contents of each of which is incorporated herein by reference in its entirety for all purposes.

[0234] In some embodiments, the immune effector cell further comprises at least one surface molecule capable of binding specifically to an antigen.

[0235] The products and methods disclosed herein may be applied to all forms of T cells, which include but not limited to T cells i) that express a chimeric antigen receptor (CAR); ii) that express an endogenous αβ TCR, which is specific for an antigen (e.g., derived from viral or tumor-associated antigens (including neoantigens)); iii) that transgenically express an αβ TCR, which is specific for an antigen (e.g., derived from viral or tumor-associated antigens (including neoantigens)); iv) that transgenically express antibodies, which recognize an antigen (e.g., derived from viral or tumor-associated antigens (including neoantigens)) / or a peptide derived from them and an activating molecule expressed on T cells such as CD3; and / or v) that are generated via stimulation with for example but not limited to peptides, antigen presenting and / or artificial antigen presenting cells (in vitro sensitized [IVS] T cell therapy).

[0236] The products and methods disclosed herein may be applied to all forms of NK cells, which include but not limited to NK cells i) that express a chimeric antigen receptor (CAR); ii) that express an endogenous αβ TCR, which is specific for an antigen (e.g., derived from viral or tumor-associated antigens (including neoantigens)); iii) that transgenically express an αβ TCR, which is specific for an antigen (e.g., derived from viral or tumor-associated antigens (including neoantigens)); iv) that transgenically express antibodies, which recognize an antigen (e.g., derived from viral or tumor-associated antigens (including neoantigens)) / or a peptide derived from them and an activating molecule expressed on NK cells such as CD3; and / or v) that are generated via stimulation with for example but not limited to peptides, antigen presenting and / or artificial antigen presenting cells (in vitro sensitized [IVS] NK cell therapy).30324953654vlAttorney Docket No: 243734.000233

[0237] In some embodiments, the antigen is selected from a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, a prion antigen, and an antigen associated with an inflammatory or an autoimmune disease.

[0238] In some embodiments, the antigen is a tumor antigen. Non-limiting examples of tumor antigens that may be targeted by the immune effector cell described herein include human epidermal growth factor receptor 2 (HER2), interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), ephrin type-A receptor 2 (EphA2), A kinase anchor protein 4 (AKAP-4), adrenoceptor beta 3 (ADRB3), anaplastic lymphoma kinase (ALK), immunoglobulin lambda- like polypeptide 1 (IGLL1), androgen receptor, angiopoi etin-binding cell surface receptor 2 (Tie 2), B7H3 (CD276), bone marrow stromal cell antigen 2 (BST2), carbonic anhydrase IX (CAIX), CCCTC-binding factor (Zinc Finger Protein)-like (BORIS), CD171, CD179a, CD24, CD300 moleculelike family member f (CD300LF), CD38, CD44v6, CD72, CD79a, CD79b, CD97, chromosome X open reading frame 61 (CXORF61), claudin 6 (CLDN6), CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, or 19A24), C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule- 1 (CLL-1), Cyclin B 1, Cytochrome P450 IB 1 (CYP1B 1), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), epidermal growth factor receptor (EGFR), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor (FCAR), Fc receptor-like 5 (FCRL5), Fms-like tyrosine kinase 3 (FLT3), Folate receptor beta, Fos-related antigen 1, Fucosyl GM1, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), ganglioside GD3, ganglioside GM3, glycoceramide (GloboH), Glypican-3 (GPC3), Hepatitis A virus cellular receptor 1 (HAVCR1), hexasaccharide portion of globoH, high molecular weight-melanoma-associated antigen (HMWMAA), human Telomerase reverse transcriptase (hTERT), interleukin 11 receptor alpha (IL-1 IRa), KIT (CD117), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), Lewis(Y) antigen, lymphocyte antigen 6 complex, locus K 9 (LY6K), lymphocyte antigen 75 (LY75), lymphocytespecific protein tyrosine kinase (LCK), mammary gland differentiation antigen (NY-BR-1), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma inhibitor of apoptosis (ML-IAP), mucin 1, cell surface associated (MUC1), N-acetyl glucosaminyl-transferase V (NA17), neural cell adhesion molecule (NCAM), o-acetyl-GD231324953654vlAttorney Docket No: 243734.000233ganglioside (0AcGD2), olfactory receptor 51E2 (OR51E2), p53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), placenta-specific 1 (PLAC1), platelet-derived growth factor receptor beta (PDGFR-beta), Polysialic acid, proacrosin binding protein sp32 (OY-TES 1), prostate stem cell antigen (PSCA), Protease Serine 21 (PRSS21), Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2), Ras Homolog Family Member C (RhoC), sarcoma translocation breakpoints, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), squamous cell carcinoma antigen recognized by T cells 3 (SART3), stage-specific embryonic antigen-4 (SSEA-4), synovial sarcoma, X breakpoint 2 (SSX2), TCR gamma alternate reading frame protein (TARP), TGS5, thyroid stimulating hormone receptor (TSHR), Tn antigen (Tn Ag), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), uroplakin 2 (UPK2), vascular endothelial growth factor receptor 2 (VEGFR2), v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), Wilms tumor protein (WT1), and X Antigen Family, Member 1A (XAGE1), or a fragment or variant thereof.

[0239] In some embodiments, the tumor antigen is associated with brain cancer, glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematologic malignancy. Non-limiting examples of the tumor antigens associated with cervical cancer or head and neck cancer include MUC1, Mesothelin, HER2, GD2, and EGFR. Non-limiting examples of tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, Mesothelin, CA125, EpCAM, EGFR, PDGFRa, Nectin-4, B7-H3 and B7-H4. Non-limiting examples of tumor antigens associated with hematological malignancies include BCMA, GPRC5D, SLAM F7, CD33, CD19, CD22, CD79, CLL1, CD123, and CD70. Non-limiting examples of tumor antigens associated with bladder cancer include Nectin-4 and SLITRK6. Non-limiting examples of tumor antigens associated with renal cancer include CD70 and FOLR1. Non-limiting examples of tumor antigen associated with glioblastoma include FGFR1, FGFR3, MET, CD70, ROBO1, IL13Ra2, HER2, EGFRvIII, EGFR, CD 133, and PDGFRA. Non-limiting examples of tumor antigen associated with liver cancer include, EpCAM, cMET, AFP, Claudin 18.2, and GPC-3.

[0240] Additional antigens that may be targeted by the extracellular antigen-binding domain include, but are not limited to, carbonic anhydrase EX, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, CA125, CD1, CDla, CD3, CD5, CD15, CD16, CD19,32324953654vlAttorney Docket No: 243734.000233CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphAl, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-I), la, IL-2, IL-6, IL-8, insulin growth factor- 1 (IGF-I), KC4-antigen, KS-1 -antigen, KS1-4, Le- Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-lA-antigen, an angiogenesis marker, an oncogene marker or an oncogene product.

[0241] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cell receptors and cells which produce “self ’-directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or disorder such as, psoriasis, vasculitis, Wegener's granulomatosis, Hashimoto's thyroiditis, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, Rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, Systemic lupus erythematosus, sarcoidosis, Type 1 diabetes mellitus, insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, or Myasthenia gravis.

[0242] In some embodiments, autoimmune antigens that may be targeted by the immune effector cell disclosed herein include but are not limited to islet cell antigen, platelet antigens, Sm antigens in snRNPs, myelin protein antigen, Rheumatoid factor, and anticitrullinated protein., glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin and 25-35 kD nuclear protein, granular proteins such as bactericidal permeability increasing protein (BPI), elastase fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclical citrullinated peptides), translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), circulating serum proteins such as RFs (IgG, IgM), fibrinogen, plasminogen, components of articular cartilage such as collagen II, IX, and XI, ferritin, nuclear components such as RA33 / hnRNP A2, Sm, stress proteins such as HSP-65, -70, -90, BiP, inflammatory / immune33324953654vlAttorney Docket No: 243734.000233factors such as B7-H1, IL-1 alpha, and IL-8, enzymes such as calpastatin, alpha-enolase, eukaryotic translation elongation factor 1 alpha 1 aldolase- A, dipeptidyl peptidase, osteopontin, cathepsin G, myeloperoxidase, proteinase 3, antigen, islet cell antigen, rheumatoid factor, histones, ribosomal P proteins platelet antigens, myelin protein, cardiolipin, vimentin, nucleic acids such as, and RNA, ribonuclear particles and proteins such as Sm antigens (including but not limited to SmD's and SmBTB), U1RNP, A2 / B1 hnRNP, Ro (SSA), and La (SSB) antigens, dsDNA, and ssDNA.

[0243] In some embodiments, the antigen targeted by immune effector cells of the present disclosure is an antigen expressed in the tumor stroma. Exemplary antigens expressed in the tumor stroma that may be targeted by immune effector cells of the present disclosure include but are not limited to oncofetal splice vari ants of fibronectin and tenascin C, tumor-specific splice variants of collagen, and fibroblast activating protein (FAP).

[0244] In some embodiments, the antigen targeted by immune effector cells of the present disclosure is an antigen expressed on endothelial cell. Exemplary antigens expressed on endothelial cells that may be targeted by immune effector cells of the present disclosure include, but are not limited to, VEGF receptors, and tumor endothelial markers (TEMs).

[0245] Exemplary infectious associated antigens that may be targeted by the immune effector cells of the present disclosure include those derived from Adenoviridae (most adenoviruses); Arena viridae (hemorrhagic fever viruses); Birnaviridae, Bimgaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Calciviridae (e.g., strains that cause gastroenteritis); Coronoviridae (e.g., coronaviruses); Filoviridae (e.g., ebola viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Hepadnaviridae (Hepatitis B virus; HBsAg); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus); Iridoviridae (e.g., African swine fever virus); Norwalk and related viruses, and astroviruses.; Orthomyxoviridae (e.g., influenza viruses); Papovaviridae (papillomaviruses, polyoma viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Parvovirida (parvoviruses); Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Poxviridae (variola viruses, vaccinia viruses, pox viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III / LAV, or HIV-III); and other isolates, such as34324953654vlAttorney Docket No: 243734.000233HIV-LP); Rhabdoviradae (e.g., vesicular stomatitis viruses, rabies viruses); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis, the agents of non-A, non-B hepatitis (i.e. Hepatitis C)).

[0246] Additional infectious antigens that may be targeted by the immune effector cells of the present disclosure include bacterial antigens, fungal antigens, parasite antigens, or prion antigens, or the like. Non-limiting examples of infectious bacteria include but are not limited to: Actinomyces israelii, Bacillus antracis, Bacteroides sp., Borelia burgdorferi, Chlamydia., Clostridium perfringers, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium sp., Enterobacter aerogenes, Enterococcus sp., Erysipelothrix rhusiopathiae, Fusobacterium nucleatum, Haemophilus influenzae, Helicobacter pyloris, Klebsiella pneumoniae, Legionella pneumophilia, Leptospira, Listeria monocytogenes, Mycobacteria sps. e.g., M tuberculosis, M avium, M gordonae, M intracellulare, M kansaif), Neisseria gonorrhoeae, Neisseria meningitidis, Pasturella multocida, pathogenic Campylobacter sp., Rickettsia, Staphylococcus aureus, Streptobacillus monihformis, Streptococcus (anaerobic sps.), Streptococcus (viridans group), Streptococcus agalactiae (Group B Streptococcus), Streptococcus bovis, Streptococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes (Group A Streptococcus), Treponema pallidium, and Treponema pertenue. Non-limiting examples of infectious fungi include: Cryptococcus neoformans, Histoplasma capsulatuin, Coccidioides immitis, Blastomyces dernatitidis, Chlamydia trachomatis and Candida albicans. Other infectious organisms (i.e., protists) include: Plasmodium such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Toxoplasma gondii and Shistosoma. Other medically relevant microorganisms have been descried extensively in the literature, e.g., see C. G. A. Thomas, “Medical Microbiology”, Bailliere Tindall, Great Britain 1983, which is hereby incorporated by reference in its entirety.

[0247] Other examples of antigens that may be targeted by the immune effector cells of the present disclosure include antigens expressed on immune and / or stem cells to deplete these cells such as CD45RA and c-kit.

[0248] Various non-limiting exemplary antigen targets are also displayed in Tables 1-3.

[0249] In some embodiments, the antigen-binding domain may comprise a VH sequence, a VL sequence, and / or CDRs thereof, such as those described in the cited publications, the contents35324953654vlAttorney Docket No: 243734.000233of each publication are incorporated herein by reference in their entirety for all purposes (Table 1)Table 1. Exemplary antigen-binding moieties comprising a VH sequence, a VL sequence, and / or CDRs thereofAntigen target Type Examples of Source Type Examples of Source Oncofetal - VL Identifier 1, 2, 7 in fibronectin US20070202103A1 VMS2 VH FIG. 1 in W02000058363 - - GCC1 VH Identifier 1 in VL Identifier 4, 2 in US20160030595A1 US20160030595A1 MVR VH Identifier 1 in VL Identifier 5 in US20160257762A1 US20160257762A1 OlfmB VH Identifier 1 in VL Identifier 2 in WO2015054441A1 WO2015054441A1 TRBC1 VH Identifier 1 in VL Identifier 2 in WO2015132598 WO2015132598 Malignant VH Identifier 1 in VL Identifier 5 in Variable WO2015133817A1 WO2015133817A1 ReceptorCD40 VH Identifier 1 in VL Identifier 2 inWO2016069919; Identifier WO2016069919; Identifier 5, 7, 8 in WO2015091655 6 in WO2015091655 CDIM VH Identifier 1, 10, 11, 12, 13, VL Identifier 28, 29, 30, 31,14, 15, 16, 17, 18, 19, 2, 20, 32, 33, 34, 35, 36, 37, 38, 21, 22, 3, 4, 5, 6, 7, 8, 9 in 39, 40, 41, 42, 43, 44, 45, W02013120012 46, 47, 48, 49 in W02013120012 ILIRAP VH Identifier 1, 10, 19, 8, 9 in VL Identifier 14, 15, 17, 18, 2,WO2016020502; Identifier 20 in W02016020502; 120, 122, 124 in Identifier 121, 123, 125 in WO2016179319A1 WO2016179319A1 ALK VH Identifier 1, 11, 13, 15, 3, 5, VL Identifier 10, 12, 14, 16, 2,7, 9 inUS20160280798Al; 4, 6, 8 inIdentifier 9, 1, 3, 5, 11, 13, US20160280798 Al;15, 7, 9 in WO2015069922 Identifier 10, 12, 14, 16, 8 in WO2015069922;Identifier 2, 4, 6 in WO2015069922LHR VH Identifier 1, 2, 3, 4, 5, 6, 7, 8 - - in WO2016160618A3MUC16 VH Identifier 1, 21, 41, 81, 61 in VL Identifier 2, 22, 42, 62, 82WO2016149368; Identifier in WO2016149368 11, 4, 6, 8 inUS2013017115236324953654vlAttorney Docket No: 243734.000233PTK7 VH Identifier 1, 25, 49 in VL Identifier 20, 22, 24, 26, US20150315293; Identifier 28, 30, 32, 34, 36, 38, 40, 21, 23, 25, 27, 29, 31, 33, 42, 44, 46, 48, 50, 52, 54, 35, 37, 39, 41, 43, 45, 47, 56, 58, 60, 62, 64, 66, 68 in 49, 51, 53, 55, 57, 59, 61, WO2012112943A1;63, 65, 67, 69 in Identifier 15, 39, 63 in WO2012112943A1 US20150315293 ANG2 VH Identifier 1, 3 in VL Identifier 2, 4 in WO2015091655 WO2015091655 CD71 VH Identifier 1, 3, 325, 4, 5, 699 VL Identifier 2,327, 329, 331, in US20160355599 333, 335, 337, 6, 650, 652,654, 656, 658, 660, 670, 671, 672, 673, 7, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784785, 786, 787, 788, 8, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 810, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 879, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895,896, 897, 898, 899, 900,37324953654vlAttorney Docket No: 243734.000233901, 902, 903, 904, 905, 906, 907, 908 in US20160355599 TEM8 VH Identifier 1, 3, 5, 7 in VL Identifier 4, 6, 8 in US20160264662A1 US20160264662A1 IGFI VH Identifier 1, 3, 7 in VL Identifier 2, 4, 6, 8 in W02007118214; Identifier W020071182147 in WO2015073575FAP VH Identifier 1, 5 in VL Identifier 2, 6 in W02015118030; Identifier WO2015118030; Identifier 170, 172 in 171, 173 in W02016120216; Identifier W02016120216; Identifier 8 in US20160326265 Al 9 in US20160326265A1 Mesothelin VH Identifier 1, 6 in VL Identifier 3, 5WO2015188141; Identifier WO2015188141; Identifier 119, 50 in 1, 2, 3 in WO2013142034; US20160333114A1; Identifier 11, 15, 19, 23, 27 Identifier 5, 6 in in US20160229919A1; WO2013142034; Identifier Identifier 120, 47, 49 in 15, 2 in US9416190B2; US20160333114A1 Identifier 13, 17, 21, 25, 29,9 in US20160229919A1Frizzled VH Identifier 10 in VL Identifier 12, 14 in Receptor WO2010037041 WO2010037041 APCDD1 VH Identifier 10, 102, 106, 110, VL Identifier 136, 100, 104,114, 118, 122, 126, 130, 108, 112, 116, 12, 120, 134, 14, 6, 98 in 124, 128, 132, 16, 8 in WO2012019061 WO2012019061CSF VH Identifier 10, 102, 14, 18, 2, VL Identifier 12, 32, 44, 48, 6022, 26, 30, 34, 38, 46, 50, in US20050059113A1 54, 58, 6, 62, 66, 70, 74, 78,82, 86, 90, 94, 98 inUS20050059113A1TIGIT VH Identifier 10, 11, 12, 124, VL Identifier 130, 131, 132,125, 126, 127, 128, 129, 13, 133, 137, 139, 145, 146, 136, 138, 14, 143, 144, 149, 151, 152, 25, 26, 27, 28, 15, 150, 16, 17, 18, 19, 20, 29, 30, 50, 51, 52, 64, 95, 8 21, 22, 23, 24, 37, 38, 39, in US20160355589 40, 41, 42, 43, 44, 45, 46,47, 63, 94, 7, 9 inUS20160355589B7H3 VH Identifier 10, 11, 12, 13, 14, VL Identifier 1, 2, 3, 4, 5, 6, 7,15, 16, 9 in WO2016033225 8 in WO2016033225 IL4R VH Identifier 10, 11, 14, 15, 9 in VL Identifier 13, 7, 8 inWO2009121847 WO200912184738324953654vlAttorney Docket No: 243734.000233Lymphotoxin VH Identifier 10, 12, 14, 16, 2 in VL Identifier 1, 15, 4, 6, 8 in beta receptor W02004002431 W02004002431 LGR5 VH Identifier 10, 12, 16, 18, 20, VL Identifier 15, 19, 21, 23,22, 24, 26, 4 in 25, 3 in US20160102146 US20160102146CD 148 VH Identifier 10, 14, 18, 2, 22, VL Identifier 12, 16, 20, 24,26, 30, 6 in WO2005118643 28, 32, 4, 8 in WO2005118643 GPC3 VH Identifier 10, 14, 2, 3, 4, 5, VL Identifier 10, 14, 18, 22,6, 7, 8, 9 in 24, 26 in US9409994B2; US20160208015 Al; Identifier 16, 31 in Identifier 22 in US20160208015 Al;WO2016049459; Identifier Identifier 23 in12, 16, 20, 37, 8 in WO2016049459 US9409994B2CSPG4 VH Identifier 10, 16, 18, 4, 6, 8 VL Identifier 7 inin WO2016077638; WO2016164429; Identifier Identifier 8 in 12, 14 in WO2016077638 WO2016164429AGR2 VH Identifier 10, 18 in VL Identifier 11, 19 in WO2016040321 W02016040321 Tissue factor VH Identifier 10, 19, 23, 27, 29, VL Identifier 25, 31 in6 in W02004094475; US20040229301A1;Identifier 38 in Identifier 12, 21, 25, 31, 8 US20160333114A1 in W02004094475;Identifier 35, 37 in US20160333114A1 SEMAPHORIN VH Identifier 10, 25, 9 in VL Identifier 17, 18, 29 in 4D US20160115240A1 US20160115240 Al PDL1 VH Identifier 10, 32, 8 in VL Identifier 22, 26, 34, 42, US20160319022; Identifier 58, 66, 74, 82, 86 in 18, 30, 38, 46, 50, 54, 62, W02016061142; Identifier 70, 78 in W02016061142; 30, 8, 9 inIdentifier 29, 7 in US20150190506; Identifier US20150190506; Identifier 7, 9 in US20160319022; 16, 18, 197, 247, 248, 250, Identifier 17, 22, 24, 249, 251, 252, 253, 254, 255, 26, 28, 309, 311, 313, 320, 256, 257, 258, 259, 260, 30, 325, 34, 340, 357, 359, 36, 308, 310, 312, 319, 32, 324, 42, 44, 58, 60, 66, 68, 74, 339, 356, 38, 40, 46, 48, 50, 76, 8, 82, 84, 86, 88 in 52, 54, 6, 62, 70, 72, 78, 80, US2016010812391, 96 in US20160108123;Identifier 358, 56, 64 inUS20160108123HLAG VH Identifier 10, 8 in VL Identifier 18, 20 inWO2016160622 A2 WO2016160622 A239324953654vlAttorney Docket No: 243734.000233B7H4 VH Identifier 100, 101, 102, VL Identifier 104, 11, 126,103, 107, 108, 109, 110, 134, 138, 19, 27, 3, 35, 55, 111, 112, 113, 114, 12, 127, 93, 95, 97, 98, 145, 146, 130, 131, 132, 133, 137, 2, 147, 148 in20, 28, 36, 37, 38, 4, 56, 99, US20160159910; Identifier 144 in US20160159910; 29, 31, 33 inIdentifier 13, 15, 17 in W02016160620 W02016160620VEGFR2 VH Identifier 100, 101, 102, VL Identifier 107, 108, 109,103, 114, 115, 116, 117, 110, 111, 112, 113, 86, 87, 118, 119, 120, 121, 122, 88, 89, 90, 91, 92, 93, 94 in 123, 124, 95, 96, 97, 98, 99 WO2017004254 in WO2017004254CD73 VH Identifier 100, 103, 107, VL Identifier 12, 20, 44, 72,109, 112, 114, 116, 119, 76, 8, 84, 92 in121, 16, 32, 4, 52, 60, 68, US20160145350; Identifier 80, 88 in US20160145350; 22, 29, 37, 4 in Identifier 135, 40, 21, 3, 28, W02016055609A1;36 in W02016055609A1 Identifier 101, 102, 104,106, 110, 117, 118, 120, 122 in US20160145350 Claudin VH Identifier 101, 103, 105, VL Identifier 114, 116, 118,107, 109, 111, 113, 115, 120, 22, 25, 29, 33, 37, 41, 117, 119, 121, 122, 123, 45, 49, 53, 57, 61, 65, 69, 124, 125, 126, 127, 128, 73, 77 in129, 130, 131, 132, 133, WO2016073649A1 134, 135, 136, 137, 138,139, 140, 141, 142, 143,144, 145, 146, 147, 148,149, 150, 23, 27, 31, 35, 39,43, 47, 51, 55, 59, 63, 67,71, 75, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99 inWO2016073649A1CLL3 VH Identifier 101, 103, 105, VL Identifier 100, 102, 104,107, 109, 111, 113, 115, 106, 108, 110, 112, 114, 117, 119, 121, 123, 125, 116, 118, 120, 122, 124, 127, 129, 131, 133, 135, 126, 128, 130, 132, 134, 137, 139, 141, 145, 147, 136, 138, 140, 144, 146, 149, 151, 153, 155, 157, 148, 150, 152, 154, 156, 159, 161, 163, 165, 167, 158, 160, 162, 164, 166, 169, 171, 173, 175, 177, 170, 172, 174, 176, 178, 179, 181, 183, 185, 187, 180, 182, 184, 186, 190, 191, 193, 195, 197, 199, 192, 194, 196, 198, 20, 201, 203, 205, 207, 209, 21, 200, 202, 204, 206, 208,211, 213, 23, 25, 27, 29, 31, 210, 212, 22, 24, 26, 28,40324953654vlAttorney Docket No: 243734.00023333, 35, 37, 39, 41, 43, 45, 30, 32, 34, 36, 38, 40, 42, 47, 49, 51, 53, 55, 57, 59, 44, 46, 48, 50, 54, 56, 58, 61, 63, 65, 67, 69, 71, 73, 60, 62, 64, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87, 74, 76, 78, 80, 82, 84, 86, 89, 91, 93, 95, 97, 99 in 88, 90, 92, 94, 96, 98 in US20170000901 US20170000901 OX40 VH Identifier 101, 103, 105, VL Identifier 10, 45, 47, 49, 8107, 109, 111, 113, 115, in US8283450; Identifier 117, 119, 121, 123, 124, 11, 7 in US9428570;125, 17, 28, 318, 37, 48, 50, Identifier 116, 120, 122, 58, 66„ 74, 85, 93, 95, 97, 30, 38, 49, 57, 65, 73, 84, 99 in WO2016196228; 86, 94, 98 inIdentifier 31, 34, 36, 38, 40, WO2016196228; Identifier 42, 44, 46, 48, 50, 53, 54, 24, 26, 27, 28, 30, 60, 8, 55, 58, 59, 61 in 81, 82, 83, 84, 85, 86, 87, US20150190506; Identifier 88, 89 in US8748585;33, 35, 37, 39, 41, 43, 45, Identifier 30, 32 in47, 49, 51, 53, 55, 57, 59, US20160137740; Identifier 61, 63, 65, 67, 71 in 32, 35, 39, 41, 43, 45, 47, US20160137740; Identifier 49, 51, 52, 56, 57, 62 in 44, 46, 48, 7, 9 in US20150190506; Identifier US8283450; Identifier 9, 15 29, 37 in US20160137740 in US9428570; Identifier19, 21, 22, 23, 29, 58, 59, 7,77, 78, 79, 80 inUS8748585MUCIN 1 VH Identifier 101, 106, 109, VL Identifier 148, 158, 162,115, 119, 123, 127, 141, 15, 167, 170, 174, 184, 190, 23, 28, 33, 39, 42, 47, 5, 57, 193, 203, 208, 211, 220, 66, 70, 75, 80, 83, 87, 92 in 225, 229, 234, 242, 246, EP3049812A2 250, 255, 261, 270, 275,279, 283, 291, 297, 303, 308, 315, 319, 323, 333, 340 in EP3049812A2 MCSF VH Identifier 102, 10, 14, 18, 2, VL Identifier 8, 32, 52, 60, 28,22, 26, 30, 34, 38, 46, 50, 36, 4, 44, 48, 56, 62, 12, 54, 58, 6, 66, 70, 74, 78, 82, 16, 20, 24 in86, 90, 94, 98 in W02005030124 W02005030124LAG3 VH Identifier 102, 106, 110, VL Identifier 32, 36, 40, 44,113, 122, 18, 30, 66, 70, 74, 48, 52, 56, 60, 84, 88, 92, 78 in US20150259420; 96, 134, 34, 38, 42, 46, 50, Identifier 100, 104, 108, 28, 54, 58, 60, 86, 90, 94, 98 in 64, 68, 72, 76, 8, 80 in US20150259420; Identifier US20150259420; Identifier 2 in WO20150422461 in WO201504224641324953654vlAttorney Docket No: 243734.000233TIM3 VH Identifier 102, 112, 12, 2,22, 32, 42, 52, 62, 72, 82, 91in US20150086574;Identifier 82 inWO2013006490; Identifier13, 21, 29, 37, 45, 5, 53, 61,69, 77, 85, 93 inWO2016179319A1;Identifier 7 inWO2013006490; Identifier107, 117, 17, 27, 37, 47, 57,67, 7, 77, 87, 97 inUS20150086574; Identifier17, 25, 33, 41, 49, 57, 65,73, 81, 89, 9, 97 inWO2016179319A1CD2 VH Identifier 103, 117, 119 in VL Identifier 102, 116 in W02016122701 WO2016122701 CD52 VH Identifier 103, 136, 137 in VL Identifier 102, 138 in WO2010132659 WO2010132659 WT1 / HLA VH Identifier 104, 111, 128, 14, VL Identifier 106, 112, 130, Bispecific 32, 50, 68, 86 in 34, 52, 70, 88 in WO2015070061 W02015070061CD3 VH Identifier 108, 112, 115 in VL Identifier 104 in WO2016122701; Identifier W02016122701; Identifier 29 in WO2014144722 A2; 13 in WO2016126213A1 Identifier 12 inWO2016126213A1PG9 VH Identifier 11 in VL Identifier 10 in EP3074419A2 EP3074419A2V2 VH Identifier 11 in - - US20160194375A1HSP70 VH Identifier 11, 12 in VL Identifier 16, 17 in W02016120217 W02016120217 CD37 VH Identifier 11, 12, 18 in VL Identifier 14, 15 in US20170000900 US20170000900 CD123 VH Identifier 11, 13, 14, 21 in VL Identifier 9, 11, 18, 19, 20,WO2015140268A1; 21, 22, 23 inIdentifier 113, 115, 57, 59, WO2016120220; Identifier 63 in W02016120216; 12, 16, 18, 19, 22 in Identifier 12, 123, 24, 25, WO2015140268A1;26, 27, 28, 29, 30, 9 in Identifier 275, 276, 277, WO2016120220; Identifier 278, 307, 308, 309, 310 in 216, 217, 218, 219, 274 in WO2016028896; IdentifierWO2016028896 5 in US20160333108A1;42324953654vlAttorney Docket No: 243734.000233Identifier 114, 116, 58, 60, 64 in W02016120216 GD3 VH Identifier 11, 13, 15, 17 in VL Identifier 12, 14, 16, 18 in WO2016185035A1 WO2016185035A1 TAG72 VH Identifier 115 in VL Identifier 116 in US20160333114A1 US20160333114A1 MCAM VH Identifier 115, 116, 117, VL Identifier 109, 110, 111,118, 119, 157, 158, 159, 112, 121, 122, 123 in 160, 161, 178, 179 in US20150259419; Identifier US20150259419; Identifier 30, 40, 50, 60, 70, 71, 72 in 35, 45, 55, 65, 77, 89 in US20150239980 US20150239980; Identifier101, 102, 103, 104, 105,106, 107 inUS20150259419CA19.9 VH Identifier 117 in VL Identifier 118 in US20160333114A1 US20160333114A1 BMPR1A VH Identifier 12 in - - WO2011116212LGR4 VH Identifier 12, 13, 5, 9 in VL Identifier 10, 11, 6 in US20160046723 US20160046723 APRIL VH Identifier 12, 14, 16. 18. 3. VL Identifier 20, 22, 24, 26,32, 34, 36, 38, 40, 42, 44, 28, 30, 4, 50 in46, 48, 52 in US20160264674 US20160264674FcRL5 VH Identifier 12, 16, 20, 24, 28, VL Identifier 11, 15, 19, 23, (FcReceptorLike 32, 36, 4, 40, 44, 48, 8, 915, 27, 3, 31, 35, 39, 43, 47, 7, 5) 919 in W02016090337 917, 921 W02016090337 CLDN18.2 VH Identifier 12, 2 in VL Identifier 13, 3 in US20160347815 Al US20160347815A1 ROR1 VH Identifier 12, 20, 28, 36, 44, VL Identifier 16, 24, 32, 40,60, 68 WO2016016343 Al; 56, 64, 72, 36, 62, 23, 49, Identifier 57, 19, 31, 45, 53, 58 WO2016016343A1; 71 in WO2016016344A1; Identifier 86, 88, 90 in Identifier 85, 87, 89 in W02016120216; Identifier W02016120216; Identifier 126, 127, 234, 235, 236, 122, 125, 175, 176, 179, 237, 238, 240, 241, 242, 180, 181, 182, 183, 184, 243, 244, 245, 246, 247, 185, 186, 187, 188, 189, 248 in US20160208018A1; 190, 191, 192, 193, 194, Identifier 56 in195, 196, 197, 197, 199, EP3083671 Al; Identifier 200, 201, 202, 203, 204, 103, 111, 127, 135, 143, 205, 206, 207, 208, 209 in 15, 151, 159, 167, 175, US20160208018A1; 183, 191, 199, 207, 215, Identifier 55 in 223, 23, 231, 239, 247,EP3083671A1; Identifier 255, 263, 271, 279, 287,43324953654vlAttorney Docket No: 243734.000233104, 112, 120, 128, 152, 16, 295, 303, 31, 311, 319, 160, 168, 176, 184, 192, 327, 335, 343, 351, 359, 200, 208, 216, 224, 232, 24, 39, 47, 55, 63, 7, 71, 79, 240, 248, 256, 264, 272, 87, 95 in280, 288, 296, 304, 312, 32, WO2016187216A1 320, 336, 344, 352, 360, 40,48, 56, 64, 72, 8, 80, 88 inWO2016187216A1B7H1 VH Identifier 12, 32, 42, 52, 72, VL Identifier 17, 37, 47, 57, 7,2, 62 in US20130034559 77, 27, 67 in US20130034559 CD32B VH Identifier 127 in VL Identifier 126 in W02016122701 W02016122701 CD64 VH Identifier 129 in VL Identifier 128 in W02016122701 W02016122701 PG16 VH Identifier 13 in VL Identifier 12 in EP3074419A2 EP3074419A2MPER VH Identifier 13 in VL Identifier 12 in US20160194375A1 US20160194375A1 CD105 VH Identifier 13, 14, 16 in VL Identifier 1, 17, 20, 22, 23WO2014039682 in WO2014039682 CLL1 VH Identifier 13, 14, 15, 17, 19, VL Identifier 16, 18, 20, 22,21, 23, 25, 27, 29, 31, 33, 35 24, 26, 28, 30, 32, 24, 36 in in W02016120219; WO2016120219; Identifier Identifier 195, 65, 66, 67, 196, 78, 79, 80, 81, 82, 83, 68, 69, 70, 71, 72, 73, 74, 84, 85, 86, 87, 88, 89, 90 in 75, 76, 77 in WO2016014535; Identifier WO2016014535; Identifier 30, 32, 35, 37, 39, 41 in 31, 33, 34, 36, 38, 40, 42, 46 US20160075787; Identifier in US20160075787; 152, 104, 106, 108, 110, Identifier 150, 103, 105, 112, 114, 116, 118 in 107, 109, 111, 113, 115, 117 WO2016179319A1 in WO2016179319A1GPRC5D VH Identifier 13, 17, 21, 25, 29, VL Identifier 10, 14, 18, 2, 22,314, 326, 33, 338, 350, 362, 26, 30, 303, 315, 327, 339, 37, 374, 386, 41, 45, 49, 5, 34, 351, 363, 375, 38, 387, 53, 57, 61, 65, 69, 73, 77, 42, 46, 50, 54, 58, 6, 62, 81, 85, 89, 93, 1, 9 in 66, 70, 74, 78, 82, 86, 94 in W02016090312 W02016090312 EFNA4 VH Identifier 13, 39 in - - US20150125472CD79 VH Identifier 131 in VL Identifier 130 in W02016122701 WO2016122701 FGFR3 VH Identifier 132, 134, 136 in VL Identifier 133, 135, 137,US9499623 139 in US949962344324953654vlAttorney Docket No: 243734.000233TCR VH Identifier 133 in VL Identifier 132 in W02016122701 W02016122701MN VH Identifier 133, 135, 137, VL Identifier 134, 136, 138,139, 141, 143, 145, 147, 140, 142, 144, 146, 148, 149, 151 in W02007070538 150, 152 in W02007070538IL33 VH Identifier 134, 136, 138, VL Identifier 135, 137, 139,185, 187, 189, 216, 218, 184, 188, 217, 219, 237, 220, 221,236, 246, 282, 284, 247, 283, 285, 287, 37, 39, 286, 36, 38, 40, 84, 86, 88 41, 87 in US20160168242 in US20160168242NKG2D VH Identifier 135, 137 in VL Identifier 134, 136 in W02016122701 W02016122701 CD30 VH Identifier 14, 16 in VL Identifier 13, 15 in WO2016134284 WO2016134284 EGFR VH Identifier 14, 50, 9 in VL Identifier 15 in WO2015143382; Identifier WO2015143382; Identifier 12, 14, 15, 21 in 14 in WO2014143765; US20100008978A1; Identifier 4051, 4052, Identifier 2123 in 4053, 4054, 4055, 4056, WO2018231759 4057, 4058, 4059, 4060,4061, 4062, 4063, 4064, 4065, 4066, 4067, 4068, 4069, 4070, 4071, 4072, 4073, 4074, 4075, 4076, 4077, 4078, 4079, 4080, 4081, 4082, 4083, 4084, 4085, 4086, 4087, 4088, 4089. 4090, 4091, 4092, 4093, 4094, 4095, 4096, 4097, 4098, 4099, 4100, 4101,4102, 4103, 4104, 4105, 4106, 4107, 4108, 4109, 4110, 4111, 4112, 4113, 4114, 4115, 4116, 4117, 4118, 4119, 4120, 4121, 4122, 4123, 4124, 4125, 4126, 4127, 4128, 4129, 4130, 4131, 4132, 4133, 4134, 4135, 4136, 4137, 4138, 4139, 4140, 4141, 4142, 4143, 4144, 4145, 4146, 4147, 4148, 4149, 4150, 4151, 4152, 4153, 4154, 4155, 4156,4157, 4158, 4159, 4160,45324953654vlAttorney Docket No: 243734.0002334161, 4162, 4163, 4164, 4165, 4166, 4167, 4168, 4169, 4170, 4171, 4172, 4173, 4174, 4175, 4176, 4177, 4178, 4179, 4180, 4181, 4182, 4183, 4184, 4185, 4186, 4187, 4188, 4189, 4190, 4191, 4192, 4193, 4194, 4195, 4196, 4197, 4198, 4199, 4200, 4201, 4202, 4203, 4204, 4205,, 4206, 4207, 4208, 4209, 4210, 4211, 4212, 4213, 4214, 4215, 4216, 4217, 4218, 4219, 4220, 4221, 4222, 4223, 4224, 4225, 4226, 4227, 4228, 4229, 4230, 4231, 4232, 4233, 4234, 4235, 4236, 4237, 4238, 4239, 4240, 4241, 4242, 4243, 4244, 4245, 4246, 4247, 4248, 4249, 4250, 4251, 4252, 4253, 4254, 4255, 4256, 4257, 4258, 4259, 4260, 4261, 4262, 4263, 4264, 4265, 4266, 4267, 4268, 4269, 4270, 4271, 4272, 4273, 4274, 4275, 4276, 4277, 4278, 4279, 42870, 4281, 4282, 42834284, 4285, 4286, 4287, 4288, 4289, 4290, 4291, 4292, 4293, 4294, 4295, 4296, 4297, 4298, 4299, 4300, 4301, 4302, 4303, 4304, 4305, 4306, 4307, 4308, 4309, 4310, 4311, 4312, 4313, 4314, 4315, 4316, 4317, 4318, 4319, 4320, 4321, 4322, 4323, 4324, 4325, 4326, 4327, 4328, 4329, 4330, 4331, 4332, 4333, 4334, 4335, 4336, 4337, 4338, 4339, 4340,4341, 4342, 4343, 4344,46324953654vlAttorney Docket No: 243734.0002334345, 4346, 4347, 4348, 4349, 4350, 4351, 4352, 4353, 4354, 4355, 4356, 4357, 4358, 4359, 4360, 4361, 4362, 4363, 4364, 4365, 4366, 4367, 4368, 4369, 4370, 4371, 4372, 4373, 4374, 4375, 4376, 4377, 4378, 4379, 4380, 4381, 4382, 4383, 4384, 4385, 4386, 4387, 4388, 4389, 4390, 4391, 4392, 4393, 4394, 4395, 4396, 4397, 4398, 4399, 4400, 4401, 4402, 4403, 4404, 4405, 4406, 4407, 4408, 4409, 4410, 4411, 4412, 4413, 4414, 4415, 4416, 4417, 4418, 4419, 4420, 4421, 4422, 4423, 4424, 4425, 4426, 4427, 4428, 4429, 4430, 4431, 4432, 4433, 4434, 4435, 4436, 4437, 4438, 4439, 4440, 4441, 4442, 4443, 4444, 4445, 4446, 4447, 4448, 4449, 4450, 4451, 4452, 4453, 4454, 4455, 4456, 4457, 4458, 4459, 4460, 4461, 4462, 4463, 4464, 4465, 4466, 4467, 4468, 4469, 4470, 4471, 4472, 4473, 4474, 4475, 4476, 4477, 4478, 4479, 4480, 4481, 4482, 4483, 4484, 4485, 4486, 4487, 4488, 4489, 4490, 4491, 4492, 4493, 4494, 4495, 4496, 4497, 4498, 4499, 4500, 4501, 4502, 4503, 4504, 4505, 4506, 4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4516, 4517, 4518, 4519, 4520, 4521, 4522, 4523, 4524,4525, 4526, 4527, 4528,47324953654vlAttorney Docket No: 243734.0002334529, 4530, 4531, 4532, 4533, 4534, 4535, 4536, 4537, 4538, 4539, 4540, 4541, 4542, 4543, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4559, 4560, 4561, 4562, 4563, 4564, 4565, 4566, 4567, 4568, 4569, 4570, 4571, 4572, 4573, 4574, 4575, 4576, 4577, 4578, 4579, 4580, 4581, 4582, 4583, 4584, 4585, 4586, 4587, 4588, 4589, 4590, 4591, 4592, 4593, 4594, 4595, 4596, 4597, 4598, 4599, 4600, 4601, 4602, 4603, 4604, 4605, 4606, 4607, 4608, 4609, 4610, 4611, 4612, 4613, 4614, 4615, 4616, 4617, 4618, 4619, 4620, 4621, 4622, 4623, 4624, 4625, 4626, 4627, 4629, 4629, 4630, 4631, 4632, 4633, 4634, 4635, 4636, 4637, 4638, 4639 in WO2018231759Her2 VH Identifier 141, 262, 264, VL Identifier 140 in266, 268, 270 in WO2016054555A2;WO2016168773A3; Identifier 261, 263, 265, Identifier 11 in US9518118; 267, 269 inIdentifier 62 in WO2016168773A3;US20160333114A1; Identifier 10, 18, 23 in Identifier 19, 24 in US9518118; Identifiers in US9518118 WO2016168769A1;Identifier 59, 61 in US20160333114A1 EFNA VH Identifier 149, 153, 157, 161 VL Identifier 151, 155, 159, in US20150125472 163 in WO2012118547;Identifier 27, 53 in US20150125472 PGT1 VH Identifier 15 in EP307441 - - Factor XII VH Identifier 15 in VL Identifier 17 inWO2014089493 WO201408949348324953654vlAttorney Docket No: 243734.000233E7MC VH Identifier 15, 16, 17, 18, 19, VL Identifier 238, 239, 240,20, 21, 22, 23, 233, 234, 241, 242, 243, 36, 37, 38, 235, 236, 237, 24, 25, 26, 39, 41, 42, 43, 44, 45, 46, 27, 28, 29, 30, 31, 32, 33, 47, 48, 49, 50, 51, 52, 53, 34, 35 in 54, 55, 56 in WO2016182957A1 WO2016182957A1 ICOS VH Identifier 15, 16, 19, 23, 7 in VL Identifier 17, 18, 20, 24, 8US20160215059 in US20160215059 CD76b VH Identifier 15, 17, 19, 23, 27, VL Identifier 16, 18, 22, 38,29, 37, 57, 59, 61 in 58, 60, 62 in US20160159906 US20160159906 MUC1C ECD VH Identifier 15, 19, 23, 60, 64, VL Identifier 17, 21, 25, 62,68, 72 in 66, 70, 75 in US20160340442A1 US20160340442A1 CD4BS VH Identifier 15, 3 in VL Identifier 14, 2 in US20160194375A1 US20160194375A1 CD7 VH Identifier 16, 20 in VL Identifier 17, 21 in WO2016126213A1 WO2016126213A1 PGT2 VH Identifier 17 in VL Identifier 16 in EP3074419A2 EP3074419A2OTK3 VH Identifier 17 in VL Identifier 18 in WO2015158868 WO2015158868GD2 VH Identifier 17 in VL Identifier 10, 2, 5, 7, 9 in WO2016134284; Identifier US20130216528; Identifier 1 in US20130216528; 11, 12 in WO2015132604; Identifier 10, 9 in Identifier 18 in WO2015132604; Identifier WO20161342843, 4, 6, 8 inUS20130216528Trophoblast VH Identifier 17, 13, 15, 11 in VL Identifier 18, 12, 14, 16 in Glycoprotein WO2016034666A1 WO2016034666A1 5T4AMC VH Identifier 17, 18, 19, 20, 21, VL Identifier 27, 28, 29, 31,22, 23, 24, 25, 26 in 32, 33, 34, 35, 36 in W02016161390 W02016161390 Factor D VH Identifier 17, 20, 27, 29, 30, VL Identifier 16, 18, 19, 26, 331, 32, 33, 4 in in US20160017052 US20160017052CD276 VH Identifier 17, 26, 7 in VL Identifier 18, 27 in US20160053017 US20160053017 B7H3 (CD276) VH Identifier 17, 26, 7 in VL Identifier 18, 27, 8 in WO2016044383 WO2016044383RAS VH Identifier 17, 47, 57, 67, 7, VL Identifier 19, 49, 59, 69,77 in WO2016154047 79, 9 in WO2016154047 DR5 VH Identifier 18, 82, 90, 98, 8 in VL Identifier 13, 23, 25, 27, 3,W02016122701 78, 86, 94, 29 in49324953654vlAttorney Docket No: 243734.000233W02016122701; Identifier 62 in W02016122701; Identifier 54 in WO2016122701; Identifier 70 in W02016122701 PGT3 VH Identifier 19 in VL Identifier 18 in EP3074419A2 EP3074419A2PD1 VH Identifier 19 in VL Identifier 2, 39, 7, 8, 9 in US20150290316; Identifier US 20160159905;25, 26, 27, 28, 29 in Identifier 21 in US20130291136; Identifier US20150290316; Identifier 29, 3, 38 in US 30, 31, 32, 33 in 20160159905; Identifier 38, US20130291136; Identifier 50 in WO2015112900; 42, 46, 54 inIdentifier 4, 4, 6 in US WO2015112900; Identifier 20160159905; Identifier 82, 58, 62, 66, 70, 74, 78 in 86 in WO2015112900; WO2015112900; Identifier Identifier 17 in 18 in WO2014055648; WO2014055648; Identifier Identifier 5 in4 in WO2016040892; WO2016040892; Identifier Identifier 12 in 13 in US20150190506 US20150190506NYBR1 VH Identifier 19 in VL Identifier 18 in US20160333422A1 US20160333422A1 CD28 VH Identifier 19 in VL Identifier 20 in WO2015158868 WO2015158868Tn Glycopeptide VH Identifier 19, 20 in - - W02015120180humanERBB3 VH Identifier 19, 29, 38, 45, 55, VL Identifier 10, 20, 30, 39,61, 9 in WO2013052745 46, 56, 62 in WO2013052745 Olfml3 VH Identifier 19, 3 in VL Identifier 20, 4 in WO2015054441A1 WO2015054441A1 RHAMM VH Identifier 2 in VL Identifier 4 in antagonist body W02000029447 W02000029447 CD38 VH Identifier 2 in VL Identifier 1, 11 in W02009080830; Identifier W0200908083010 in WO2015121454PD1 (Nivolumab) VH Identifier 2 in VL Identifier 11 in WO2016040892; Identifier US2015019050610 in US20150190506PDK1 VH Identifier 2 in VL Identifier 9 in WO2016090365 WO2016090365IL21 VH Identifier 2, 3 in - -US2016014533250324953654vlAttorney Docket No: 243734.000233ERBB2 VH Identifier 2, 4 in VL Identifier 1 inUS20110129464; Identifier US20110129464; Identifier 10, 2, 26, 30, 38, 4, 40, 42, 12, 16, 20, 24, 32, 36, 44, 52, 54, 56, 57, 58, 6 in 50, 51, 53, 8 inUS20130089544; Identifier US20130089544; Identifier 8 in US20130266564; 7 in US20130266564;Identifier 1 in Identifier 3 inUS20150104443 US201101294645T4 VH Identifier 2, 4 in VL Identifier 1, 3 in WO2016022939 WO2016022939 GPDL1 VH Identifier 20 in - - US20160108123GM2 VH Identifier 20, 22, 23, 26, 27, VL Identifier 21, 24, 25, 31,28, 29, 30 in 32, 33, 34, 35 in US20090028877 US20090028877 EphA2 receptor VH Identifier 20, 22, 24, 32, 34, VL Identifier 26, 28, 30, 47,36, 37, 38, 40, 42, 43, 45, 48, 49, 50, 52, 78, 80 in 74, 76 in US20150274824 US20150274824KDR VH Identifier 20, 24, 26, 29, 31, VL Identifier 22 in33 in W02003075840 W02003075840 PGT4 VH Identifier 21 in VL Identifier 20 in EP3074419A2 EP3074419A2CD324 VH Identifier 21, 23, 25, 27, 29, VL Identifier 20, 22, 24, 26,31, 33, 35, 37, 39, 41, 43, 28, 30, 32, 34, 36, 38, 40, 45, 47, 49, 51, 53, 55, 57, 42, 44, 46, 48, 50, 52, 54, 59, 61, 63, 65, 67, 69, 71 in 56, 58, 60, 62, 64, 66, 68, US9534058 70 in US9534058 AXL VH Identifier 21, 3, 45 in VL Identifier 22, 4 in W02016097370 WO2016097370 collagen VH Identifier 21, 4, 15, 17, 18, VL Identifier 11, 12, 14, 23,19, 20, 5, 6, 7, 1, 2, 3 in 25, 26, 27, 8, 9 in W02007024921 W02007024921 EGFR VH Identifier 2124 in VL 4640, 4641, 4642, 4643, (EGFRvIII) WO2018231759; Identifier 4644, 4645, 4646, 4647,13 in W02016016341; 4648, 4649, 4650, 4651, Identifier 24 in 4652, 4653, 4654, 4655, WO2016168773 A3; 4656, 4657, 4658 in Identifier 34 in WO2018231759; Identifier US20160304615; Identifier 14 in W02016016341; 2 in US20160200819A1; Identifier 23 in Identifier 91, 93 in WO2016168773 A3;W02016120216 Identifier 42 inUS20160304615; Identifier 1 in US20160200819A1; Identifier 92, 94 inW0201612021651324953654vlAttorney Docket No: 243734.000233IL3 alpha VH Identifier 22 in VL Identifier 27, 37 in WO2008127735 WO2008127735CS1 VH Identifier 22 in VL Identifier 104, 106, 108 in WO2016168773 A3; 103, WO2016120216; Identifier 105, 107, 109 in 14, 16, 18, 20, 22 in W02016120216; Identifier WO2015166056A1;13, 15, 17, 19 in Identifier 39, 41, 43, 45, 47 WO2015166056A1; in WO2015121454;Identifier 38, 40, 42, 44, 46 Identifier 110, 112 in in WO2015121454; W02016120216 Identifier 26 inUS20160075784A1KMA VH Identifier 22 in VL Identifier 2, 21 in WO2016172703 A2 WO2016172703 A2 PGT5 VH Identifier 23 in VL Identifier 22 in EP3074419A2 EP3074419A2CD45 VH Identifier 24 in VL Identifier 25 in WO2016126213A1 WO2016126213A1 leukocytegenA2 VH Identifier 25 in - - WO2010065962 A2CD16 VH Identifier 25 in VL Identifier 26 in WO2015158868 WO2015158868BCM A VH Identifier 26 in VL Identifier 25 in WO2016168773 A3; WO2016168773 A3;Identifier 142, 148, 154, Identifier 42 in160, 166, 172, 178, 184, WO2016097231; Identifier 190, 196, 202, 208, 214, 143, 149, 155, 161, 167, 220, 226, 232, 238, 244, 173, 179, 185, 191, 197, 250, 256, 262, 268, 274, 203, 209, 215, 221, 227, 280, 286, 292, 298, 304, 233, 239, 245, 251, 257, 310, 316, 322, 328, 334, 263, 269, 275, 281, 287, 340, 346, 352 in 293, 299, 305, 311, 317, WO2016168595A1; 323, 329, 335, 341, 347, Identifier 8 in 353 in WO2016168595 Al; WO2016094304 A3; Identifier 192, 193, 194, Identifier 171, 172, 173, 195, 196, 197, 198, 199, 174, 175, 176, 177, 178, 200, 201, 204, 205, 207, 179, 180, 181, 182, 183, 208, 211, 259, 260, 84, 85, 184, 185, 186, 187, 190, 86, 87, 88, 89, 90, 91, 92, 255, 257, 258, 69, 70, 71, 93, 94, 95, 96, 97, 98 in 72, 73, 74, 75, 76, 77, 78, WO2016014565; Identifier 79, 80, 81 WO2016014565; 53 in WO2016187349A1; Identifier 38 in Identifier 7 in EP3057994A1; Identifier 55 WO2016094304 A3; in WO2016187349A1; Identifier 10, 14, 18, 2, 22,Identifier 1, 13, 17, 21, 25, 26, 30, 34, 38, 42, 46, 50,52324953654vlAttorney Docket No: 243734.00023329, 33, 37, 41, 45, 49, 5, 53, 54, 58, 6, 62, 66 in57, 61, 65, 9 in WO2016090320; Identifier WO2016090320; Identifier 100, 102, 175, 96, 98 in 101, 743, 174, 758, 95, 759, WO2016120216; Identifier 97, 760, 99 in 12, 14, 16, 18 in W02016120216; Identifier WO2015158671A1;11, 741, 17 in Identifier 7, 8, 9 in WO2015158671A1; WO2016014789; Identifier Identifier 10, 11, 12, 13, 14 14 in WO2016168766A1 in WO2016014789;Identifier 15 inWO2016168766A1humanCD79b VH Identifier 27 in VL Identifier 28, 30 in WO2016112870 WO2016112870 humanCD79b VH 2304Identifier 29 inWO2016112870B2MG VH Identifier 28 in VL Identifier 29 in WO2016126213A1 WO2016126213A1 CD19H 803 VH Identifier 28, 29, 32, 33, 34,35 in WO2016168773 A3;Identifier 51 inWO2016187349A1;Identifier 20 inUS20160039942; Identifier1 in WO2014184143;Identifier 5 inUS20160145337A1;Identifier 166, 167, 168,172, 176, 177, 181, 183,184, 185, 62 inUS20160152723; Identifier15 US20160319020;Identifier 17, 33, 34, 35 inEP3057994A1; Identifier 62in WO2016097231;Identifier 12 inWO2016134284; Identifier111, 113 inUS20160333114A1NOTCH 2 / 3 VH Identifier 29 in VL Identifier 31 in WO2013074596 WO2013074596KIR (Lirilumab) VH Identifier 3 in VL Identifier 5 inUS20150290316; 2371 US20150290316; Identifier Identifier 1 in 2 in WO2014055648WO201405564853324953654vlAttorney Docket No: 243734.000233CD22 VH Identifier 3 in VL Identifier 17, 8, 14, 15 in WO2013059593; Identifier US20150239974; Identifier 10, 11, 12, 7, 9, 8 in 7 in US20150299317;US20150299317; Identifier Identifier 681 in201 in WO2016164731; WO2016164731; Identifier CD22 VH 869 Identifier 682 in WO2016164731; 671 in WO2016164731; Identifier 683, 2020 in Identifier 672 in WO2016164731; Identifier WO2016164731; Identifier 684 in WO2016164731; 673 in WO2016164731; Identifier 685 in Identifier 676 in WO2016164731; Identifier WO2016164731; Identifier 686 in WO2016164731; 678 in WO2016164731; Identifier 687 in Identifier 679 in WO2016164731; Identifier WO2016164731; Identifier 688 in WO2016164731; 680 in WO2016164731; Identifier 690 in Identifier 700 in WO2016164731; Identifier WO2016164731; Identifier 740 in WO2016164731; 701 in WO2016164731; Identifier 741 in Identifier 702 in WO2016164731; Identifier WO2016164731; Identifier 742 in WO2016164731; 703 in WO2016164731; Identifier 743 in Identifier 704 in WO2016164731; Identifier WO2016164731; Identifier 744 in WO2016164731; 705 in WO2016164731; Identifier 745 in Identifier 706 in WO2016164731; Identifier WO2016164731; Identifier 746 in WO2016164731; 707 in WO2016164731; Identifier 747 in Identifier 708 in WO2016164731; Identifier WO2016164731, Identifier 748 in WO2016164731; 709 in WO2016164731; Identifier 749 in Identifier 711 in WO2016164731; Identifier WO2016164731; Identifier 750 in WO2016164731; 712 in WO2016164731; Identifier 752 in Identifier 713 in WO2016164731; Identifier WO2016164731; Identifier 753 in WO2016164731; 714 in W 02016164731; Identifier 754 in Identifier 715 in WO2016164731; Identifier WO2016164731; Identifier 755 in WO2016164731; 716 in WO2016164731; Identifier 756 in Identifier 717 in WO2016164731; Identifier WO2016164731; Identifier 757 in WO2016164731; 718 in WO2016164731; Identifier 758 in Identifier 719 in WO2016164731; Identifier WO2016164731; Identifier 759 in WO2016164731;720 in WO2016164731; Identifier 760 in54324953654vlAttorney Docket No: 243734.000233Identifier 721 in WO2016164731; Identifier WO2016164731; Identifier 761 in WO2016164731; 722 in WO2016164731; Identifier 762 in Identifier 723 in WO2016164731; Identifier WO2016164731; Identifier 763 in WO2016164731; 724 in WO2016164731; Identifier 764 in Identifier 725 in WO2016164731; Identifier WO2016164731; Identifier 765 in WO2016164731; 726 in WO2016164731; Identifier 766 in Identifier 727 in WO2016164731; Identifier WO2016164731; Identifier 767 in WO2016164731; 728 in WO2016164731; Identifier 768 in Identifier 729 in WO2016164731; Identifier WO2016164731; Identifier 769 in WO2016164731; 730 in WO2016164731; Identifier 770 in Identifier 731 in WO2016164731; Identifier WO2016164731; Identifier 771 in WO2016164731; 732 in W 02016164731; Identifier 772 in Identifier 733 in WO2016164731; Identifier WO2016164731; Identifier 773 in WO2016164731; 734 in WO2016164731; Identifier 774 in Identifier 735 in WO2016164731; Identifier WO2016164731; Identifier 775 in WO2016164731; 736 in WO2016164731; Identifier 776 in Identifier 737 in WO2016164731; Identifier WO2016164731; Identifier 777 in WO2016164731; 738 in WO2016164731 Identifier 124 in W02016122701 CTLA4 VH Identifiers, 31, 32, 33, 34, VL Identifier 36, 37, 38, 39,35, 41, 42, 43, 44, 45, 7 in 40, 46, 47, 48, 49, 50, 8, 4 US20140105914; Identifier in US20140105914;4 in US8697845; Identifier Identifier 2 in US8697845; 19 in US20150283234; Identifier 20 in Identifier 17 in US20150283234; Identifier WO2014066532 18 in WO2014066532 Integrin VH Identifier 3, 4, 5 in US VL Identifier 10, 11, 8, 9 in US 20140161794 20140161794IL13 VH Identifier 302 in VL Identifier 303 in US20160168242 US20160168242 Human collagen VH Identifier 31 in VL Identifier 32 inVII WO2016112870 WO2016112870 NKG2A VH Identifier 32 in VL Identifier 33 in WO2016126213A1; WO2016126213A1;Identifier 2, 3, 4, 5, 6 in Identifier 7 inWO2016041947 WO201604194755324953654vlAttorney Docket No: 243734.000233Campath 1 VH Identifier 34 in VL Identifier 31, 33 in US20160333114A1 US20160333114A1 KIR2DL 1 VH Identifier 36 in VL Identifier 37 in WO2016126213A1 WO2016126213A1 KIR2DL 2 / 3 VH Identifier 36 in VL Identifier 37 in WO2016126213A1 WO2016126213A1 VISTA VH Identifier 37, 38, 39, 40 in VL Identifier 41, 42, 43, 44, 45WO2015097536 in WO2015097536 CD46 VH Identifier 39, 47, 59, 15, 19, VL Identifier 41, 61, 21, 25,23, 27, 31, 35, 43, 51, 55, 29, 33, 37, 45, 49, 53, 57, 63, 67, 71, 75, 79, 83, 69, 65, 69, 73, 77, 81, 85, 17, 71, 83 in WO2012031273; 73, 77 in WO2012031273; Identifier 1, 10, 11, 12, 13, Identifier 23, 24, 25, 26, 14, 15, 16, 17, 3, 5, 6, 7, 9, 27, 28, 29, 30, 31, 32, 33, 18, 19, 20, 21 in 34, 35, 36, 37, 38, 39, 40, WO2016040683 41, 42 in WO2016040683 RHAMM VH Identifier 4 in - - US20020127227A1VEGF VH Identifier 4, 8, in VL Identifier 2, 6 in W02000034337; Identifier W02000034337; Identifier 12, 20, 4, 44 in 9 in US20030175276A1; W02006012688A1; Identifier 11, 19, 27, 28, 3, Identifier 7 in 43 in W02006012688 Al; US20030175276A1; Identifier 160, 161, 162, Identifier 152, 153, 154, 163, 164, 165, 166, 167 in 155, 156, 157, 158, 159 in US20160090427 US20160090427Endoglin VH Identifier 41, 42, 43, 71, 73, VL Identifier 103, 88, 89, 90,75, 88, 89, 90, 91, 92 in 91, 92, 93, 94, 95, 96, 97, US20160009811 102, 100 inWO2011041441; Identifier 100, 102, 103, 3, 4, 5, 70, 72, 74, 93, 94, 95, 96, 97 in US20160009811PRP VH Identifier 42 in VL Identifier 39, 41 in US20160333114A1 US20160333114A1 Lysyloxidase- VH Identifier 42, 44 in VL Identifier 43, 45 in like 2 W02011097513 WO2011097513 PSMA VH Identifier 43 in VL Identifier 44 in WO2016097231 WO2016097231; Identifier 44 in WO2016097231 PDL2 VH Identifier 43, 44, 56, 46 in VL Identifier 47, 48, 49, 50, 51US20130291136 in US20130291136 Daclizumab VH Identifier 44, 46 in VL Identifier 43, 45 inUS20160333114A1 US20160333114A156324953654vlAttorney Docket No: 243734.000233CD20 VH Identifier 45 in VL Identifier 46 in WO2016097231; Identifier WO2016097231; Identifier 11, 13, 14, 15, 16, 17, 18, 10, 12, 8 in19, 20, 21, 22, 23, 24, 25, WO2017004091; Identifier 26, 27, 28, 29, 30, 31, 32, 51 inUS20160333114Al 33, 7, 9 in WO2017004091;Identifier 26 inUS20170000900; Identifier54 inUS20160333114Al;Identifier 25 inUS20170000900; Identifier24 in US20170000900;Identifier 23 inUS20170000900Klon43 VH Identifier 47 in VL Identifier 48 in WO2016097231 WO2016097231 MUC1 VH Identifier 5 in VL Identifier 7 inUS20160130357; Identifier US20160130357; Identifier 2, 14 in WO2013023162; 16, 7 in WO2013023162; Identifier 15, 19, 23, 60, 64, Identifier 17, 21, 25, 62, 68 in WO2015116753 66, 70 in WO2015116753 CD4i VH Identifier 5 in VL Identifier 4 in US20160194375A1 US20160194375A1 BAT1 VH Identifier 5, 6, 7, 8, 9 in VL Identifier 1, 2, 3, 4 in WO2013014668 WO2013014668 PRAME VH Identifier 50, 52, 54, 56, 58, VL Identifier 49, 51, 53, 55,60, 62 in 57, 59, 61 in WO2016191246A2 WO2016191246A2 CD19 VH Identifier 53, 55 in VL Identifier 27, 31 in W02016120216 WO2016168773 A3;Identifier 49 in WO2016187349A1;Identifier 11 in WO2016134284; Identifier 194 in US20140134142A1; Identifier 54, 56 in W02016120216; Identifier 13, 14, 15, 16, 17, 186, 187, 188, 189, 192, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 64, 66, 67, 68, 69, 70, 71, 91 US20160152723; Identifier 22 in US20160039942; 3361 Identifier 63 inWO2016097231; Identifier57324953654vlAttorney Docket No: 243734.0002333 in US20160145337A1; Identifier 112 in US20160333114A1;Identifier 114 in US20160333114A1;Identifier 13, 6US20160319020 Notum VH Identifier 56, 331 in VL Identifier 332, 58 in WO2012027723 WO2012027723 NOTCH 1 VH Identifier 58 in VL Identifier 16, 20 in US20160333114A1; WO2013074596; Identifier Identifier 12 in 55, 57 in WO2013074596 US20160333114A1 Osteonectin VH Identifier 58 in VL Identifier 59 in WO2016112870 WO2016112870 CD74 VH Identifier 6 in VL Identifier 25, 29, 31, 35 in US20100284906A1; US20130171064; Identifier Identifier 10, 11, 9 in 12, 13, 14, 11, 4 in US20040115193A1; US20040115193 Al Identifier 23, 27, 30, 33 inUS20130171064CD33 VH Identifier 65, 67, 69, 71, 77, VL Identifier 12, 14, 16, 18 in 79, 81, 83, 84 in WO2015150526A2;W02016120216; Identifier Identifier 66, 68, 70, 72, 11, 13, 15; 17 in 78, 80, 82 in WO2015150526A2; WO2016120216; Identifier Identifier 57, 58, 59, 60, 61, 66, 67, 68, 69, 70, 71, 72, 62, 63, 64, 65 in 73, 74 in WO2016014576 WO2016014576Rituximab VH Identifier 66 in VL Identifier 63, 65 in US20160333114A1 US20160333114A1 GAH VH Identifier 7 in VL Identifier 8 in US20060057147A1 US20060057147A1 Glyco epitope VH Identifier 7 in VL Identifier 10 inand ErbBB I W02012007167A1 W02012007167A1 SpecificCD3s VH Identifier 7 in VL Identifier 8 inWO2014144722 A2 WO2014144722 A2 IGFR1 VH Identifier 7 in VL Identifier 8 in WO2015073575 A2 WO2015073575 A2 IL13Ra2 VH Identifier 7, 8 in - - WO2016123143N Glycan VH Identifier 7, 9 in VL Identifier 6, 8 in US20160194375A1 US20160194375A1 N Upar VH Identifier 72 in VL Identifier 71, 73 inUS20160333114A1 US20160333114A158324953654vlAttorney Docket No: 243734.000233CXCR4 VH Identifier 72, 73, 74, 75, 84 VL Identifier 76, 77, 78, 79, in US20110020218 80, 81, 82, 87, 88, 90, 91,92, 93 in US20110020218 Tie VH Identifier 723 in VL Identifier 724 in US20060057138A1 US20060057138A1 CEA VH Identifier 8 in US8287865 VL Identifier 10, 38, 39, 7, 9 in US8287865Herl / her3 VH Identifier 8 of VL Identifier 4 of WO2016073629 WO2016073629 CD70 VH Identifier 81, 85, 89 in VL Identifier 83, 87, 91 in WO2015121454 WO2015121454; Identifier 83 in WO2015121454; Identifier 87 in WO2015121454; Identifier 91 in WO2015121454 leukocytegen A VH Identifier 9 in VL Identifier 24 inW02010065962 A2 WO2010065962 A2

[0250] In some embodiments, the antigen-binding moiety may comprise an scFv derived from an antibody or antigen-binding fragment thereof that binds to an antigen target such as those described in the cited publications, the contents of each publication are incorporated herein by reference in their entirety for all purposes (Table 2).Table 2. Exemplary antigen-binding moieties comprising an scFv derived from an antibody or antigen-binding fragment thereof that binds to an antigen target Antigen Target Examples of SourceCEA Identifier 1 in US20160303166A1; Identifier 22 in US20140242701A1 GPC3 Identifier 1 in WO2016049459; Identifier 12 in US20160208015A1 CS1 Identifier 1 of W02016090369; Identifier 17 in WO2014179759A1 VEGFR2 Identifier 1, 2 in US20120213783TSLPR Identifier 1, 2 inUS20160311910Al; Identifier 1, 2 in WO2015084513 B7H4 Identifier 1, 2, 3, 4 in WO2013067492; Identifier 1 in US9422351B2 CD276 Identifier 10, 19, 28 in US20160053017GPRC5D Identifier 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,301, 313, 325, 337, 349, 361, 373, 385 in W02016090312WTI / HLA Identifier 108, 113, 18, 36, 54, 72, 90 in W02015070061bi specificCMet Identifier 11, 12, 13, 14, 15, 16, 17, 18, 19, 2, 21, 22, 23, 25, 26, 27, 28, 3,30, 31, 33, 34, 35, 36, 37, 38, 39, 4; 40, 41, 42, 43, 44, 48, 49, 5, 50, 51, 52, 53, 54, 55, 56, 57, 58, 6, 60, 7, 9, 29 in US20040166544; Identifier 26, 27, 28, 29, 30, 32 in US20150299326; Identifier 32 in US20130034559 FcRL Identifier 11, 15, 19, 23, 27, 31, 35, 39, 3, 43, 7, 594, 596, 598, 600, 602,604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632,59324953654vlAttorney Docket No: 243734.000233634, 636, 638, 640, 642, 644, 646, 648, 652, 654, 656; 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 680, 682, 684, 686, 688, 690, 692, 694, 696, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844; 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 650, 678 in W02016090337 EGFR Identifier 11, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83,88, 91, 94 in WO2014130657Claudin Identifier 11, 5, 7, 9 in WO2016073649A1; Identifier 17 in WO2014179759A1PSMA diabody Identifier 12, 13, 14, 15 in WO2011069019TRBC1 Identifier 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 3 in WO2015132598 CD19 / CD22 Identifier 1303, 1307 in WO2016164731 A2BispecificCD46 Identifier 1-42 in W02016040683MUC1 Identifier 15 in US20160130357MUC3 Identifier 15 in US20160130357Folate receptor Identifier 15 in US20170002072A1PDK1 Identifier 15 in WO2016090365Folate receptor Identifier 15, 23 in WO2012099973alphaBCM A Identifier 152, 158, 176, 185, 188, 200, 212, 218, 224, 284, 290, 296, 302,314, 326, 344, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 263, 264, 265, 266, 271, 273, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 64, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 263, 264, 265, 266, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 in WO2016014565; Identifier 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 251 in US20160311907A1CD 123 Identifier 157, 158, 159, 160, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 478, 480, 483, 485 in WO2016028896; Identifier 36 in W02015092024A2; Identifier 57 in WO2016115482A1; Identifier 36 in EP3083691A2; Identifier 157 in US20160311907A1CD 124 Identifier 158 in US20160311907A1CD125 Identifier 159 inUS20160311907AlCD5 Identifier 16 in WO2016138491CD 126 Identifier 160 inUS20160311907AlCD127 Identifier 161 inUS20160311907AlCD128 Identifier 162 inUS20160311907Al60324953654vlAttorney Docket No: 243734.000233CD 129 Identifier 163 inUS20160311907AlCD130 Identifier 164 inUS20160311907AlClaudin6 Identifier 164 in WO2016115482A1CD131 Identifier 165 in US20160311907A1Claudin7 Identifier 165 in WO2016115482A1GCN4 Identifier 165, 166, 167, 168, 169, 170 in WO2016168773 A3 Claudin8 Identifier 166 in WO2016115482A1VEGF Identifier 168, 169, 170, 171, 172, 173, 174, 175 in US20160090427;Identifier 498, 500, 502, 504, 506, 508 in US20110177074A1 MUC2 Identifier 17 in US20160130357MUC4 Identifier 17 in US20160130357CD44 Identifier 17 in WO2016042461 AlIL4 Identifier 17, 16 in WO2009121847ALK Identifier 17, 18, 19, 20, 21, 22, 23 in WO2015069922; Identifier 17, 18,19, 20, 21, 22, 23, 24 in US20160280798 Al; Identifier 24 in WO23015069922ESKAVT Identifier 173 in WO2016115482A1GD2 Identifier 19, 20, 21, in WO2016134284; Identifier 8 in WO2015132604 PSMA Identifier 19, 21, 30, 31, 34, 35 in WO2012145714TOSO Identifier 2 in EP3098237A1CSPG4 Identifier 2 in W02015080981; Identifier 2 in EP3074025A1 CLDN6 Identifier 2 in WO2016150400Integrin bivalent Identifier 2, 1 in W02009070753CD30 Identifier 20 in WO2016116035 Al; Identifier 2 in US20160200824A1 NYBR1 Identifier 21 in US20160333422A1; Identifier 21, 18, 19 in WO2015112830CD37 Identifier 21, 22 in US20170000900PDL1 nanobody Identifier 22, 23, 24, 25, 26, 27 in US20110129458Radiation Identifier 22, 24 in W02005042780A1inducibleneoantigenE7MC Identifier 223, 224, 225, 226, 227, 228, 229, 230, 231, 232 in WO2016182957A1TRBC2 Identifier 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 in WO2015132598 GPC4 Identifier 24 in WO2016049459ERBB2 Identifier 26, 27 in US20110059076A1; Identifier 1, 2 in US7244826 CD33 Identifier 262, 263, 264, 265, 266, 267, 268, 39, 40, 41, 42, 43, 44, 45, 46,47 in WO2016014576; Identifier 37 in W02015092024 A2; Identifier 37 in EP3083691A2; Identifier 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 in WO2016115482A1PDL2 nanobody Identifier 28, 29, 30, 31, 32, 33 in US201101294586462O-acetylated GD2 Identifier 29, 31 in US20150140023ganglioside0X40 Identifier 33 in US2015019050661324953654vlAttorney Docket No: 243734.000233CD79b Identifier 33 in US20160208021Human CD79b Identifier 33 in WO2016112870CD33 / CD3s Identifier 33, 34, 84 in WO2014144722 A2bispecifcRORI Identifier 34 in EP3083691A2; Identifier 249, 250251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268 in US20160208018A1; Identifier 57 in EP3083671A1; Identifier 1, 2 in US20160304619A1; Identifier 34 in WO2015092024 A2Human collagen Identifier 34 in WO2016112870VIITrastuzumab Identifier 35 in US20160208021; Identifier 35 in WO2016112870 Rituximab Identifier 36 in US20160208021; Identifier 36 in WO2016112870 CD138 Identifier 36 in WO2016130598A1Centuxiamb Identifier 37 in WO2016112870; Identifier 37 in US20160208021 Nivolumab Identifier 38 in US20160208021; Identifier 38 in WO2016112870 Ipilimumab Identifier 39 in US20160208021; Identifier 39 in WO2016112870 PD1 Identifier 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61 in US20160311917A1CLL1 Identifier 39, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51 in WO2016014535;Identifier 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 in US20160311907A1CLDN7 Identifier 4 in WO2016150400Ranibizuman Identifier 40 in US20160208021; Identifier 40 in WO2016112870 Adalimunab Identifier 41 in US20160208021; Identifier 41 in WO2016112870 Teplizumab Identifier 42 in US20160208021(mutated)Teplizumab Identifier 42 in WO2016112870CD3 Identifier 46, 47 in WO2015153912A1EGFR VIII Identifier 5 in US20140037628; Identifier 174 in US20160311907A1;Identifier 38 in US9394368B2; Identifier 5 in US20160200819A1 CD22 Identifier 5, 6 in WO2013059593; Identifier 9 in US20150299317;Identifier 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 203, 209, 215, 221, 227, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, 328, 334, 340, 346, 353, 358, 364, 370, 376, 383, 388, 394, 400, 406, 412, 418, 423 in WO2016164731A2CD19 Identifier 53, 54, 37 in EP3083671A1; Identifier 1, 10, 11, 12, 2 in WO2015157252; Identifier 10, 2, 206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 4, 45, 47, 49, 51, 53, 55, 57, 51, 53, 55, 57, 59, 6, 8, 87 in W02016033570; Identifiers, 4, 5, 59, 6, 7, 8, 9 in WO2015157252; 5754 Identifier 5 in WO2015155341A1; Identifier 7 in WO2014184143; Identifier 9 in WO2016139487; Identifier 10, 2, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 4, 45, 47, 49, 51, 53, 55, 57, 59, 6, 8, 87, 89 in US20160152723; Identifier 32, 35, 38 in EP3083691A2; Identifier 174 in WO2016115482A1; Identifier 20 inW02012079000; Identifier 32, 33, 35, 38 in W02015092024A2;62324953654vlAttorney Docket No: 243734.000233Identifier 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 in W02016109410; Identifier 5, 6 in WO2015155341A1; Identifier 7, 9 in US20160145337A1; Identifier 20 in US9499629B2; Identifier 73 in WO2016164580; Identifier 10, 2, 206, 207, 209, 210, 212, 216, 218, 219, 220, 221, 222, 223, 224, 225, 4, 45, 47, 49, 51, 53, 55, 57, 59, 6, 8, 87, 89 in US20160152723; Identifier 5 in WO2016055551END 0180 Identifier 6 in WO2013098813CLDN8 Identifier 6 in WO2016150400PRAME Identifier 63, 64, 65, 66, 67, 68, 69 in WO2016191246A2CD20 Identifier 691 in W02016164731A100; Identifier 692 in WO2016164731A101; Identifier 693 in WO2016164731A102; Identifier 694 in WO2016164731A103; Identifier 695 in WO2016164731A104; Identifier 696 in WO2016164731A105; Identifier 175 inWO2016115482A1Mesothelin Identifier 7 WO2015188141; Identifier NO 47, 46, 57, 48, 49, 50, 51, 53,54, 55, 56, 58, 59, 62, 64, 65, 66, 67, 68, 69, 70, 52, 60, 61, 63 in W02016090034; Identifier 10, 11, 12 in WO2013142034; Identifier 11 in WO2013063419CCR4 Identifier 7, 9 in WO2015191997Mec / CD3s Identifier 78 in WO2014144722 A2bispecificActivated alpha v Identifier 8, 2, 4 in US20090117096A1beta 3RAS Identifier 81 in WO2016154047CXCR4 Identifier 83, 85, 86, 89 in US20110020218HER2 / CD3 Identifier 9 in WO2014144722 A2FOLR1 / CD3S Identifier 90 in WO2014144722 A2bispecificB7H3 Identifier 99, 100, 101, 102, 103, 104, 102, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 87, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98 in WO2016033225

[0251] In some embodiments, the antigen-binding moiety may comprise an antigen-binding moiety derived from a CAR that binds to an antigen target, such as those described in the cited publications, the contents of each publication are incorporated herein by reference in their entirety for all purposes (Table 3).Table 3. Exemplary antigen-binding moieties comprising an antigen-binding moiety derived from a CAR that binds to an antigen target Antigen Target Examples of SourceCAR or gate (DC 19 or Identifier 1 in US20160296562DC33)CD19 or CD33 Identifier 1 in WO2015075468 which recognizes CD19 OR CD33CD19 / IL13 bispecific Identifier 10 in US20160340649A163324953654vlAttorney Docket No: 243734.000233CAT 19, campana Identifier 10 in WO2016139487architectureCD2 Identifier 10, 11 in WO2016138491CD70 Identifier 100, 93, 94, 96, 101, 95, 97, 98 in WO2015121454 VNAR Identifier 105, 106, 107, 108, 109, 110 in US20160333094A1 FcRL5 Identifier 11 in US20170008963A1CAT 19 CAR with Identifier 11 in WO20161394870X40 zetaendodomainFolate receptor Identifier 12 in US20170008963A1GD2 Identifier 12 in US9446105B2; Identifier 273, 274 in WO2016168773A3; Identifier 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 in WO2015132604; WO2016134284 (no Identifier) CD19 Identifier 12 in US9499629B2; Identifier 24 in US20160333108A1;Identifier 25, 29 in US20160333108A1; Identifier 27 in US20160333108A1; Identifier 1 in EP2997134A4; Identifier 19, 20 in EP3071687A1; Identifier 181 in WO2016168773A3; Identifier 2 in WO2015157399A9; Identifier 56, 62 in WO2016174409A1;Identifier 145, 293, 294, 295, 296, 297, 298 in WO2016179319A1; Identifier 73 in WO2013176915A1; Identifier 73 in WO2013176916A1; Identifier 73 in US20130315884A1; Identifier 73 in US20140134142A1; Identifier 73 in US20150017136A 1;Identifier 73 in US20150203817A1; Identifier 73 inUS20160120905 Al; Identifier 73 in US20160120906A1; Identifier 8, 5 in WO2015124715; Identifier 73 in WO2014184744; Identifier 73 in WO2014184741; Identifier 14, 15 in US20160145337A1;Identifier 14, 15 in WO2014184143; Identifier 15, 16 in WO2015075175; Identifier 16 in US20160145337A1; Identifier 16 in WO2014184143; Identifier 12 in W02012079000; Identifier 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 58 in WO2016164580;Identifier 14, 15 in US20160296563A1; Identifier 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 in WO2015157252; Identifier 14, 15 in WO2016139487; Identifier 53, 54, 55, 56, 57, 58 in US20160250258A1; Identifier 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 in WO2015187528; Identifier 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 58 in WO2015157252; Identifier 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 in WO2014153270; WO2016134284 (no Identifier); Identifier 13 in WO2016139487CD3 Identifier 12 in WO2016138491CAT 19 CAR with Identifier 12 in WO2016139487CD28 zetaendodomainFRa Identifier 13, 14 in US20120213783CD4 Identifier 13, 14 in WO2016138491FR beta Identifier 13, 22 in US9402865B2; Identifier 2, 4, 6 in US9446105B264324953654vlAttorney Docket No: 243734.000233CD19 / CD20 bi specific Identifier 1308 in WO2016164731 A2; Identifier 2, 8, 11 in US9447194B2CLL1 Identifier 148 in WO2016179319A1; Identifier 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 in W02016120218; Identifier 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 197 in WO2016014535CD5 Identifier 15, 13 in WO2016138491EGFR vIII Identifier 15, 16, 17, 18, 24, 25, 26, 27 in WO2016016341; Identifier 5, 10, 12, 8, 31, 30, 3 in US20160311907A1; Identifier 10 in US20160200819A1; Identifier 43, 49, 55, 61, 67, 73, 79, 85, 90, 96 in US9394368B2; Identifier 49, 55, 61, 67, 73, 79, 85, 90, 2, 1 in US20170008963 Al; Identifier 10, 11 in US20140037628CD7 Identifier 17 in WO2016138491CD52 Identifier 18 in WO2016138491BCM A Identifier 180, 162, 168, 174, 144, 150, 186, 192, 198, 204, 210, 156,216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276330, 282, 300, 306, 336, 354, 288, 312, 294, 342, 324, 318, 348 in WO2016168595A1; Identifier 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 in WO2015158671A1; Identifier 124, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 267, 268, 269, 270 in WO2016014565; Identifier 1, 2, 3, 4, 5, 20 in WO2015052538;Identifier 1, 2, 3, 4, 5, 6 in US20160237139A1; Identifier 9 in W02016094304 A3; Identifier 4, 5, 6, 8, 9, 10, 11, 12 in WO2013154760; Identifier 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 71, 73 in WO2016014789; Identifier 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 145, 146, 147, 148, 149, 150 in W02016120216; Identifier 102, 106, 107, 108, 109, 110, 111, 112, 129, 130, 131, 132, 133, 134, 135, 136, 113, 114, 115, 116, 117, 118, 101, 100, 137, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 103, 104, 105, 213 in WO2016097231 Tan Identifier 19 in US20160311907A1 recognizes CD19 AND CD33 using a CD45 phosphatase; Identifier 5 in WO2016026742A1 recognizes CD19 AND CD33 using a CD148 phosphatase; Identifier 6 in WO2016026742A1 which recognizes CD 19 AND NOT CD33 and is based on an HIM containing endodomain from LAIR1;Identifier 3 in WO2015075468 which recognizes CD19 AND NOT CD33 based on PTPN6 phosphatase; Identifier 2 in WO2015075468 which recognizes CD 19 AND NOT CD33 and recruits aPTPN6 / CD148 fusion protein to an ITIM containing endodomain65324953654vlAttorney Docket No: 243734.000233GD3 Identifier 19 in WO2016185035A1; Identifier 20, 21, 22, 23, 24, 25,26 in WO2016185035A1; WO2016134284 (no Identifier)CAR and gate (CD 19 Identifier 2 in US20160296562and CD33) CD148phosphataseCD30 Identifier 20 in W02016008973A1; Identifier 1 inWO2016116035 Al; WO2016134284 (no Identifier); Identifier 2 in W02016008973CD44 Identifier 21, 22, 23, 24, 25, 26, 27, 28, 31, 32, 33, 34, 35 in WO2016042461A1ROR1 Identifier 216, 217, 215 in WO2016097231; Identifier 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 127, 128, 129, 130, 131,132, 133,134, 1335, 136, 137, 138, 97, 98, 99, 100, 101, 102, 121, 122, 123, 124, 125, 126 in WO2016016344A1; Identifier 386, 387, 388, 389, 390, 391, 392, 393, 394 in WO2016187216A1FR Identifier 22 in US20170002072A1CLDN6 Identifier 22, 23, 24 in W02016150400PD1 Identifier 23 in WO2016014565; Identifier 26 in WO2015142675 Herl / Her3 bispecific Identifier 23, 24 in US20160215261A1HER2 Identifier 25 in US20160215261 Al; Identifier 9, 10 of WO2016073629; Identifier 17, 28, 98, 110 in US20160333114A1; Identifier 271, 272 in WO2016168773A3; Identifier 5 in WO2016168769A1CD20 Identifier 25 in WO2015157399A9; Identifier 177, 181, 182, 183,184, 185, 186, 187, 205, 206, 207, 208, 209, 210, 211, 188, 189, 190, 191, 192, 193, 176, 212, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 178, 179, 180 in WO2016097231P5A Identifier 26, 29, 60 in US20160333422A1GFR alpha Identifier 27 in WO2016185035A1GPC3 Identifier 28, 29 in WO2016185035A1; Identifier 3, 27, 10, 29, 14,30, 31, 18, 33 in WO2016049459; Identifier 22 in US20160215261A1CD22 / CD19 bispecific Identifier 29, 30 in WO2016149578; Identifier 1304 in WO2016164731A2CAR and gate (CD 19 Identifier 3 in US20160296562or CD33) CD45phosphataseEGFR Identifier 3, 2in WO2014130657; Identifier 36, 37, 38, 39, 35 in US20140242701 A; Identifier 43, 96, 49, 55, 61, 67, 73, 79, 85, 90, 1 in WO2014130657IL13 Identifier 30, 31, 32 in W02016120217Acid / base leucine Identifier 34, 35 in WO2016124930zipperP5AC1 Identifier 343, 344, 345, 346 in US20160297884A166324953654vlAttorney Docket No: 243734.000233P5AC16 Identifier 347, 396, 348 in US20160297884A1P6AP Identifier 349, 350, 351 in US20160297884A1PC1C12 Identifier 352, 353, 354 in US20160297884A1PD1 Identifier 355, 356, 357 in US20160297884A1; Identifier 119 in WO2014153270; Identifier 121 in WO2014153270; Identifier 22, 24, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 in US20160311917A1; Identifier 26, 39 in WO2016172537A1; Identifier 40 in US20160311907A1; Identifier 121, 119 in WO2015157252; Identifier 24 in WO2016014565;Identifier 22 in WO2016014565COM22 Identifier 358, 359, 360 in US20160297884A1DDD1 / AD1 -based zip Identifier 36 in WO2016124930PCI Identifier 361, 362, 363 in US20160297884A1P6DY Identifier 364, 365, 366 in US20160297884A1DDD1 / AD1 zip Identifier 37 in WO2016124930CD22 Identifier 380, 204, 260, 266, 272, 278, 284, 290, 296, 302, 308, 341,213, 320, 326, 332, 338, 347, 350, 356, 362, 368, 374, 219, 386, 392, 398, 404, 410, 416, 421, 427, 225, 230, 1109, 236, 242, 248, 254 in WO2016164731A2; Identifier 15, 16, 17, 18, 19, 20, 32 in WO2013059593; Identifier 22, 23, 24 in US20150299317PSMA Identifier 39 in WO2015142675; Identifier 28, 29 in US20160311907A1; Identifier 140, 144, 145, 146, 147, 148, 149, 150, 167, 168, 169, 170, 171, 172, 173, 174, 151, 152, 153, 154, 155, 156, 139, 138, 175, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 141, 142, 143, 214 in WO2016097231CD276 Identifier 39, 40, 41, 42, 43, 44, 45, 46, 47, 122, 123, 124, 125, 126,127, 128, 129, 130 in US20160053017SNAP Identifier 395 in WO2016187216A1SSEA4 Identifier 396, 397 in WO2016187216A1CEA Identifier 4 in W02016008973A1; Identifier 29, 30 in US20140242701ACAR and not gate Identifier 4, 5 in US20160296562(CD19 and not CD33)IL13Ra2specific Identifier 4, 5, 6 in WO2016089916A1; Identifier 47, 49 in WO2016123143; Identifier 51, 53, 55 in WO2016123143; Identifier 1, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 in US20160340649A1TSLPR Identifier 40, 41, 42 in WO2016034666A1; Identifier 39, 40, 41, 42,43, 44, 45, 46 in WO2015084513; Identifier 39, 40, 41, 42, 43 in US20160311910A1CAR and gate (CD 19 Identifier 41 in US20160296562and GD2)CAR and gate (CD 19 Identifier 43 in US20160296562and CD5)61324953654vlAttorney Docket No: 243734.000233ALK Identifier 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90 in WO2015069922 VEGFR2 Identifier 44, 45, 46 in US20160311910A1; Identifier 10, 11, 12 in US20120213783CAR and gate (CD 19 Identifier 45 in US20160296562and EGFR VIII)KMA Identifier 46 in US20160340649A1Mesothelin Identifier 47, 48 in US20160340649A1; Identifier 48 in US20160340649A1; Identifier 27 in WO2016172703A2; Identifier 18, 19, 20, 21, 22, 23 in WO2013142034; Identifier 3 inWO2013067492CAR and not gate 1 Identifier 48 in US20160296562HIV Env Identifier 48, 49 in WO2016168766A1; Identifier 4 in EP2997134A4; Identifier 7, 9, 47, 49 in WO2015077789CD33 Identifier 48, 49, 50, 51, 52, 53, 54, 55, 83 in WO2016014576;Identifier 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 in WO2015150526A2CAR and not gate 2 Identifier 49 in US20160296562CD435 Identifier 5 in EP3074419A2TOSO Identifier 5, 4 in WO2015075468MUC1 Identifier 5, 7 in WO2013063419; Identifier 51 in US20160340406A1; Identifier 30, 32, 34 in US20160130357;Identifier 295, 298, 301, 304, 307, 607, 609, 611, 613 in WO2016130726CAR and not gate 3 Identifier 50 in US20160296562CD8 stalk APRIL Identifier 51 in US20160296562A1HSP70 Identifier 51, 53, 5 in WO2015077789; Identifier 21, 22, 23, 24, 25,26, 27, 28, 29 in W02016120217APRIL-based CAR Identifier 53 in US20160296562A1; Identifier 52 in US20160296562A1CS1 Identifier 55, 57, 60, 54, 56, 48, 49, 50, 51, 52, 53, 58, 59, 61, 62 in WO2015121454; Identifier 28 in WO2014179759A1CAR and not gate Identifier 6 in US20160296562A1(CD 19 and not CD33)Trophoblast Identifier 6 in WO2015075468; Identifier 4 in US20160347854A1; Glycoprotein 5T4 Identifier 4 in EP3098237A1; Identifier 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 in WO2016034666A1HERVK Identifier 6 in WO2016168769A1NCAR with RQR82 Identifier 615 in WO2016130726ACD 1968324953654vlAttorney Docket No: 243734.000233NYBR1 Identifier 617, 619 in WO2016130726; Identifier 218 inWO2016097231; Identifier 26, 29, 60 in WO2015112830; Identifier 1 in US20160333422A1CD 123 Identifier 69 in WO2016142532; Identifier 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 48 in WO2015140268A1; Identifier 9, 10, 11, 12 in US20140271582; Identifier 56, 57, 58, 59, 60, 61 in WO2016097231; Identifier 98, 99, 100, 101, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 in WO2016028896; Identifier 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197 in W02016120220; Identifier 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 142 in W02016120216CD410 Identifier 7 in EP3074419A2CD38 Identifier 70, 71, 72, 64, 65, 66, 67, 68, 69 in WO2016097231;Identifier 35, 36, 37 in WO2015121454GCN4 Identifier 8, 10 in US9446105B2CD4-DDY3 Identifier 9 in EP3074419A2Fra Identifier 959 in W02016090337; Identifier 13 inUS20170002072A1CD70 Identifier 99 in WO2015121454

[0252] In some embodiments, the tumor antigen targeted by the immune effector cell is B7 Homolog 3 (B7-H3), disialoganglioside variant 2 (GD2), IL13Ra2, erythropoietin-producing human hepatocellular receptor A2 (EphA2), or human epidermal growth factor receptor 2 (HER2).

[0253] In some embodiments, the tumor antigen targeted by the immune effector cell is B7-H3. B7-H3, also known as CD276, is a type I transmembrane glycoprotein in the B7 / CD28 family of immunoglobulin superfamily molecules, encoded by the CD276 gene on chromosome 15. Its extracellular region consists of immunoglobulin-like domains, with two main isoforms in humans containing either two (21g) or four (41g) Ig-like domains due to exon duplication. B7-H3 is expressed at low levels on some normal tissues and various immune cells (such as antigen-69324953654vlAttorney Docket No: 243734.000233presenting cells) but is frequently upregulated on a wide range of malignant cells and cells within the tumor microenvironment.

[0254] In some embodiments, the tumor antigen targeted by the immune effector cell is GD2. GD2 is a glycosphingolipid (ganglioside) composed of a ceramide backbone linked to an oligosaccharide chain containing two sialic acid residues, typically located in the outer leaflet of the plasma membrane. GD2 is physiologically expressed at low levels in the central nervous system and peripheral nerves and is overexpressed on several tumor types.

[0255] In some embodiments, the tumor antigen targeted by the immune effector cell is IL13Ra2. Interleukin- 13 receptor subunit alpha-2 (IL13Ra2), also referred to as CD213A2 (cluster of differentiation 213 A2), is a membrane bound protein that in humans is encoded by the IL13RA2 gene.

[0256] In some embodiments, the tumor antigen targeted by the immune effector cell is EphA2. Erythropoietin-producing human hepatocellular receptor A2, also referred to as ephrin type-A receptor 2 (EphA2), also referred to as Eck (epithelial cell kinase), Myk2, or Sek2, is a member of the Eph receptor tyrosine kinase family which binds Ephrins Al, 2, 3, 4, and 5.

[0257] In some embodiments, the tumor antigen targeted by the immune effector cell is HER2. HER2, also referred to as HER2 / neu, receptor tyrosine-protein kinase erbB-2, CD340 (cluster of differentiation 340), proto-oncogene Neu, or ERBB2, is a membrane tyrosine kinase and oncogene that is overexpressed in some types of cancer.

[0258] In some embodiments, the at least one surface molecule capable of binding specifically to an antigen is a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof.Chimeric Antigen Receptor (CAR)

[0259] In some embodiments, the immune effector cell further comprises a chimeric antigen receptor (CAR).

[0260] CARs are primarily comprised of 1) an antigen-binding moiety, such as but not limited to a single-chain variable fragment (scFv) derived from an antigen-specific monoclonal antibody, and 2) a lymphocyte activation domain, such as but not limited to the ζ-chain from the T-cell receptor CD3. These two regions are fused together via a transmembrane domain. A hinge domain is usually required to provide more flexibility and accessibility between the antigenbinding moiety and the transmembrane domain. Upon transduction, the lymphocyte expresses70324953654vlAttorney Docket No: 243734.000233the CAR on its surface, and upon contact and ligation with the target antigen, it signals through the lymphocyte activation domain (e.g., CD3ζ chain) inducing cytotoxicity and cellular activation.

[0261] Constructs with only the antigen-specific binding region together with the lymphocyte activation domain are termed first-generation CARs. While activation of lymphocytes through a lymphocyte activation domain such as CD3 is sufficient to induce tumor-specific killing, such CARs fail to optimally induce T cell proliferation and survival in vivo. The second-generation CARs added co-stimulatory polypeptides to boost the CAR-induced immune response. For example, the co-stimulating polypeptide CD28 signaling domain was added to the CAR construct. This region generally contains the transmembrane region of the co-stimulatory peptide (in place of the CD3ζ transmembrane domain) with motifs for binding other molecules such as PI3K and Lek. T cells expressing CARs with only CD3^ vs CARs with both CD3(^ and a co-stimulatory domain (e g., CD28) demonstrated the CARs expressing both domains achieve greater activity. The most commonly used co-stimulating molecules include CD28 and 4- IBB, which promotes both T cell proliferation and cell survival. The third-generation CAR includes three signaling domains (e.g., CD3ζ, CD28, and 4-1BB), which further improves lymphocyte cell survival and efficacy. Examples of third-generation CARs include CD 19 CARs, most notably for the treatment of chronic lymphocytic leukemia (Milone, M. C., et al., (2009) Mol. Ther. 17:1453-1464; Kalos, M., et al., Sci. Transl. Med. (2011) 3:95ra73; Porter, D., et al., (2011) N. Engl. J. Med. 365: 725-533, each of which is herein incorporated by reference in their entirety for all purposes). Studies in three patients showed impressive function, expanding more than 1000-fold in vivo, and resulted in sustained remission in all three patients.

[0262] In some embodiments, the CAR comprises (i) an extracellular antigen-binding domain and (ii) a transmembrane domain. In some embodiments, the CAR further comprises a cytoplasmic domain. Each domain is fused in frame. In some embodiments, the CAR expressed by an immune effector cell described herein is a second-generation CAR.Extracellular Antigen-Binding Domain of the CAR

[0263] The choice of antigen-binding domain depends upon the type and number of antigens that define the surface of a target cell. For example, the antigen-binding domain may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the CARs can be genetically modified to target a71324953654vlAttorney Docket No: 243734.000233tumor antigen of interest by way of engineering a desired antigen-binding domain that specifically binds to an antigen (e.g., on a cancer cell). Non-limiting examples of cell surface markers that may act as targets for the antigen-binding domain in the CAR include those associated with viral, bacterial and parasitic infections, autoimmune disease, and cancer cells.

[0264] In some embodiments, the extracellular antigen-binding domain comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the extracellular antigen-binding domain comprises an antigen-binding polypeptide or functional variant thereof that binds to an antigen. In some embodiments, the antigen-binding polypeptide is an antibody or an antigenbinding fragment thereof that binds to an antigen. An example of an antigen that may be targeted by the extracellular antigen-binding domain includes, but is not limited to, the B7 homolog 3 (B7-H3) antigen.

[0265] In some embodiments, the antigen-binding polypeptide can be monomeric or multimeric e.g., homodimeric or heterodimeric), or associated with multiple proteins in a non-covalent complex. In some embodiments, the extracellular antigen-binding domain may consist of an Ig heavy chain. In some embodiments, the Ig heavy chain can be covalently associated with Ig light chain (e.g., via the hinge and optionally the CHI region). In some embodiments, the Ig heavy chain may become covalently associated with other Ig heavy / light chain complexes (e.g., by the presence of hinge, CH2, and / or CH3 domains). In the latter case, the heavy / light chain complex that becomes joined to the chimeric construct may constitute an antibody with a specificity distinct from the antibody specificity of the chimeric construct. In some embodiments, the entire chain may be used. In some embodiments, a truncated chain may be used, where all or a part of the CHI, CH2, or CH3 domains may be removed or all or part of the hinge region may be removed. Non-limiting examples of antigen-binding polypeptides include antibodies and antigen-binding fragments thereof such as e.g., murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, single chain variable fragments (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, or diabodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, single domain antibody variable domains, nanobodies (VHHs), and camelized antibody variable domains. In some embodiments, the antigen-binding polypeptide include a single-chain variable fragment (scFv).72324953654vlAttorney Docket No: 243734.000233

[0266] In some embodiments, the extracellular antigen-binding domain is an scFv. In some embodiments, the scFv comprises a linker between the VH and VL. Non-limiting examples of the linker sequence that may be used in the scFvs described herein include, GGGGSGGGGSGGGGS ((G4S)3; SEQ ID NO: 34), GGGGS (SEQ ID NO: 35), GGGGSGGGGS ((G4S)2; SEQ ID NO: 36), GGGGSGGGGSGGGGSGGGGS ((G4S)4; SEQ ID NO: 37), KESGSVSSEQLAQFRSLD (SEQ ID NO: 38), EGKSSGSGSESKST (SEQ ID NO: 39), EGKSSGSGSESKSTQ (SEQ ID NO: 40), GSTSGSGKSSEGKG (SEQ ID NO: 41), SSADDAKKDDAKKDDAKKDDAKKDG (SEQ ID NO: 42), EGKSSGSGSESKVD (SEQ ID NO: 43), or ESGSVSSEELAFRSLD (SEQ ID NO: 44), or SGGGGSGGGGSGGGGS ((SG4)3 linker; SEQ ID NO: 14)) or a functional variant thereof. Additional linkers include those described in, e.g., Whitlow and Filpula, Methods, Volume 2, Issue 2, April 1991, Pages 97-105, the content of which is incorporated herein by reference in its entirety.

[0267] In some embodiments, the linker sequence comprises the amino acid sequence SGGGGSGGGGSGGGGS (SEQ ID NO: 14), or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 14. In certain embodiments, the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 14, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 14. In certain embodiments, the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 45. In certain embodiments, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45.

[0268] In some embodiments, the extracellular antigen-binding domain is specific for B7-H3, or a fragment or variant thereof. In some embodiments, the extracellular antigen-binding domain is specific for GD2, or a fragment or variant thereof. In some embodiments, the extracellular antigen-binding domain is specific for IL13Ra2, or a fragment or variant thereof. In some73324953654vlAttorney Docket No: 243734.000233embodiments, the extracellular antigen-binding domain is specific for EphA2, or a fragment or variant thereof. In some embodiments, the extracellular antigen-binding domain is specific for HER2, or a fragment or variant thereof.

[0269] In some embodiments, B7-H3 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 13. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 13, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 13. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 52, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 52. In certain embodiments, the VH of B7-H3 scFv comprises the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 52.

[0270] In some embodiments, B7-H3 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 15. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 15. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 53, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 53. In certain embodiments, the VL of B7-H3 scFv comprises the amino74324953654vlAttorney Docket No: 243734.000233acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 53.

[0271] In some embodiments, the extracellular antigen-binding domain comprises an scFv capable of binding to B7-H3. In some embodiments, the scFv capable of binding to B7-H3 comprises the amino acid sequence of SEQ ID NO: 2. The scFv capable of binding to B7-H3 comprises the amino acid sequence of SEQ ID NO: 2, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the anti-B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 2. In certain embodiments, the nucleotide sequence that encodes the B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 54, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 54. In some embodiments, the anti-B7-H3 scFv comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the nucleotide sequence that encodes the B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 54.

[0272] In some embodiments, B7-H3 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 59, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 59. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 59, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 59. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 60, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80,75324953654vlAttorney Docket No: 243734.000233at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 60. In certain embodiments, the VH of B7-H3 scFv comprises the amino acid sequence set forth in SEQ ID NO: 59. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 60.

[0273] In some embodiments, B7-H3 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 61, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 61, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 62, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 62. In certain embodiments, the VL of B7-H3 scFv comprises the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 62.

[0274] In some embodiments, the extracellular antigen-binding domain comprises an scFv capable of binding to B7-H3. In some embodiments, the scFv capable of binding to B7-H3 comprises the amino acid sequence of SEQ ID NO: 63. The scFv capable of binding to B7-H3 comprises the amino acid sequence of SEQ ID NO: 63, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 63. In some embodiments, the nucleotide sequence encoding the anti-B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 63, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the B7-H376324953654vlAttorney Docket No: 243734.000233scFv comprises the nucleotide sequence set forth in SEQ ID NO: 64, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 64. In some embodiments, the anti-B7-H3 scFv comprises the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 64.

[0275] In some embodiments, B7-H3 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 65, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 65, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 66, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 66. In certain embodiments, the VH of B7-H3 scFv comprises the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments, the nucleotide sequence that encodes the VH of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 66.

[0276] In some embodiments, B7-H3 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 67, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 67. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 67, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 67. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 68, or a nucleotide77324953654vlAttorney Docket No: 243734.000233sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 68. In certain embodiments, the VL of B7-H3 scFv comprises the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, the nucleotide sequence that encodes the VL of B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 68.

[0277] In some embodiments, the extracellular antigen-binding domain comprises an scFv capable of binding to B7-H3. In some embodiments, the scFv capable of binding to B7-H3 comprises the amino acid sequence of SEQ ID NO: 69. The scFv capable of binding to B7-H3 comprises the amino acid sequence of SEQ ID NO: 69, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 69. In some embodiments, the nucleotide sequence encoding the anti-B7-H3 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 69, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 69. In certain embodiments, the nucleotide sequence that encodes the B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 70, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 70. In some embodiments, the anti-B7-H3 scFv comprises the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the nucleotide sequence that encodes the B7-H3 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 70.

[0278] In some embodiments, the extracellular antigen-binding domain comprises an scFv capable of binding to GD2. In some embodiments, the extracellular antigen-binding domain comprises an scFv capable of binding to IL13Ra2. In some embodiments, the extracellular antigen-binding domain comprises an scFv capable of binding to EphA2. In some embodiments, the extracellular antigen-binding domain comprises an scFv capable of binding to HER2.

[0279] In some embodiments, IL13Ra2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 55, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or78324953654vlAttorney Docket No: 243734.000233at least 99%, sequence identity with SEQ ID NO: 55. In certain embodiments, the nucleotide sequence that encodes the IL13Ra2 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 55, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 55. In certain embodiments, the nucleotide sequence that encodes the IL13Ra2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 56, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 56. In certain embodiments, the IL13Ra2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 55. In certain embodiments, the nucleotide sequence that encodes the IL13Ra2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 56.

[0280] In some embodiments, EphA2 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 46, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 46. In certain embodiments, the nucleotide sequence that encodes the VH of EphA2 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 46. In certain embodiments, the nucleotide sequence that encodes the VH of EphA2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 47, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 47. In certain embodiments, the VH of EphA2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the nucleotide sequence that encodes the VH of EphA2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 47.

[0281] In some embodiments, EphA2 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 48, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID79324953654vlAttorney Docket No: 243734.000233NO: 48. In certain embodiments, the nucleotide sequence that encodes the VL of EphA2 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 48. In certain embodiments, the nucleotide sequence that encodes the VL of EphA2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 49, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 49. In certain embodiments, the VL of EphA2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the nucleotide sequence that encodes the VL of EphA2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 49.

[0282] In some embodiments, EphA2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 50, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 50. In certain embodiments, the nucleotide sequence that encodes the EphA2 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 50, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 50. In certain embodiments, the nucleotide sequence that encodes the EphA2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 51, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 51. In certain embodiments, the EPHA2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the nucleotide sequence that encodes the EphA2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 51.

[0283] In some embodiments, HER2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 57, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 57. In certain embodiments, the nucleotide sequence that encodes the HER2 scFv comprises the nucleotide sequence that encodes the amino acid80324953654vlAttorney Docket No: 243734.000233sequence of SEQ ID NO: 57, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 57. In certain embodiments, the nucleotide sequence that encodes the HER2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 58, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 58. In certain embodiments, the HER2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, the nucleotide sequence that encodes the HER2 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 58.

[0284] In some embodiments, the extracellular antigen-binding domain further comprises a leader sequence. The leader sequence may be located at the amino-terminus of the extracellular antigen-binding domain. The leader sequence may be optionally cleaved from the antigenbinding moiety during cellular processing and localization of the CAR to the cellular membrane.

[0285] In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the leader comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 3. In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the nucleotide sequence that encodes the leader sequence comprises the nucleotide sequence set forth in SEQ ID NO: 72, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 72. In certain embodiments, the nucleotide sequence that encodes the leader sequence comprises the nucleotide sequence set forth in SEQ ID NO: 72.81324953654vlAttorney Docket No: 243734.000233Transmembrane Domain of the CAR

[0286] In some embodiments, the CARs expressed by the immune effector cell comprise a transmembrane domain. The transmembrane domain may be fused in frame between the extracellular antigen-binding domain and the cytoplasmic domain.

[0287] The transmembrane domain may be derived from the protein contributing to the extracellular antigen-binding domain, the protein contributing the signaling or co-signaling domain, or by a totally different protein. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid binding of proteins naturally associated with the transmembrane domain. In some embodiments, the transmembrane domain includes additional amino acids to allow for flexibility and / or optimal distance between the domains connected to the transmembrane domain.

[0288] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) the α, β or ζ chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan and / or valine can be found at each end of a synthetic transmembrane domain.

[0289] In some embodiments, it will be desirable to utilize the transmembrane domain of the ζ, η or FcεR1γ chains which contain a cysteine residue capable of disulfide bonding, so that the resulting chimeric protein will be able to form disulfide linked dimers with itself, or with unmodified versions of the ζ, η or FcεR1γ chains or related proteins. In some instances, the transmembrane domain will be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it will be desirable to employ the transmembrane domain of ζ, η or FcεR1γ and -β, MB1 (Igα.), B2982324953654vlAttorney Docket No: 243734.000233or CD3- γ, ζ, or η, in order to retain physical association with other members of the receptor complex.

[0290] In some embodiments, the transmembrane domain is derived from CD3ζ, CD28, CD4,or CD8α.

[0291] In a specific embodiment, the transmembrane domain is derived from the CD3(^ transmembrane domain. In some embodiments, the transmembrane domain is derived from CD3ζ and comprises the amino acid sequence SEQ ID NO: 4. In some embodiments, the CD3(^ transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 4, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 4. In some embodiments, the nucleotide sequence that encodes the CD3ζ transmembrane domain comprisesthe nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 4, or a variantthereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, atleast 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 4. In some embodiments, thenucleotide sequence that encodes the CD3ζ transmembrane domain comprises the nucleotidesequence set forth in SEQ ID NO: 25, or a nucleotide sequence having at least 50, at least 55, atleast 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least92, at least 93, at least 94, at least 95, at least 96, at least97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 25. In some embodiments, the CD3ζ transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the nucleotide sequence that encodes the CD3ζ transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 25.

[0292] In a specific embodiment, the transmembrane domain is derived from the CD28 transmembrane domain. In some embodiments, the transmembrane domain is derived from CD28 and comprises the amino acid sequence SEQ ID NO: 5. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 5, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 5. In some83324953654vlAttorney Docket No: 243734.000233embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 5, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 5. In some embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 26, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 26. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 26.

[0293] In a specific embodiment, the transmembrane domain is derived from the CD8α transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8α and comprises the amino acid sequence SEQ ID NO: 6 or SEQ ID NO: 17. In some embodiments, the CD8α transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 17, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 6 or SEQ ID NO: 17. In some embodiments, the nucleotide sequence that encodes the CD8α transmembrane domain comprises the nucleotide sequence that encodesthe amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17, or a variant thereof having at least50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or atleast 99%, sequence identity with SEQ ID NO: 6 or SEQ ID NO: 17. In some embodiments, thenucleotide sequence that encodes the CD8α transmembrane domain comprises the nucleotidesequence set forth in SEQ ID NO: 27, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 27. In some embodiments, the CD8a transmembrane domain84324953654vlAttorney Docket No: 243734.000233comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 17. In someembodiments, the nucleotide sequence that encodes the CD8α transmembrane domain comprisesthe nucleotide sequence set forth in SEQ ID NO: 27.

[0294] In a specific embodiment, the transmembrane domain is derived from the CD4 transmembrane domain. In some embodiments, the transmembrane domain is derived from CD4 and comprises the amino acid sequence SEQ ID NO: 7. In some embodiments, the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 7, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 7. In some embodiments, the nucleotide sequence that encodes the CD4 transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 7. In some embodiments, the nucleotide sequence that encodes the CD4 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 28, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 28. In some embodiments, the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleotide sequence that encodes the CD4 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 28.

[0295] In some embodiments, the CAR further comprises a linker domain between the extracellular antigen-binding domain and the transmembrane domain. In some embodiments, the antigen-binding domain, linker, and the transmembrane domain are in frame with each other.

[0296] The term “linker domain” as used herein generally means any oligo- or polypeptide that functions to link the antigen-binding moiety to the transmembrane domain. A linker domain can be used to provide more flexibility and accessibility for the antigen-binding moiety. A linker domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. A linker domain may be derived from all or part of naturally85324953654vlAttorney Docket No: 243734.000233occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the linker domain may be a synthetic sequence that corresponds to a naturally occurring linker domain sequence, or may be an entirely synthetic linker domain sequence. Non-limiting examples of linker domains which may be used in accordance with the invention include a part of human CD8a chain, partial extracellular domain of CD28, FcyRllla receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof. In some embodiments, additional linking amino acids are added to the linker domain to ensure that the antigen-binding moiety is an optimal distance from the transmembrane domain. In some embodiments, when the linker is derived from an Ig, the linker may be mutated to prevent Fc receptor binding.

[0297] In some embodiments, the linker domain comprises a hinge region. In some embodiments, the hinge domain is a CD8a hinge domain. Other hinge regions suitable for use in the present invention may be derived from an immunoglobulin IgG hinge or functional fragment, including IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera or variant thereof.

[0298] In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 8. In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 8, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the hinge region comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the hinge region comprises the sequence set forth in SEQ ID NO: 29, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 29. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 8. In some86324953654vlAttorney Docket No: 243734.000233embodiments, the nucleotide sequence encoding the hinge region comprises the nucleotide sequence set forth in SEQ ID NO: 29.

[0299] In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 9. In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 9, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the hinge region comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the hinge region comprises the sequence set forth in SEQ ID NO: 30, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 30. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the hinge region comprises the nucleotide sequence set forth in SEQ ID NO: 30.

[0300] In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 16. In some embodiments, the hinge region comprises the amino acid sequence SEQ ID NO: 16, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the hinge region comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 16.87324953654vlAttorney Docket No: 243734.000233

[0301] In some embodiments, the linker domain comprises the amino acid sequence SEQ ID NO: 10. In some embodiments, the linker domain comprises the amino acid sequence SEQ ID NO: 10, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the linker domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the linker domain comprises the sequence set forth in SEQ ID NO: 31, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 31. In some embodiments, the linker domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the linker domain comprises the nucleotide sequence set forth in SEQ ID NO: 31.Cytoplasmic Domain of the CAR

[0302] In some embodiments, the CAR expressed by the immune effector cell described herein further comprises a cytoplasmic domain. In some embodiments, the CAR cytoplasmic domain comprises one or more lymphocyte activation domains.

[0303] The cytoplasmic domain, which comprises the lymphocyte activation domain of the CAR, is responsible for activation of at least one of the normal effector functions of the lymphocyte in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “lymphocyte activation domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire lymphocyte activation domain is present, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term88324953654vlAttorney Docket No: 243734.000233intracellular signaling domain is thus meant to include any truncated portion of the lymphocyte activation domain sufficient to transduce the effector function signal.

[0304] In some embodiments, the lymphocyte activation domain is derived from DAP10, DAP12, Fc epsilon receptor I γ chain (FCER1G), CD3δ, CD3ε, CD3γ, CD3ζ, CD27, CD28, CD40, CD134, CD137, CD226, CD79A, ICOS, or MyD88.

[0305] In some embodiments, the lymphocyte activation domain is the ^-chain from the T-cell receptor CD3. In some embodiments, the lymphocyte activation domain is derived from CD3ζ and comprises the amino acid sequence SEQ ID NO: 11. In some embodiments, the CD3^ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 11. In some embodiments, the nucleotide sequence that encodes the CD3ζ signaling domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 11. In some embodiments, the nucleotide sequence that encodes the CD3ζ signaling domain comprises the nucleotide sequence set forth in SEQ ID NO: 32, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 32. In some embodiments, the CD3ζ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleotide sequence that encodes the CD3ζ signaling domain comprises the nucleotide sequence set forth in SEQ ID NO: 32.

[0306] In some embodiments, the CAR cytoplasmic domain comprises one or more costimulatory domains. CAR constructs may also include co-stimulatory polypeptides to boost the CAR-induced immune response. The most commonly used co-stimulating molecules include CD28 and 4-1BB, which promotes both T-cell proliferation and cell survival. Another example of co-stimulatory domains is a MyD88 / CD40 molecule that can be used with or without the use of a separate dimerization agent. Additional CAR constructs may also include three signaling89324953654vlAttorney Docket No: 243734.000233domains (e.g., CD3ζ, CD28, and 4-1BB), which further improves lymphocyte cell survival and efficacy.

[0307] Non-limiting examples of co-stimulatory domains that may be used in the CARs described herein include those derived from 4-1BB (CD137), CD28, ICOS, CD134 (OX-40), BTLA, CD27, CD30, GITR, CD226, and HVEM.

[0308] In some embodiments, the co-stimulatory domain is derived from CD28 and comprises the amino acid sequence SEQ ID NO: 12. In some embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 12. In some embodiments, the nucleotide sequence that encodes the CD28 co-stimulatory domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 12, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 12. In some embodiments, the nucleotide sequence that encodes the CD28 co-stimulatory domain comprises the nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 33. In some embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the nucleotide sequence that encodes the CD28 co-stimulatory domain comprises the nucleotide sequence set forth in SEQ ID NO: 33.

[0309] In some embodiments, the cytoplasmic domain comprises both the CD3 lymphocyte activation domain and the CD28 co-stimulatory domain, which are fused in frame. The CD3ζ lymphocyte activation domain and the CD28 co-stimulatory domain can be in any order. In some embodiments, the CD3ζ lymphocyte activation domain is downstream of the CD28 co-stimulatory domain.

[0310] In addition to the CAR construct, the CAR may further comprise an accessory gene that encodes an accessory peptide. Examples of accessory genes can include a transduced host cell90324953654vlAttorney Docket No: 243734.000233selection marker, an in vivo tracking marker, a cytokine, a suicide gene, or some other functional gene. In some embodiments, the functional accessory gene can increase the safety of the CAR.

[0311] Non-limiting examples of classes of accessory genes that can be used to increase the effector function of CAR containing immune effector cells, include i) secretable cytokines (e.g., but not limited to, IL-7, IL- 12, IL- 15, IL- 18), ii) membrane bound cytokines (e.g., but not limited to, IL- 15), iii) chimeric cytokine receptors (e.g., but not limited to, IL-2 / IL-7, IL-4 / IL-7), iv) constitutive active cytokine receptors (e.g., but not limited to, C7R), v) dominant negative receptors (DNR; e g., but not limited to TGFRII DNR), vi) ligands of costimulatory molecules (e.g., but not limited to, CD80, 4-1 BBL), vii) antibodies, including fragments thereof and bispecific antibodies (e.g., but not limited to, bispecific T-cell engagers (BiTEs)), or vii) a second CAR.

[0312] In some embodiments, the CAR can be encoded by one polynucleotide chain. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the nucleotide sequence that encodes the CAR comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 1. In some embodiments, the nucleotide sequence that encodes the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 71, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 71. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleotide sequence that encodes the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 71.

[0313] In some embodiments, the immune effector cell is an allogeneic cell.

[0314] In some embodiments, the immune effector cell is an autologous cell.91324953654vlAttorney Docket No: 243734.000233

[0315] The immune effector cells may be autologous / autogeneic (“self’) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). In some embodiments, the immune effector cells are obtained from a mammalian subject. In other embodiments, the immune effector cells are obtained from a primate subject. In some embodiments, the immune effector cells are obtained from a human subject.

[0316] In some embodiments, the immune effector cell is isolated from a subject having a disease.

[0317] In some embodiments, the disease is a cancer, an infectious disease, an inflammatory disorder, or an autoimmune disease.

[0318] In some embodiments, the cancer is a cancer expressing B7-H3, GD2, IL13Ra2, or EphA2, or HER2.

[0319] In some embodiments, the immune effector cell is derived from a blood, marrow, tissue, or a tumor sample.

[0320] Lymphocytes can be obtained from sources such as, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Lymphocytes may also be generated by differentiation of stem cells. In some embodiments, lymphocytes can be obtained from blood collected from a subject using techniques generally known to the skilled person, such as sedimentation, e.g., FICOLL™ separation.

[0321] In some embodiments, cells from the circulating blood of a subject are obtained by apheresis. An apheresis device typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. The cells can be washed with PBS or with another suitable solution that lacks calcium, magnesium, and most, if not all other, divalent cations. A washing step may be accomplished by methods known to those in the art, such as, but not limited to, using a semiautomated flowthrough centrifuge (e.g., Cobe 2991 cell processor, or the Baxter CytoMate). After washing, the cells may be resuspended in a variety of biocompatible buffers, cell culture medias, or other saline solution with or without buffer.92324953654vlAttorney Docket No: 243734.000233

[0322] In some embodiments, immune effector cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes. As an example, the cells can be sorted by centrifugation through a PERCOLL™ gradient. In some embodiments, after isolation of PBMC, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and / or genetic modification.

[0323] In some embodiments, T lymphocytes can be enriched. For example, a specific subpopulation of T lymphocytes, expressing one or more markers such as, but not limited to, CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD27, CD28, CD34, CD36, CD45RA, CD45RO, CD56, CD62, CD62L, CD122, CD123, CD127, CD235a, CCR7, HLA-DR or a combination thereof using either positive or negative selection techniques. In some embodiments, the T lymphocytes for use in the compositions of the invention do not express or do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0324] In some embodiments, NK cells can be enriched. For example, a specific subpopulation of T lymphocytes, expressing one or more markers such as, but not limited to, CD2, CD 16, CD56, CD57, CD94, CD 122 or a combination thereof using either positive or negative selection techniques.Methods for genetically modifying immune effector cells

[0325] In one aspect, provided herein is an immune effector cell, wherein the transcriptional activity of transcription factor(s) ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof is inhibited in the immune effector cell as compared to a control cell in which the transcriptional activity of transcription factor(s) is not inhibited. In certain embodiments, the immune effector cell is a T cell, an NK cell, or an iPSC capable of differentiating into an immune cell. In certain embodiments, the immune effector cell comprises a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof. In certain embodiments, the immune effector cell is a CAR T cell.

[0326] In some embodiments, the transcription factor(s) are DNMT1, FLI1, LEF1, or a combination thereof.93324953654vlAttorney Docket No: 243734.000233

[0327] In some embodiments, a transcription factor gene is deleted or defective so that no detectable functional transcription factor is expressed.

[0328] In some embodiments, the enzymatic activity of the transcription factor(s) is inhibited in the immune effector cell.

[0329] In some embodiments, the enzymatic activity of the transcription factor(s) is inhibited by exposing the cell to a transcription factor inhibitor.

[0330] In some embodiments, enzymatic activity of one or more transcription factors described herein is inhibited by exposing the cell to a transcription factor inhibitor. Such transcription factor inhibitors may be, for example, a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a peptidomimetic, a fusion peptide, a protein, an oligonucleotide, an aptamer, a natural product, a carbohydrate, an avimer, an anticalin, or a speigelmer.

[0331] In some embodiments, a transcription factor inhibitor described herein may be a small molecule. A small molecule can enter cells easily because it has a low molecular weight (typically, up to about 1 kDa). Once inside the cells, it can affect cellular proteins, such as one or more transcription factors described herein, and may, for example, bind directly to the catalytic (active) site of a transcription factor and inhibit the enzymatic activity of the transcription factor. Such small molecules may mimic a natural substrate or cofactor of a transcription factor and block the enzymatic activity of the transcription factor. Alternatively, a small molecule may bind to a site distinct from the active site and induce a conformational change that reduces or abolishes enzymatic activity of a transcription factor.

[0332] In some embodiments, bifunctional small molecules (e.g., proteolysis targeting chimeras (PROTACs), or molecular glues) may be used as a transcription factor inhibitor in the present disclosure. Such bifunctional small molecules can recruit an E3 ligase to the transcription factor, leading to its ubiquitination and proteasomal degradation, thereby eliminating its enzymatic activity.

[0333] In some embodiments, a transcription factor inhibitor described herein may be an antibody or antigen-binding fragment thereof. Antibodies or antigen-binding fragments can bind to the transcription factor and block the active site, allosteric sites, or critical interaction surfaces. Antibodies or antigen-binding fragments that can act as transcription factor inhibitors of the present disclosure may of any one of various antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is IgGl, IgG2, IgG3, or IgG4 isotype.94324953654vlAttorney Docket No: 243734.000233In some embodiments, the antibody isotype is IgA1 or IgA2. Antibody or antigen-binding fragment thereof specificity is largely determined by the amino acid sequence, and arrangement, of the complementarity -determining regions (CDRs). The CDRs of one isotype may be transferred to another isotype without altering antigen specificity. Alternatively, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without altering antigen specificity. Accordingly, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.

[0334] In some embodiments, the antibody or antigen-binding fragment that can act as transcription factor inhibitors of the present disclosure is a human antibody, a monoclonal antibody, a humanized antibody, a single-chain Fv (scFv), a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, a Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a tribody, or a tetrabody.

[0335] In some embodiments, a transcription factor inhibitor described herein may be a peptide, or peptidomimetic. Peptides or peptidomimetics may mimic substrates, cofactors, or interaction motifs of transcription factors, and block catalytic sites or disrupt necessary intramolecular or interm olecular interactions required for enzymatic activity.

[0336] In some embodiments, a transcription factor inhibitor described herein may be an oligonucleotide. The oligonucleotide may be a DNA or RNA. Synthetic oligonucleotides may mimic a transcription factor’ s binding sites (on DNA or RNA) and can sequester it away from its physiological substrates or chromatin loci. If substrate engagement is necessary for catalysis, this approach can effectively inhibit enzymatic function at the relevant genomic sites.

[0337] In some embodiments, a transcription factor inhibitor described herein may be a structured oligonucleotide such as an aptamer. Aptamers may block catalytic domains, disrupt conformational changes, or prevent interactions with cofactors or substrates. An aptamer described herein may comprise any nucleic acid which specifically binds specifically to a target, e.g., a transcription factor described herein. In some embodiments, the aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, a nucleic acid aptamer may comprise a single-stranded RNA (ssDNA or ssRNA) or DNA. In certain embodiments, a single-stranded nucleic acid aptamer may form loop(s) and / or helice(s) structures. The nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, modified95324953654vlAttorney Docket No: 243734.000233nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleotides, or a combination of thereof.

[0338] In some embodiments, a transcription factor inhibitor described herein may interfere with the nuclear localization signals (NLS) or nuclear export signals (NES) of a transcription factor described herein, leading to mis-localization of the transcription factor. If the enzymatic activity of a transcription factor is required in the nucleus, cytoplasmic retention or forced export indirectly inhibits its functional enzymatic role on chromatin.

[0339] In some embodiments, a transcription factor inhibitor described herein may employ an engineered protease or a chemical strategy to selectively cleave a transcription factor described herein, removing or disrupting its catalytic domain. Cleavage of a transcription factor may structurally disable its enzymatic activity even if some binding functions remain.

[0340] In some embodiments, the transcription factor gene is mutated so that the enzymatic activity of the transcription factor is inhibited.

[0341] In some embodiments, the catalytic domain of the transcription factor is mutated.

[0342] The level of enzymatic activity of the transcription factor in the immune effector cell may be decreased by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The level of enzymatic activity of the transcription factor in the immune effector cell may be decreased by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%. In some embodiments, the level of enzymatic activity of the transcription factor in the immune effector cell is decreased by 50% or more.

[0343] In another aspect, provided herein is an immune effector cell, wherein the transcriptional activity of transcription factor(s) ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof is activated in the immune effector cell as compared to a control cell in which the transcriptional activity of transcription factor(s) is not96324953654vlAttorney Docket No: 243734.000233activated. In certain embodiments, the immune effector cell is a T cell, an NK cell, or an iPSC capable of differentiating into an immune cell. In certain embodiments, the immune effector cell comprises a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof. In certain embodiments, the immune effector cell is a CAR T cell,

[0344] In some embodiments, the enzymatic activity of the transcription factor(s) is activated in the immune effector cell.

[0345] In some embodiments, the enzymatic activity of the transcription factor(s) is activated by exposing the cell to a transcription factor activator.

[0346] In some embodiments, enzymatic activity of one or more transcription factors described herein is activated by exposing the cell to a transcription factor activator. Such transcription factor activators may be, for example, a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a peptide analog, a fusion peptide, a protein, an oligonucleotide, an aptamer, a natural product, a carbohydrate, an avimer, an anticalin, or a speigelmer.

[0347] In some embodiments, a transcription factor activator described herein may be a small molecule. A small molecule activator may activate a transcription factor through different mechanisms. For example, a small molecule activator may bind to regulatory (non-catalytic) sites and stabilize an active conformation of the transcription factor’s enzymatic domain, increasing its catalytic efficiency. A small molecule activator may also promote or stabilize interactions between the transcription factor and its co-activators, scaffolds, or regulatory subunits required for enzymatic activity. A small molecule activator may also inhibit repressors (e.g., phosphatases, deacetylases, inhibitory kinases) that normally keep the transcription factor in a less active state, thereby indirectly activating it. A small molecule activator may also increase the availability or effectiveness of essential cofactors (e.g., ATP, NAD+, acetyl-CoA, metal ions) to enhance activity of enzymatic domains. Additionally, a small molecule activator may enhance nuclear import or inhibit nuclear export, increasing access of the transcription factor to chromatin and substrates.

[0348] In some embodiments, a transcription factor activator described herein may be a peptide, or peptidomimetic. For example, peptide or peptidomimetics may mimic natural activating motifs from co-activators or regulatory partners and promote assembly or activation of the transcription factor’s enzymatic machinery. Peptide or peptidomimetics may also bind97324953654vlAttorney Docket No: 243734.000233intramolecular inhibitory domains of a transcription factor (if present), disrupting autoinhibitory interactions and shifting the transcription factor to an active state.

[0349] In some embodiments, a transcription factor activator described herein may be an antibody or antibody fragment. Antibody or antibody fragments may stabilize an active conformation or promote activating protein-protein interactions. Antibodies or antigen-binding fragments that can act as transcription factor activators of the present disclosure may of any one of various antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is IgGl, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody isotype is IgAl or IgA2. Antibody or antigen-binding fragment thereof specificity is largely determined by the amino acid sequence, and arrangement, of the complementarity-determining regions (CDRs). The CDRs of one isotype may be transferred to another isotype without altering antigen specificity. Alternatively, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without altering antigen specificity. Accordingly, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.

[0350] In some embodiments, the antibody or antigen-binding fragment that can act as transcription factor activators of the present disclosure is a human antibody, a monoclonal antibody, a humanized antibody, a single-chain Fv (scFv), a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, a Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a tribody, or a tetrabody.

[0351] In some embodiments, a transcription factor activator described herein may be an oligonucleotide. The oligonucleotide may be a DNA or RNA. Oligonucleotide activator may recruit both the transcription factor and co-activators or modifying enzymes to the same locus, thereby promoting its enzymatic activation.

[0352] In some embodiments, a transcription factor inhibitor described herein may be a structured oligonucleotide such as an aptamer. Aptamers may bind the transcription factor and stabilize its active state rather than block the catalytic or regulatory sites.

[0353] In further embodiments, gene expression of transcription factors may be activated by a genetic engineering system, such as a CRISPR activation (CRISPR) system. For example, CRISPRa systems (such as dCas9-VP64, dCas9-p300, SunTag-based activators, dCas9-VPR, or SAM-based activators) can be targeted to the endogenous promoter or enhancer regions of the98324953654vlAttorney Docket No: 243734.000233transcription factor gene, recruiting transcriptional activators to increase its native transcription without altering the DNA sequence. In additional embodiments, gene activation may be achieved using other programmable DNA-binding platforms, including but not limited to transcription activator-like effector (TALE)-based activators (e.g., TALE-VP64, TALE-VPR) or zinc finger protein (ZFP)-based activators (e.g., ZFP-VP16, ZFP-p300), which are engineered to recognize specific genomic sequences in promoter or enhancer regions and are fused to one or more transcriptional activation domains to enhance transcription factor expression. In certain embodiments, epigenome-editing systems comprising catalytically inactive or nickase variants of genome-targeting proteins fused to chromatin-modifying enzymes (for example, histone acetyltransferases, demethylases, or other epigenetic regulators) may be used to modify regulatory regions of the transcription factor gene to establish a chromatin state permissive for transcriptional activation, thereby increasing endogenous expression without introducing doublestrand breaks or permanent sequence changes.

[0354] In still further embodiments, expression of transcription factors may be upregulated by delivery of exogenous expression cassettes via viral vectors (such as lentiviral, adenoviral, or adeno-associated viral vectors), non-viral plasmid vectors, transposon-based systems (such as Sleeping Beauty or PiggyBac), or recombinase-mediated cassette exchange systems, thereby increasing the cellular levels of the transcription factor and mimicking or enhancing its physiological function.

[0355] In some embodiments, the transcription factor gene is mutated so that the enzymatic activity of the transcription factor is increased.

[0356] In some embodiments, the transcription factor gene is mutated to result in deletion of an N-terminal inhibitory domain of the transcription factor. In some embodiments, the transcription factor gene is mutated to result in deletion of a C-terminal inhibitory domain of the transcription factor. In some embodiments, the transcription factor gene is mutated to result in one or more substitutions at one or more activating residues of the transcription factor.

[0357] The level of enzymatic activity of the transcription factor in the immune effector cell may be increased by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80%99324953654vlAttorney Docket No: 243734.000233to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The level of enzymatic activity of the transcription factor in the immune effector cell may be increased by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or even 100%. In some embodiments, the level of enzymatic activity of the transcription factor in the immune effector cell is increased by 50% or more.

[0358] In another aspect, provided herein is an immune effector cell, wherein the transcriptional activity of transcription factor(s) ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof is inhibited in the immune effector cell as compared to a control cell in which the transcriptional activity of transcription factor(s) is not inhibited and the transcriptional activity of transcription factor(s) ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof is activated in the immune effector cell as compared to a control cell in which the transcriptional activity of transcription factor(s) is not activated. In certain embodiments, the immune effector cell is a T cell, an NK cell, or an iPSC capable of differentiating into an immune cell. In certain embodiments, the immune effector cell comprises a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof. In certain embodiments, the immune effector cell is a CAR T cell.

[0359] In a further aspect, provided herein is a method for generating the immune effector cell as described herein, said method comprising (a) deleting or modifying the gene or gene product of ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell so that the transcription factor activity is decreased and / or (b) overexpressing or modifying the gene or gene product of ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell so that the transcription factor activity is increased.

[0360] In some embodiments, the transcription factor gene in the immune effector cell is deleted, overexpressed, or modified as a result of an activity of a site-specific nuclease. The term “site-specific nuclease” as used herein refers to a nuclease capable of specifically recognizing and cleaving a nucleic acid (DNA or RNA) sequence. Suitable site-specific nucleases for use in100324953654vlAttorney Docket No: 243734.000233the present invention include, but are not limited to, RNA-guided endonuclease (e.g., CRISPR-associated (Cas) proteins), zinc finger nuclease, a TALEN nuclease, or mega-TALEN nuclease.

[0361] Site-specific nucleases may create double-strand breaks or single-strand breaks (i.e., nick) in a genomic DNA of a cell. Although not wishing to be bound by theory, these breaks are typically repaired by the cell using one of two mechanisms: non-homologous end joining (NHEJ) and homology-directed repair (HDR). In NHEJ, the double-strand breaks are repaired by direct ligation of the break ends to one another. As a result, no new nucleic acid material is inserted into the site, although a few bases may be lost or added, resulting in a small insertion and deletion (indel). In HDR, a donor polynucleotide with homology to the cleaved target DNA sequence is used as a template to repair the cleaved target DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the target DNA. As such, new nucleic acid material may be inserted or copied into the cleavage site. In some cases, an exogenous donor polynucleotide can be provided to the cell. The modifications of the target DNA due to NHEJ and / or HDR may lead to, for example, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, sequence replacement, etc. Accordingly, cleavage of DNA by a site-directed nuclease may be used to delete nucleic acid material from a target DNA sequence by cleaving the target DNA sequence and allowing the cell to repair the sequence in the absence of an exogenously provided donor polynucleotide. Thus, the methods can be used to knock out a gene (resulting in complete lack of transcription or altered transcription) or to knock in genetic material (e.g., a transgene) into a locus of choice in the target DNA.

[0362] In some embodiments, the site-specific nuclease is an RNA-guided endonuclease. In particular, a group of RNA-guided endonucleases known as CRISPR-associated (Cas) proteins may be employed to genetically modify the immune effector cell. A Cas protein may form an RNA-protein complex (referred to as RNP) with a guide RNA (gRNA) and is capable of cleaving a target site bearing sequence complementarity to a short sequence (typically about 20-40nt) in the gRNA.

[0363] Examples of Cas proteins useful in the methods of the present disclosure include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Cpfl, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2,101324953654vlAttorney Docket No: 243734.000233Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[0364] In some embodiments, the RNA-guided endonuclease is a Cas9 protein. The Cas9 protein may be from S. pyogenes, Streptococcus thermophilus, Neisseria meningitidis, F. novicida, S. mutans or Treponema denticola. The Cas9 may be a native or modified Cas9 protein.

[0365] Cas proteins useful in the methods of the present disclosure can be wild type proteins (i.e., those that occur in nature), modified Cas proteins ( / .e., Cas protein variants), or fragments of wild type or modified Cas proteins. Cas proteins can also be active variants or fragments with respect to catalytic activity of wild type or modified Cas proteins. Active variants or fragments with respect to catalytic activity can comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the wild type or modified Cas protein or a portion thereof, wherein the active variants retain the ability to cut at a desired cleavage site and hence retain nick-inducing or double-strand-break-inducing activity.

[0366] A “guide RNA” or “gRNA” is an RNA molecule that binds to a Cas protein e.g., Cas9 protein), or functional fragment or derivative thereof, and targets the Cas protein to a specific location within a target DNA. In some embodiments, the guide RNA is a single guide RNA (sgRNA). For Cas9, for example, a single-guide RNA can comprise a crRNA fused to a tracrRNA (e.g., via a linker). In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the ARID1A protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the DNMT1 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the ELF5 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the FLI1 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the GRHL3 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the GTF2IRD1 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the LEF1 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the MEF2B protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the SON protein. In some embodiments, the102324953654vlAttorney Docket No: 243734.000233gRNA is designed to target a locus within or near the gene encoding for the ZFHX3 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the ATF6 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the EN1 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the HMGA2 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the IRF1 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the POU3F4 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the REL protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the SRY protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the TFAP4 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the USF2 protein. In some embodiments, the gRNA is designed to target a locus within or near the gene encoding for the YY1 protein.

[0367] The Cas9 protein may be programmed with a guide RNA (gRNA) that targets a locus with or near the transcription factor gene. In some embodiments, the Cas9 protein is programmed with a gRNA that comprises a nucleotide sequence encoded by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0368] In some embodiments, the site-specific nuclease is a zinc finger nuclease, a TALEN nuclease, or mega-TALEN nuclease.

[0369] In some embodiments, the transcription gene product in the immune effector cell is deleted or modified as a result of an activity of an RNA interference (RNAi) molecule or an antisense oligonucleotide. RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by small interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951). Any small nucleic acid molecules capable of mediating RNAi, such as a short interfering nucleic acid (siNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a micro-RNA (miRNA), and a short hairpin RNA (shRNA), may be to inhibit the expression of the DNMT3A gene. An antisense oligonucleotide (ASO) is a short nucleotide sequence that can hybridize or bind (e.g., by Watson-Crick base pairing) in a complementary fashion to its target sequence.103324953654vlAttorney Docket No: 243734.000233

[0370] In some embodiments, the RNAi molecule is a small interfering RNA (siRNA) or a small hairpin RNA (shRNA). siRNAs, also known as short interfering RNA or silencing RNA, are a class of double-stranded RNA molecules, 20-25 base pairs in length, and operating within the RNA interference (RNAi) pathway. shRNAs or short hairpin RNAs are a group of artificial RNA molecules with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi).

[0371] In some embodiments, the site-specific nuclease or the RNAi molecule or the antisense oligonucleotide is introduced into the immune effector cell via a viral vector, a non-viral vector or a physical means.

[0372] In some embodiments, modifying one or more gene(s) or gene product(s) in the immune effector cell comprises silencing an mRNA transcribed from the one or more gene(s) that encode for a protein (e.g, ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3) with an RNA interference (RNAi) molecule or an antisense oligonucleotide. In some embodiments, the RNAi molecule is a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

[0373] In some embodiments, modifying one or more gene(s) or gene product(s) in the immune effector cell comprises inhibiting a protein expressed by the one or more gene(s) that encode for the protein (e.g., ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3) with one or more of a small molecule inhibitor, a peptide, an antibody or antibody fragment, or an aptamer.

[0374] In some embodiments, modifying one or more gene(s) or gene product(s) in the immune effector cell comprises activating an mRNA transcribed from the one or more gene(s) that encode for a protein (e.g, ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1) with a small activating RNA (saRNA) molecule or an antisense oligonucleotide.

[0375] In some embodiments, modifying one or more gene(s) or gene product(s) in the immune effector cell comprises activating a protein expressed by the one or more gene(s) that encode for the protein (e.g, ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1) with one or more of a small molecule activator, a peptide, an antibody or antigen-binding fragment thereof, or an aptamer.

[0376] The genetically modifying step may be conducted ex vivo or in vivo. In some embodiments, the genetically modifying step is conducted ex vivo. The method may further104324953654vlAttorney Docket No: 243734.000233include activation and / or expansion of the host cell ex vivo before, after and / or during the genetic modification. The genetically-modified immune effector cells do not require, but may be grown on the hydrogel substrate matrix (e.g., the slow-relaxing hydrogel substrate matrix) as described herein. The genetically modifying step may be conducted independently of the growing of the immune effector cells on the hydrogel substrate matrix. The genetically modifying step may be conducted in combination with the growing of the immune effector cells on the hydrogel substrate matrix.

[0377] In some embodiments, the immune effector cell is further engineered to express a chimeric antigen receptor (CAR) as described herein. In some embodiments, the CAR, antigen specific T-cell receptor, or antibody or antigen-binding fragment thereof is expressed from a transgene introduced into the immune effector cell. In some embodiments, the CAR-, antigen specific T-cell receptor-, or antibody- or antigen-binding fragment-expressing transgene is introduced into the immune effector cell using a viral vector, a non-viral vector or a physical means.

[0378] In order to reach sufficient therapeutic doses of immune effector cell compositions, immune effector cells are often subjected to one or more rounds of stimulation / activation. In some embodiments, a method of producing immune effector cells for administration to a subject comprises stimulating the immune effector cells to become activated in the presence of one or more stimulatory signals or agents (e.g., compound, small molecule, e.g., small organic molecule, nucleic acid, polypeptide, or a fragment, isoform, variant, analog, or derivative thereof). In some embodiments, a method of producing immune effector cells for administration to a subject comprises stimulating the immune effector cells to become activated and to proliferate in the presence of one or more stimulatory signals or agents.

[0379] Immune effector cells (e.g., T lymphocytes andNK cells) can be activated by inducing a change in their biologic state by which the cells express activation markers, produce cytokines, proliferate and / or become cytotoxic to target cells. All these changes can be produced by primary stimulatory signals. Co-stimulatory signals amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity.

[0380] T cells can be activated generally using methods as described, for example, in U. S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;105324953654vlAttorney Docket No: 243734.0002337,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0381] In some embodiments, the T cell based immune effector cells can be activated by binding to an agent that activates CD3ζ.

[0382] In other embodiments, a CD2-binding agent may be used to provide a primary stimulation signal to the T cells. For example, and not by limitation, CD2 agents include, but are not limited to, CD2 ligands and anti-CD2 antibodies, e.g., the T1 1.3 antibody in combination with the T1 1.1 or T1 1.2 antibody (Meuer, S. C. et al. (1984) Cell 36:897-906) and the 9.6 antibody (which recognizes the same epitope as TI 1.1) in combination with the 9-1 antibody (Yang, S. Y. et al. (1986) J. Immunol. 137:1097-1100, which is incorporated herein by reference in its entirety). Other antibodies which bind to the same epitopes as any of the above described antibodies can also be used.

[0383] In some embodiments, the immune effector cells are activated by administering phorbol myristate acetate (PMA) and ionomycine. In some embodiments, the immune effector cells are activated by administering an appropriate antigen that induces activation and then expansion. In some embodiments, PMA, ionomycin, and / or appropriate antigen are administered with CD3 induce activation and / or expansion.

[0384] In general, the activating agents used in the present invention includes, but is not limited to, an antibody, an antigen-binding fragment thereof and a proteinaceous binding molecule with antibody-like functions. Examples of (recombinant) antigen-binding fragments are Fab fragments, Fv fragments, single-chain Fv fragments (scFv), a divalent antigen-binding fragment such as an (Fab)2'-fragment, diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441, which is incorporated herein by reference in its entirety), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94, which is incorporated herein by reference in its entirety) and other domain antibodies (Holt, L. J., et al., Trends Biotechnol. (2003), 21, 11, 484-490, which is incorporated herein by reference in its entirety). The divalent antigen-binding fragment may be an (Fab)2'-fragment, or a divalent single-chain Fv fragment while the monovalent antigen-binding fragment may be selected from the group consisting of a Fab fragment, a Fv fragment, and a single-chain Fv fragment (scFv).

[0385] In some embodiments, one or more binding sites of the CD3ζ agents may be a bivalent proteinaceous artificial binding molecule such as a dimeric lipocalin mutein (z.e., duocalin). In106324953654vlAttorney Docket No: 243734.000233some embodiments the receptor binding reagent may have a single second binding site, (z.e., monovalent). Examples of monovalent agents include, but are not limited to, a monovalent antigen-binding fragment, a proteinaceous binding molecule with antibody-like binding properties or an MHC molecule. Examples of monovalent antigen-binding fragments include, but are not limited to a Fab fragment, a Fv fragment, and a single-chain Fv fragment (scFv), including a divalent single-chain Fv fragment.

[0386] The agent that specifically binds CD3 includes, but is not limited to, an anti-CD3-antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 -antibody, and a proteinaceous CD3-binding molecule with antibodylike binding properties. A proteinaceous CD3-binding molecule with antibody-like binding properties can be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, and an avimer. It also can be coupled to a bead.

[0387] In some embodiments, the activating agent (e.g., CD3-binding agents) can be present in a concentration of about 0.1 to about 10 pg / ml. In some embodiments, the activating agent (e.g., CD3 -binding agents) can be present in a concentration of about 0.2 pg / ml to about 9 pg / ml, about 0.3 pg / ml to about 8 pg / ml, about 0.4 pg / ml to about 7 pg / ml, about 0.5 pg / ml to about 6 pg / ml, about 0.6 pg / ml to about 5 pg / ml, about 0.7 pg / ml to about 4 pg / ml, about 0.8 pg / ml to about 3 pg / ml, or about 0.9 pg / ml to about 2 pg / ml. In some embodiments, the activating agent (e.g., CD3-binding agents) is administered at a concentration of about 0.1 pg / ml, about 0.2 pg / ml, about 0.3 pg / ml, about 0.4 pg / ml, about 0.5 pg / ml, about 0.6 pg / ml, about 0.7 pg / ml, about 0.8 pM, about 0.9 pg / ml, about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pM, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, or about 10 pg / ml. In some embodiments, the CD3 -binding agents can be present in a concentration of 1 pg / ml.

[0388] NK cells can be activated generally using methods as described, for example, in U. S. Patents 7,803,376, 6,949,520, 6,693,086, 8,834,900, 9,404,083, 9,464,274, 7,435,596, 8,026,097, and 8,877,182; U. S. Patent Applications US2004 / 0058445, US2007 / 0160578, US2013 / 0011376, US2015 / 0118207, and US2015 / 0037887; and PCT Patent Application WO2016 / 122147, each of which is incorporated herein by reference in its entirety.

[0389] In some embodiments, the NK based immune effector cells can be activated by, for example and not limitation, inhibition of inhibitory receptors on NK cells (e.g., KIR2DL1,107324953654vlAttorney Docket No: 243734.000233KIR2DL2 / 3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E or LILRB5 receptor).

[0390] In some embodiments, the NK based immune effector cells can be activated by, for example and not limitation, feeder cells (e.g., native K562 cells or K562 cells that are genetically modified to express 4-1BBL and cytokines such as IL15 or IL21).

[0391] In other embodiments, interferons or macrophage-derived cytokines can be used to activate NK cells. For example and not limitation, such interferons include but are not limited to interferon alpha and interferon gamma, and such cytokines include but are not limited to IL- 15, IL-2, IL-21.

[0392] In some embodiments, the NK activating agent can be present in a concentration of about 0.1 to about 10 pg / ml. In some embodiments, the NK activating agent can be present in a concentration of about 0.2 pg / ml to about 9 pg / ml, about 0.3 pg / ml to about 8 pg / ml, about 0.4 pg / ml to about 7 pg / ml, about 0.5 pg / ml to about 6 pg / ml, about 0.6 pg / ml to about 5 pg / ml, about 0.7 pg / ml to about 4 pg / ml, about 0.8 pg / ml to about 3 pg / ml, or about 0.9 pg / ml to about 2 pg / ml. In some embodiments, the NK activating agent is administered at a concentration of about 0.1 pg / ml, about 0.2 pg / ml, about 0.3 pg / ml, about 0.4 pg / ml, about 0.5 pg / ml, about 0.6 pg / ml, about 0.7 pg / ml, about 0.8 pM, about 0.9 pg / ml, about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pM, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, or about 10 pg / ml. In some embodiments, the NK activating agent can be present in a concentration of 1 pg / ml.

[0393] In some embodiments, the activating agent is attached to a solid support such as, but not limited to, a bead, an absorbent polymer present in culture plate or well or other matrices such as, but not limited to, Sepharose or glass; may be expressed (such as in native or recombinant forms) on cell surface of natural or recombinant cell line by means known to those skilled in the art.

[0394] In some embodiments, the immune effector cells are genetically modified by introducing polynucleotides and / or polypeptide (e.g., a CAR, a signaling molecule, site-specific nuclease, an RNAi molecule or an antisense oligonucleotide, or polynucleotides encoding the same). The immune effector cells can be genetically modified after stimulation / activation. In some embodiments, the immune effector cells are modified within 12 hours, 16 hours, 24 hours, 36 hours, or 48 hours of stimulation / activation. In some embodiments, the cells are modified108324953654vlAttorney Docket No: 243734.000233within 16 to 24 hours after stimulation / activation. Tn some embodiments, the immune effector cells are modified within 24 hours.

[0395] In order to genetically modify the immune effector cell, the polynucleotides and / or polypeptide (e.g., a CAR, a signaling molecule, site-specific nuclease, an RNAi molecule or an antisense oligonucleotide, or polynucleotides encoding the same) must be transferred into the host cell. Polynucleotide and / or polypeptide transfer may be via viral, non-viral gene delivery methods, or a physical method. Suitable methods for polynucleotide and / or polypeptide delivery for use with the current methods include any method known by those of skill in the art, by which a polynucleotide and / or polypeptide can be introduced into an organelle, cell, tissue or organism.

[0396] In various embodiments, polypeptides or polynucleotides (e.g., a CAR, a signaling molecule, site-specific nuclease, an RNAi molecule or an antisense oligonucleotide, or polynucleotides encoding the same) described in the present invention are introduced to the immune effector cell via a recombinant vector.

[0397] In some embodiments, the recombinant vector encoding a CAR described herein comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the recombinant vector encoding a CAR comprises the nucleotide sequence set forth in SEQ ID NO: 71, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 71. In some embodiments, the recombinant vector encoding a CAR comprises the nucleotide sequence set forth in SEQ ID NO: 71.

[0398] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, or a baculoviral vector. In some embodiments, the retroviral vector is a lentiviral vector.

[0399] In some embodiments, the immune effector cells can be transduced via retroviral transduction. References describing retroviral transduction of genes are Anderson et al., U. S. Pat. No. 5,399,346; Mann et al., Cell 33:153 (1983); Temin et al., U. S. Pat. No. 4,650,764; Temin et109324953654vlAttorney Docket No: 243734.000233al., U. S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62:1120 (1988); Temin et al., U. S. Pat. No. 5,124,263; International Patent Publication No. WO 95 / 07358, published Mar. 16, 1995, by Dougherty et al.; and Kuo et al., Blood 82:845 (1993), each of which is incorporated herein by reference in its entirety.

[0400] One method of genetic modification includes ex vivo modification. Various methods are available for transfecting cells and tissues removed from a subject via ex vivo modification. For example, retroviral gene transfer in vitro can be used to genetically modify cells removed from the subject and the cell transferred back into the subject. See e.g., Wilson et al., Science, 244:1344-1346, 1989 and Nabel et al., Science, 244(4910): 1342-1344, 1989, both of which are incorporated herein by reference in their entirety. In some embodiments, the immune effector cells may be removed from the subject and transfected ex vivo using the polynucleotides (e g., expression vectors) of the invention. In some embodiments, the immune effector cells obtained from the subject can be transfected or transduced with the polynucleotides (e.g., expression vectors) of the invention and then administered back to the subject.

[0401] In some embodiments, polynucleotides and / or polypeptides are transferred to the cell in a non-viral vector. In some embodiments, the non-viral vector is a transposon. In some embodiments, the transposon is a sleeping beauty transposon or PiggyBac transposon.

[0402] Nucleic acid vaccines may also be used to transfer polynucleotides into the immune effector cells. Such vaccines include, but are not limited to non-viral polynucleotide vectors, “naked” DNA and RNA, and viral vectors. Methods of genetically modifying cells with these vaccines, and for optimizing the expression of genes included in these vaccines are known to those of skill in the art.

[0403] In some embodiments, the polynucleotide(s) is operatively linked to at least one regulatory element for expression of the gene product (e.g., a CAR, a signaling molecule, sitespecific nuclease, an RNAi molecule). The regulatory element can be capable of mediating expression of the gene product in the host cell (e.g., immune effector cell). Regulatory elements include, but are not limited to, promoters, enhancers, initiation sites, polyadenylation (poly A) tails, IRES elements, response elements, and termination signals. In some embodiments, the regulatory element regulates expression of the gene product. In some embodiments, the regulatory element increased the expression of the gene product. In some embodiments, the regulatory element increased the expression of the gene product once the host cell (e.g., immune110324953654vlAttorney Docket No: 243734.000233effector cell) is activated. Tn some embodiments, the regulatory element decreases expression of the gene product. In some embodiments, the regulatory element decreases expression of the gene product once the host cell (e.g., immune effector cell) is activated.

[0404] In various embodiments, polypeptides or polynucleotides (e.g., a CAR, a signaling molecule, site-specific nuclease, an RNAi molecule or an antisense oligonucleotide, or polynucleotides encoding the same) are introduced into the modified immune effector cell using a physical means. In some embodiments, the physical means is electroporation, microinjection, magnetofection, ultrasound, a ballistic or hydrodynamic method, or a combination thereof.

[0405] Electroporation is a method for polynucleotide and / or polypeptide delivery. See e.g., Potter et al., (1984) Proc. Nat’ I Acad. Set. USA, 81, 7161-7165 and Tur-Kaspa et al., (1986) Mol. Cell Biol., 6, 716-718, both of which are incorporated herein in their entirety for all purposes. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some embodiments, cell wall-degrading enzymes, such as pectindegrading enzymes, can be employed to render the immune effector cells more susceptible to genetic modification by electroporation than untreated cells. See e.g., U. S. Pat. No. 5,384,253, incorporated herein by reference in its entirety for all purposes.

[0406] In vivo electroporation involves a basic injection technique in which a vector is injected intradermally in a subject. Electrodes then apply electrical pulses to the intradermal site causing the cells localized there (e.g., resident dermal dendritic cells), to take up the vector. These tumor antigen-expressing dendritic cells activated by local inflammation can then migrate to lymph-nodes.

[0407] Methods of electroporation for use with this invention include, for example, Sardesai, N. Y., and Weiner, D. B., Current Opinion in Immunotherapy 23:421-9 (2011) and Ferraro, B. et al., Human Vaccines 7: 120-127 (2011), both of which are hereby incorporated by reference herein in their entirety for all purposes.

[0408] Another method for polynucleotide and / or polypeptide transfer includes injection. In some embodiments, a polypeptide, a polynucleotide or viral vector may be delivered to a cell, tissue, or organism via one or more injections (e.g., a needle injection). Non-limiting methods of injection include injection of a composition (e.g., a saline based composition). Polynucleotides and / or polynucleotides can also be introduced by direct microinjection. Non-limiting sites of injection include, subcutaneous, intradermal, intramuscular, intranodal (allows for direct delivery111324953654vlAttorney Docket No: 243734.000233of antigen to lymphoid tissues), intravenous, intraprotatic, intratumor, intralymphatic (allows direct administration of DCs) and intraperitoneal. It is understood that proper site of injection preparation is necessary (e.g., shaving of the site of injection to observe proper needle placement).

[0409] Additional methods of polynucleotide and / or polypeptide transfer include liposome-mediated transfection (e.g., polynucleotide entrapped in a lipid complex suspended in an excess of aqueous solution. See e.g., Ghosh and Bachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, pp. 87-104). Also contemplated is a polynucleotide and / or polypeptide complexed with Lipofectamine, or Superfect); DEAE-dextran (e.g., a polynucleotide is delivered into a cell using DEAE-dextran followed by polyethylene glycol. See e.g., Gopal, T. N., Mol Cell Biol. 1985 May; 5(5): 1188-90); calcium phosphate (e.g., polynucleotide is introduced to the cells using calcium phosphate precipitation. See e.g., Graham and van der Eb, (1973) Virology, 52, 456-467; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987), and Rippe et al., Mol. Cell Biol., 10:689-695, 1990); sonication loading (introduction of a polynucleotide by direct sonic loading. See e.g., Fechheimer et al., (1987) Proc. Nat'l Acad. Sci. USA, 84, 8463-8467); microprojectile bombardment (e.g., one or more particles may be coated with at least one polynucleotide and / or polypeptide and delivered into cells by a propelling force. See e.g., U. S. Pat. No. 5,550,318; U. S. Pat. No. 5,538,880; U. S. Pat. No. 5,610,042; and PCT Application WO 94 / 09699; Klein et al., (1987) Nature, 327, 70-73, Yang et al., (1990) Proc. Nat'l Acad. Sci. USA, 87, 9568-9572); and receptor-mediated transfection (e.g., selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell using cell type-specific distribution of various receptors. See e.g., Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994; Myers, EPO 0273085; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993; Nicolau et al., (1987) Methods Enzymol., 149, 157-176), each reference cited here is incorporated by reference in their entirety for all purposes.

[0410] In some embodiments, the immune effector cell is activated and / or expanded ex vivo.

[0411] After the immune effector cells are activated and transduced, the cells are cultured to proliferate. T cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.112324953654vlAttorney Docket No: 243734.000233

[0412] Agents that can be used for the expansion of T cells can include interleukins, such as IL-2, IL-7, IL-15, or IL-21 (see for example Cornish et al. 2006, Blood. 108(2):600-8, Bazdar and Sieg, 2007, Journal of Virology, 2007, 81(22): 12670-12674, Battalia et al, 2013, Immunology, 139(1): 109-120, each of which is incorporated by reference in their entirety for all purposes). Other illustrative examples for agents that may be used for the expansion of T cells are agents that bind to CD8, CD45 or CD90, such as aCD8, aCD45 or aCD90 antibodies.Illustrative examples of T cell population including antigen-specific T cells, T helper cells, cytotoxic T cells, memory T cell (an illustrative example of memory T cells are CD62L+CD8+ specific central memory T cells) or regulatory T cells (an illustrative example of Treg are CD4+CD25+CD45RA+ Treg cells).

[0413] Additional agents that can be used to expand T lymphocytes includes methods as described, for example, in U. S. Patents 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; and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0414] In some embodiments, the agent(s) used for expansion (e.g., IL-7, IL-15) are administered at about 20 units / ml to about 200 units / ml. In some embodiments, the agent(s) used for expansion (e.g., IL-7, IL-15) are administered at about 25 units / ml to about 190 units / ml, about 30 units / ml to about 180 units / ml, about 35 units / ml to about 170 units / ml, about 40 units / ml to about 160 units / ml, about 45 units / ml to about 150 units / ml, about 50 units / ml to about 140 units / ml, about 55 units / ml to about 130 units / ml, about 60 units / ml to about 120 units / ml, about 65 units / ml to about 110 units / ml, about 70 units / ml to about 100 units / ml, about 75 units / ml to about 95 units / ml, or about 80 units / ml to about 90 units / ml. In some embodiments, the agent(s) used for expansion (e.g., IL-7, IL-15) are administered at about 20 units / ml, about 25 units / ml, about 30 units / ml, 35 units / ml, 40 units / ml, 45 units / ml, about 50 units / ml, about 55 units / ml, about 60 units / ml, about 65 units / ml, about 70 units / ml, about 75 units / ml, about 80 units / ml, about 85 units / ml, about 90 units / ml, about 95 units / ml, about 100 units / ml, about 105 units / ml, about 110 units / ml, about 115 units / ml, about 120 units / ml, about 125 units / ml, about 130 units / ml, about 135 units / ml, about 140 units / ml, about 145 units / ml, about 150 units / ml, about 155 units / ml, about 160 units / ml, about 165 units / ml, about 170 units / ml, about 175 units / ml, about 180 units / ml, about 185 units / ml, about 190 units / ml, about113324953654vlAttorney Docket No: 243734.000233195 units / ml, or about 200 units / ml. In some embodiments, the agent(s) used for expansion (e.g., IL-7, IL-15) are administered at about 5 ng / ml to about 10 ng / ml. In some embodiments, the agent(s) used for expansion (e.g., IL-7, IL-15) are administered at about 5.5 ng / ml to about 9.5 ng / ml, about 6 ng / ml to about 9 ng / ml, about 6.5 ng / ml to about 8.5 ng / ml, or about 7 ng / ml to about 8 ng / ml. In some embodiments, the agent(s) used for expansion (e.g., IL-7, IL- 15) are administered at about 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9, ng / ml, or 10 ng / ml.

[0415] After the immune effector cells are activated and transduced, the cells are cultured to proliferate. NK cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.

[0416] Agents that can be used for the expansion of natural killer cells can include agents that bind to CD 16 or CD56, such as for example aCD16 or aCD56 antibodies. In some embodiments, the binding agent includes antibodies (see for example Hoshino et al, Blood. 1991 Dec. 15;78(12):3232-40.). Other agents that may be used for expansion of NK cells may be IL- 15 (see for example Vitale et al. 2002. The Anatomical Record. 266:87-92, which is incorporated by reference in their entirety for all purposes).

[0417] Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media (MEM), RPMI Media 1640, Lonza RPMI 1640, Advanced RPMI, Clicks, AIM-V, DMEM, a-MEM, F-12, TexMACS, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion).

[0418] Examples of other additives for immune effector cell expansion include, but are not limited to, surfactant, piasmanate, pH buffers such as HEPES, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol, Antibiotics (e.g., penicillin and streptomycin), are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2).Pharmaceutical compositions

[0419] In some embodiments, the compositions comprise one or more polypeptides, polynucleotides, vectors comprising same, and cell compositions, as disclosed herein.114324953654vlAttorney Docket No: 243734.000233Compositions include, but are not limited to pharmaceutical compositions. In some embodiments, the compositions of the present invention comprise an amount of immune effector cells manufactured by the methods disclosed herein.

[0420] In another aspect, provided herein is a pharmaceutical composition comprising the immune effector cell as described herein and a pharmaceutically acceptable carrier and / or excipient.

[0421] Examples of pharmaceutical carriers include but are not limited to sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

[0422] Compositions comprising immune effector cells disclosed herein may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0423] Compositions comprising immune effector cells disclosed herein may comprise one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0424] In some embodiments, the compositions are formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural or intramuscular administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile. In some embodiments, the composition is reconstituted from a lyophilized preparation prior to administration.115324953654vlAttorney Docket No: 243734.000233

[0425] In some embodiments, the immune effector cells may be mixed with substances that adhere or penetrate then prior to their administration, e.g., but not limited to, nanoparticles.

[0426] In some aspects, the method(s) for genetically modifying immune effector cells is used in combination with the method(s) for manufacturing immune effector cells with improved efficacy, comprising mechanically priming the immune effector cells in the presence of a viscoelastic hydrogel substrate matrix comprising crosslinked hydrogels. The methods, including individual steps within the methods, may be applied simultaneously or sequentially, in any order, and with any length of time between them. The methods, including individual steps within the methods, may be performed at the same time to maximize efficiency, or one after the other if the situation requires it, without specifying which must occur first. There is also no restriction on timing as one method, including individual steps within the method, may follow the other method, including individual steps within the method, immediately, after a brief pause, or after an extended interval, depending on operational needs, resource availability, strategic considerations, or any number of factors. In some embodiments, the genetically modified immune effector cells do not require mechanical priming of the immune effector cells to serve purposes as intended.Methods for treating disease

[0427] In another aspect, provided herein is a method of treating a disease in a subject in need thereof comprising administering to the subject an effective amount of the immune effector cells as described herein or the pharmaceutical composition as described herein. In some embodiments, the immune effector cells are prepared by the methods as disclosed herein.

[0428] Immune effector cells can be activated and / or expanded ex vivo for use in adoptive cellular immunotherapy in which infusions of such cells have been shown to have anti-disease reactivity in a disease-bearing subject. The compositions and methods of this invention can be used to generate a population of immune effector cells (e.g., T lymphocyte or natural killer cells) with enhanced immune cell function for use in immunotherapy in the treatment of the disease.

[0429] In some embodiments, the immune effector cell is an autologous cell. In some embodiments, the immune effector cell is an allogeneic cell.

[0430] In some embodiments, the disease is a cancer, an infectious disease, an inflammatory disorder, or an autoimmune disease.116324953654vlAttorney Docket No: 243734.000233

[0431] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The term “cancer” includes, for example, the soft tissue tumors (e.g., lymphomas), and tumors of the blood and blood-forming organs (e.g., leukemias), and solid tumors, which is one that grows in an anatomical site outside the bloodstream (e.g., carcinomas). Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (e.g., osteosarcoma or rhabdomyosarcoma), and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), adenosquamous cell carcinoma, lung cancer (e.g., including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (e.g., including gastrointestinal cancer, pancreatic cancer), cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, primary or metastatic melanoma, multiple myeloma and B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, brain (e.g., high grade glioma, diffuse pontine glioma, ependymoma, neuroblastoma, or glioblastoma), as well as head and neck cancer, and associated metastases. Additional examples of cancer can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online edition at internet website of Merck Manuals); and SEER Program Coding and Staging Manual 2016, each of which are incorporated by reference in their entirety for all purposes.

[0432] The term “tumor” refers to a benign or malignant abnormal growth of tissue. The term “tumor” includes cancer.

[0433] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is breast, prostate, urinary bladder, skin, lung, ovary, sarcoma, or brain cancer.

[0434] In some embodiments, the cancer is a central nervous system (CNS) tumor.

[0435] In some embodiments, the CNS tumor is a pediatric CNS malignancy.117324953654vlAttorney Docket No: 243734.000233

[0436] In some embodiments, the pediatric CNS malignancy is Diffuse Midline Glioma (DMG).

[0437] In some embodiments, the CNS tumor is medulloblastoma.

[0438] In some embodiments, the CNS tumor is an ependymoma.

[0439] In some embodiments, the cancer is a cancer expressing B7-H3, GD2, IL13Ra2, EphA2, or HER2. The methods described herein may be used to treat a cancer expressing B7-H3, GD2, IL13Ra2, EphA2, or HER2.

[0440] Cancers expressing B7-H3 may include, but are not limited to, central nervous system tumors such as glioblastoma, other high-grade gliomas, medulloblastoma, ependymoma, Diffuse Midline Glioma (DMG), and diffuse intrinsic pontine glioma (DIPG); pediatric solid tumors such as neuroblastoma, Ewing sarcoma, rhabdomyosarcoma, and Wilms tumor; lung cancers including non-small cell lung cancer (both adenocarcinoma and squamous cell) and small cell lung cancer; breast cancers, especially triple-negative breast cancer but also some HER2-positive and hormone receptor-positive tumors; prostate adenocarcinoma, particularly in advanced or castration-resistant disease; gastrointestinal and hepatobiliary cancers including colorectal cancer, gastric cancer, pancreatic ductal adenocarcinoma, esophageal cancer, cholangiocarcinoma, and hepatocellular carcinoma; genitourinary and gynecologic cancers such as bladder (urothelial) carcinoma, renal cell carcinoma, ovarian cancer, and endometrial cancer; head and neck and other solid tumors including head and neck squamous cell carcinoma, salivary gland tumors, melanoma, and various soft tissue sarcomas; and certain hematologic malignancies such as subsets of acute myeloid leukemia, acute lymphoblastic leukemia, and some lymphomas (including T-cell and NK / T-cell lymphomas).

[0441] Cancers expressing GD2 may include, but are not limited to, neuroblastoma, osteosarcoma, Ewing sarcoma, soft tissue sarcomas (including rhabdomyosarcoma), malignant melanoma, small cell lung cancer, glioblastoma and other high-grade gliomas, medulloblastoma and other pediatric brain tumors, retinoblastoma, and certain other pediatric and adult solid tumors.

[0442] Cancers expressing IL13Ra2 may include, but are not limited to, brain cancers such as glioblastoma, colon cancer, renal cell carcinoma, pancreatic cancer, melanoma, head and neck cancer, mesothelioma, and ovarian cancer.118324953654vlAttorney Docket No: 243734.000233

[0443] Cancers expressing EphA2 may include, but are not limited to, sarcomas such as rhabdomyosarcoma, osteosarcoma, and Ewings sarcoma; breast, prostate, urinary bladder, skin cancers including melanoma, lung cancer, liver cancer, ovarian cancer, stomach cancer, colorectal cancer, thyroid cancer, head and neck cancer, cervical cancer, pancreatic cancer, endometrial cancer, and brain cancers.

[0444] Cancers expressing HER2 may include, but are not limited to, sarcomas such as angiosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, or synovial sarcoma; brain cancers such as glioblastoma; breast, prostate, lung, and colon cancers or epithelial cancers / carcinomas such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer; cancers of the genitourinary tract such as ovarian cancer, endometrial cancer, cervical cancer and kidney cancer; lung cancer, gastric cancer, cancer of the small intestine, liver cancer, pancreatic cancer, gall bladder cancer, cancers of the bile duct, esophagus cancer, cancer of the salivary glands and cancer of the thyroid gland.

[0445] In some embodiments, the method comprises:a) isolating an immune effector cell from the subject or a donor;b) modifying a transcription factor gene or gene product in the immune effector cell; and c) introducing the immune effector cell into the subject.

[0446] In some embodiments, step b) comprises (i) deleting or modifying the gene or gene product of ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell so that the transcription factor activity is decreased and / or (i) overexpressing or modifying the gene or gene product of ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell so that the transcription factor activity is increased.

[0447] In some embodiments, the method further comprises genetically modifying the immune effector cell to express a chimeric antigen receptor (CAR), antigen specific T-cell receptor, or antibody or antigen-binding fragment thereof that is capable of binding specifically to an antigen.

[0448] In cases where the immune effector cell is isolated from a donor, the method may further include a method to prevent graft vs host disease (GVHD) and the immune effector cell rejection.119324953654vlAttorney Docket No: 243734.000233

[0449] In some embodiments of any of the methods described herein, the composition is administered in a therapeutically effective amount. The dosages of the composition administered in the methods of the invention will vary widely, depending upon the subject’s physical parameters, the frequency of administration, the manner of administration, the clearance rate, and the like. The initial dose may be larger, and might be followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. It is contemplated that a variety of doses will be effective to achieve in vivo persistence of immune effector cells. It is also contemplated that a variety of doses will be effective to improve in vivo effector function of immune effector cells.

[0450] In some embodiments, the composition is administered to a subject (e.g., a mammal) over a defined time course. In some embodiments, the compositions are administered to a subject in multiple doses. The multiple doses of the composition may include sequential administrations. The sequential administrations may include the administration of a composition to the subject (e.g., a mammal) at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). In some embodiments, the methods comprise sequentially administering to the subject (e.g., a mammal) a single initial dose of a composition, followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition. The initial, secondary, and tertiary doses may all contain the same amount of the composition. The initial, secondary, and tertiary doses may differ from one another in terms of frequency of administration. In some embodiments, the amount of composition contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In some embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses followed by subsequent doses that are administered on a less frequent basis (e.g., maintenance doses). In some embodiments, the amount of a composition is gradually escalated over a defined time course to avoid or reduce the possibility of severe side effects.

[0451] The optimal therapeutically effective amount of a composition of this invention may be determined experimentally, taking into consideration the form in which the drug is administered, the indication toward which the administration is directed, the exact mode of120324953654vlAttorney Docket No: 243734.000233administration, the subject involved (e g., body weight, health, age, sex, etc.), and the preference and experience of the physician or veterinarian in charge.

[0452] For any pharmaceutical composition used in the methods as described herein, doseresponse curves derived from animal systems can be used to determine testing doses for administration to humans, veterinary animals, or companion animals (for example and not limitation, e.g., cats, dogs, horses, cows, sheep, pigs, amphibians, reptiles, avian species, etc.). In safety determinations for each composition, the frequency and dose of administration should meet or exceed those anticipated for use in any clinical trial.

[0453] In some embodiments, a composition comprising the immune effector cells manufactured by the methods described herein may be administered at a dosage of 102to 1010cells / kg body weight, 105to 109cells / kg body weight, 105to 108cells / kg body weight, 105to 107cells / kg body weight, 107to 109cells / kg body weight, or 107to 108cells / kg body weight, including all integer values within those ranges. The number of immune effector cells will depend on the therapeutic use for which the composition is intended for.

[0454] Immune effector cells may be administered multiple times at dosages listed above. The immune effector cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.

[0455] The compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth.

[0456] It is also contemplated that when used to treat various diseases, the compositions and methods of the present disclosure can be utilized with other therapeutic methods / agents suitable for the same or similar diseases / disorders. Such other therapeutic methods / agents can be coadministered (simultaneously or sequentially) to generate additive or synergistic effects. Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0457] The term “therapeutic agent” refers to a chemical compound or biological molecule that includes, without limitation, small molecules, peptides, proteins, and antibodies that can be used to treat a condition, disease, or disorder or reduce the symptoms of the condition, disease, or disorder.121324953654vlAttorney Docket No: 243734.000233

[0458] In some embodiments of any of the methods described herein, the method further comprises administering to the subject one or more additional compounds selected from the group consisting of immuno-suppressives, biologicals, probiotics, prebiotics, and cytokines (e.g., IFN or IL-2).

[0459] As a non-limiting example, the invention can be combined with other therapies that block inflammation (e.g., via blockage of IL1, INFa / p, IL6, TNF, IL23, etc.).

[0460] The methods and compositions of the invention can be combined with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GV AX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 4- IBB, 0X40, etc.). The methods of the invention can be also combined with other treatments that possess the ability to modulate NKT function or stability, including but not limited to CD Id, CD Id-fusion proteins, CD Id dimers or larger polymers of CD Id either unloaded or loaded with antigens, CD 1 d-chimeric antigen receptors (CDld-CAR), or any other of the five known CD1 isomers existing in humans (CDla, CDlb, CDlc, CDle). The methods of the invention can also be combined with other treatments such as midostaurin, enasidenib, or a combination thereof.

[0461] Methods of the invention can be combined with additional immunotherapies and therapies. For example, when used for treating cancer, the compositions of the invention can be used in combination with conventional cancer therapies, such as, e g., surgery, radiotherapy, chemotherapy or combinations thereof, depending on type of the tumor, patient condition, other health issues, and a variety of factors. In certain aspects, other therapeutic agents useful for combination cancer therapy with the inhibitors of the invention include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, e.g., TNP-470, platelet factor 4, thrombospondin- 1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000). In one embodiment, the immune effector cells of the invention can be used in combination with a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocki...

Claims

Attorney Docket No: 243734.000233WHAT IS CLAIMED IS:

1. An immune effector cell, wherein the transcriptional activity of transcription factor(s) ARID1A, DNMT1, ELF5, FLT1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof is inhibited in the immune effector cell as compared to a control cell in which the transcriptional activity of transcription factor(s) is not inhibited.

2. The immune effector cell of claim 1, wherein the transcription factor(s) are DNMT1, FLI1, LEF1, or a combination thereof.

3. The immune effector cell of claim 1 or claim 2, wherein a transcription factor gene is deleted or defective so that no detectable functional transcription factor is expressed.

4. The immune effector cell of claim 1 or claim 2, wherein the enzymatic activity of the transcription factor(s) is inhibited in the immune effector cell.

5. The immune effector cell of claim 4, wherein the enzymatic activity of the transcription factor(s) is inhibited by exposing the cell to a transcription factor inhibitor.

6. The immune effector cell of claim 4, wherein the transcription factor gene is mutated so that the enzymatic activity of the transcription factor is inhibited.

7. The immune effector cell of claim 6, wherein the catalytic domain of the transcription factor is mutated.

8. The immune effector cell of any one of claims 4-7, wherein the level of enzymatic activity of the transcription factor in the immune effector cell is decreased by 50% or more.

9. An immune effector cell, wherein the transcriptional activity of transcription factor(s) ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof170324953654vlAttorney Docket No: 243734.000233is activated in the immune effector cell as compared to a control cell in which the transcriptional activity of transcription factor(s) is not activated.

10. The immune effector cell of claim 9, wherein the enzymatic activity of the transcription factor(s) is activated in the immune effector cell.

11. The immune effector cell of claim 10, wherein the enzymatic activity of the transcription factor(s) is activated by exposing the cell to a transcription factor activator.

12. The immune effector cell of claim 9, wherein the transcription factor gene is mutated so that the enzymatic activity of the transcription factor is increased.

13. The immune effector cell of any one of claims 10-12, wherein the level of enzymatic activity of the transcription factor in the immune effector cell is increased by 50% or more.

14. The immune effector cell of any one of claims 1-13, wherein the immune effector cell is a T cell.

15. The immune effector cell of claim 14, wherein the T cell is selected from a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell,an T cell receptor (TCR) T cell, a natural killer T (NKT) cell, a y8 T cell, a memory T cell, a T-helper cell, and a regulatory T cell (Treg).

16. The immune effector cell of any one of claims 1-13, wherein the immune effector cell is a stem cell that is capable of differentiating into an immune cell.

17. The immune effector cell of claim 16, wherein the stem cell is an induced pluripotent stem cell (iPSC).

18. The immune effector cell of any one of claims 1-13, wherein the immune effector cell is a natural killer (NK) cell.171324953654vlAttorney Docket No: 243734.00023319. The immune effector cell of any one of claims 1-18, wherein the immune effector cell further comprises at least one surface molecule capable of binding specifically to an antigen.

20. The immune effector cell of claim 19, wherein the antigen is selected from a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, a prion antigen, and an antigen associated with an inflammation or an autoimmune disease.

21. The immune effector cell of claim 20, wherein the tumor antigen is B7 Homolog 3 (B7-H3), disialoganglioside variant 2 (GD2), IL13Ra2, erythropoietin-producing human hepatocellular receptor A2 (EphA2), or human epidermal growth factor receptor 2 (HER2).

22. The immune effector cell of any one of claims 1-18, wherein the cell further comprises a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigenbinding fragment thereof.

23. The immune effector cell of claim 22, wherein the cell further comprises a chimeric antigen receptor (CAR).

24. The immune effector cell of claim 23, wherein the CAR comprises (i) an extracellular antigen-binding domain and (ii) a transmembrane domain.

25. The immune effector cell of claim 24, wherein the CAR further comprises a cytoplasmic domain.

26. The immune effector cell of claim 24 or claim 25, wherein the extracellular antigen-binding domain comprises an antibody or an antigen-binding fragment.

27. The immune effector cell of claim 26, wherein the extracellular antigen-binding domain comprises an scFv capable of binding to B7 Homolog 3 (B7-H3).172324953654vlAttorney Docket No: 243734.00023328. The immune effector cell of claim 27, wherein the scFv capable of binding to B7-H3 comprises the amino acid sequence of SEQ ID NO: 2.

29. The immune effector cell of any one of claims 22-28, wherein the extracellular antigenbinding domain further comprises a leader sequence.

30. The immune effector cell of claim 29, wherein the leader sequence comprises the amino acid sequence of SEQ ID NO: 3.

31. The immune effector cell of any one of claims 22-30, wherein the transmembrane domain is derived from CD3ζ, CD28, CD4, or CD8α.

32. The immune effector cell of claim 31, wherein the transmembrane domain is derived from CD3ζ and comprises the amino acid sequence SEQ ID NO: 4.

33. The immune effector cell of claim 31, wherein the transmembrane domain is derived from CD28 and comprises the amino acid sequence SEQ ID NO: 5.

34. The immune effector cell of claim 31, wherein the transmembrane domain is derived from CD8α and comprises the amino acid sequence SEQ ID NO: 6 or SEQ ID NO: 17.

35. The immune effector cell of claim 31, wherein the transmembrane domain is derived from CD4 and comprises the amino acid sequence SEQ ID NO: 7.

36. The immune effector cell of any one of claims 22-35, wherein the CAR further comprises a linker domain between the extracellular antigen-binding domain and the transmembrane domain.

37. The immune effector cell of claim 36, wherein the linker domain comprises a hinge region.

38. The immune effector cell of claim 37, wherein the hinge region comprises the amino acid sequence SEQ ID NO: 8.173324953654vlAttorney Docket No: 243734.00023339. The immune effector cell of claim 37, wherein the hinge region comprises the amino acid sequence SEQ ID NO: 9.

40. The immune effector cell of claim 37, wherein the hinge region comprises the amino acid sequence SEQ ID NO: 16.

41. The immune effector cell of any one of claims 36-40, wherein the linker domain comprises the amino acid sequence SEQ ID NO: 10.

42. The immune effector cell of any one of claims 22-41, wherein the CAR cytoplasmic domain comprises one or more lymphocyte activation domains.

43. The immune effector cell of claim 42, wherein the lymphocyte activation domain is derived from DAP 10, DAP 12, Fc epsilon receptor I y chain (FCER1G), CD35, CD3e, CD3y, CD3C, CD27, CD28, CD40, CD134, CD137, CD226, CD79A, ICOS, orMyD88.

44. The immune effector cell of claim 43, wherein the lymphocyte activation domain is derived from CD3ζ and comprises the amino acid sequence SEQ ID NO: 11.

45. The immune effector cell of any one of claims 22-44, wherein the CAR cytoplasmic domain comprises one or more co-stimulatory domains.

46. The immune effector cell of claim 45, wherein the co-stimulatory domain is derived from CD28 and comprises the amino acid sequence SEQ ID NO: 12.

47. The immune effector cell of any one of claims 1-46, wherein the immune effector cell is an allogeneic cell.

48. The immune effector cell of any one of claims 1-46, wherein the immune effector cell is an autologous cell.174324953654vlAttorney Docket No: 243734.00023349. The immune effector cell of any one of claims 1-48, wherein the immune effector cell is isolated from a subject having a disease.

50. The immune effector cell of claim 49, wherein the disease is a cancer, an infectious disease, an inflammatory disorder, or an autoimmune disease.

51. The immune effector cell of claim 50, wherein the cancer is a cancer expressing B7-H3, GD2, IL13Ra2, orEphA2, or HER2.

52. The immune effector cell of any one of claims 1-51, wherein the immune effector cell is derived from a blood, marrow, tissue, or a tumor sample.

53. A pharmaceutical composition comprising the immune effector cell of any one of claims 1-52 and a pharmaceutically acceptable carrier and / or excipient.

54. A method for generating the immune effector cell of any one of claims 1-52, said method comprising (a) deleting or modifying the gene or gene product of ARID! A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell so that the transcription factor activity is decreased and / or (b) overexpressing or modifying the gene or gene product of ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell so that the transcription factor activity is increased.

55. The method of claim 54, wherein the transcription factor gene in the immune effector cell is deleted, overexpressed, or modified as a result of an activity of a site-specific nuclease.

56. The method of claim 55, wherein the site-specific nuclease is an RNA-guided endonuclease.175324953654vlAttorney Docket No: 243734.00023357. The method of claim 56, wherein the RNA-guided endonuclease is a Cas9 protein, Cpfl (Cast 2a) protein, C2cl protein, C2c3 protein, or C2c2 protein.

58. The method of claim 57, wherein the RNA-guided endonuclease is a Cas9 protein.

59. The method of claim 58, wherein the Cas9 protein is programmed with a guide RNA (gRNA) that comprises a nucleotide sequence encoded by SEQ ID NOs: 18-24.

60. The method of claim 55, wherein the site-specific nuclease is a zinc finger nuclease, a TALEN nuclease, or mega-TALEN nuclease.

61. The method of claim 54, wherein the transcription gene product in the immune effector cell is deleted or modified as a result of an activity of an RNA interference (RNAi) molecule or an antisense oligonucleotide.

62. The method of claim 61, wherein the RNAi molecule is a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

63. The method of any one of claims 55-62, wherein the site-specific nuclease or the RNAi molecule or the antisense oligonucleotide is introduced into the immune effector cell via a viral vector, a non-viral vector or a physical means.

64. The method of any one of claims 54-63, wherein the method further comprises genetically modifying the immune effector cell to express a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof that is capable of binding specifically to an antigen.

65. The method of claim 64, wherein the CAR, antigen specific T-cell receptor, or antibody or antigen-binding fragment thereof is expressed from a transgene introduced into the immune effector cell.176324953654vlAttorney Docket No: 243734.00023366. The method of claim 65, wherein the CAR-, antigen specific T-cell receptor-, or antibody-or antigen-binding fragment-expressing transgene is introduced into the immune effector cell using a viral vector, a non-viral vector or a physical means.

67. The method of claim 63 or 66, wherein the viral vector is a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, or a baculoviral vector.

68. The method of claim 67, wherein the retroviral vector is a lentiviral vector.

69. The method of claim 63 or 66, wherein the non-viral vector is a transposon.

70. The method of claim 69, wherein the transposon is a sleeping beauty transposon or PiggyBac transposon.

71. The method of claim 63 or 66, wherein the physical means is electroporation, microinjection, magnetofection, ultrasound, a ballistic or hydrodynamic method, or a combination thereof.

72. The method of any one of claims 54-71, wherein the immune effector cell is activated and / or expanded ex vivo.

73. A method of treating a disease in a subject in need thereof comprising administering to the subject an effective amount of the immune effector cells of any one of claims 1-52 or the pharmaceutical composition of claim 53.

74. The method of claim 73, wherein the immune effector cell is an autologous cell.

75. The method of claim 73, wherein the immune effector cell is an allogeneic cell.

76. The method of any one of claims 73-75, wherein the disease is a cancer, an infectious disease, an inflammatory disorder, or an autoimmune disease.177324953654vlAttorney Docket No: 243734.00023377. The method of claim 76, wherein the cancer is a solid tumor.

78. The method of claim 76, wherein the cancer is breast, prostate, urinary bladder, skin, lung, ovary, sarcoma, or brain cancer.

79. The method of claim 78, wherein the cancer is a central nervous system (CNS) tumor.

80. The method of claim 79, wherein the CNS tumor is a pediatric CNS malignancy.

81. The method of claim 80, wherein the pediatric CNS malignancy is Diffuse Midline Glioma (DMG).

82. The method of claim 79, wherein the CNS tumor is medulloblastoma.

83. The method of claim 79, wherein the CNS tumor is an ependymoma.

84. The method of claim 78, wherein the cancer is a cancer expressing B7-H3, GD2, IL13Ru2, EphA2, or HER2.

85. The method of any one of claims 73-84, wherein the method comprises:a) isolating an immune effector cell from the subject or a donor;b) modifying a transcription factor gene or gene product in the immune effector cell; and c) introducing the immune effector cell into the subject.

86. The method of claim 85, wherein step b) comprises (i) deleting or modifying the gene or gene product of ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell so that the transcription factor activity is decreased and / or (b) overexpressing or modifying the gene or gene product of ATF6,178324953654vlAttorney Docket No: 243734.000233EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell so that the transcription factor activity is increased.

87. The method of claim 85 of claim 86, wherein the method further comprises genetically modifying the immune effector cell to express a chimeric antigen receptor (CAR), antigen specific T-cell receptor, or antibody or antigen-binding fragment thereof that is capable of binding specifically to an antigen.

88. The method of any one of claims 73-86, wherein the subject is a human.

89. A method of enhancing an antitumor activity of an immune effector cell, comprising: a) inhibiting the transcriptional activity of transcription factor(s) ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell and / orb) activating the transcriptional activity of transcription factor(s) ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell.

90. A method of inhibiting mechanical suppression of an immune effector cell, comprising: a) inhibiting the transcriptional activity of transcription factor(s) ARID1A, DNMT1, ELF5, FLI1, GRHL3, GTF2IRD1, LEF1, MEF2B, SON, ZFHX3, or a combination thereof in the immune effector cell and / or91. b) activating the transcriptional activity of transcription factor(s) ATF6, EN1, HMGA2, IRF1, POU3F4, REL, SRY, TFAP4, USF2, YY1, or a combination thereof in the immune effector cell.

92. The method of claim 89 or claim 90, wherein the method further comprises genetically modifying the immune effector cell to express a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof that is capable of binding to an antigen.179324953654vlAttorney Docket No: 243734.00023393. The method of any one of claims 89-92, wherein the method comprises activation and / or expansion of the immune effector cell ex vivo.

94. The method of any one of claims 89-93, wherein the immune effector cell is a T cell, NK cell, or an induced pluripotent stem cell (iPSC).

95. A method for manufacturing an immune effector cell with improved efficacy, comprising mechanically priming the immune effector cell in the presence of a viscoelastic hydrogel substrate matrix comprising crosslinked hydrogels.

96. A method for manufacturing an immune effector cell resistant to mechanical suppression, comprising mechanically priming the immune effector cell in the presence of a viscoelastic hydrogel substrate matrix comprising crosslinked hydrogels.

97. The method of claim 95 or claim 96, wherein the viscoelastic hydrogel substrate matrix comprises poly(ethylene-glycol)-based hydrogels, alginate-based hydrogels, collagen-I based platforms, hyaluronic acid and collagen-I hydrogels, and / or elastin-like protein (ELP) based hydrogel systems.

98. The method of any one of claims 95-97, wherein the crosslinked hydrogel is a poly(ethylene-glycol)-based hydrogel, optionally an 8-arm poly(ethylene-glycol) macromer functionalized with reactive end groups.

99. The method of any one of claims 95-98, wherein the viscoelastic hydrogel substrate matrix comprises dynamic crosslinks (e.g., alkyl-hydrazone crosslinks) and / or stable crosslinks (e.g., benzyl-hydrazone crosslinks).

100. The method of any one of claims 95-99, wherein the viscoelastic hydrogel substrate matrix comprises a poly(ethylene-glycol)-based hydrogel comprising benzyl-hydrazone crosslinks and wherein the immune effector cell experiences enhanced antitumor efficacy or resistance to mechanical suppression as compared to an immune effector cell with no mechanical priming or180324953654vlAttorney Docket No: 243734.000233mechanical priming with a poly(ethylene-glycol)-based hydrogel comprising alkyl-hydrazone crosslinks.

101. The method of claim 100, wherein the poly(ethylene-glycol)-based hydrogel comprising benzyl-hydrazone crosslinks is prepared by:a) mixing poly(ethylene-glycol)-hydrazine in a buffer;b) mixing the mixture of step a) with a collagen;c) adding a neutralization buffer to the mixture of step b);d) adding poly(ethylene-glycol)-benzyl-aldehyde to the mixture of step c);e) casting the mixture of step d) in a cell culture dish, a mold, or coverslip;f) incubating the cell culture dish, a mold, or coverslip containing the mixture of step d) until the mixture gels;g) adding buffer or culture medium to fully cover the gel of step f); andh) seeding the cell culture dish, a mold, or coverslip with the immune effector cell.

102. The method of claim 101, wherein a molar ratio of hydrazine:aldehyde in the mixture of step d) is about 2: 1 to about 1:2.

103. The method of claim 102, wherein a molar ratio of hydrazine:aldehyde in the mixture of step d) is about 1:1.

104. The method of any one of claims 101-103, wherein the buffer comprises an osmolarity of about 270-300 mOsm.

105. The method of claim 104, wherein the buffer is phosphate buffered saline.

106. The method of any one of claims 101-105, wherein the collagen is present in the mixture of step b) at a concentration of about 2.0 mg / mL to about 5 mg / mL, or about 2.5 mg / mL.

107. The method of claim 106, wherein the collagen is collagen-1.181324953654vlAttorney Docket No: 243734.000233108. The method of any one of claims 101-107, wherein the neutralization buffer is added to the mixture of step b) in an amount effective to adjust the pH of the mixture to about pH 7.0 to about pH 7.4.

109. The method of claim 108, wherein the neutralization buffer comprises sodium hydroxide..

110. The method of any one of claims 101-109, wherein the mixture of step d) comprises a total polyethylene glycol concentration of about 2.0 % w / v to about 5.0 % w / v, or about 2.5% w / v.

111. The method of any one of claims 101-110, wherein the mixture of step d) is mixed by pipetting.

112. The method of any one of claims 101-111, wherein the mixture of step d) is not mixed for more than 30 seconds.

113. The method of any one of claims 101-112, wherein the incubation step of step f) is conducted at 37° C.

114. The method of any one of claims claim 100-113, wherein the hydrogel comprises 100% benzyl-hydrazone crosslinks.

115. The method of any one of claims 95-98, wherein the viscoelastic hydrogel substrate matrix comprises a poly(ethylene-glycol)-based hydrogel comprising alkyl-hydrazone crosslinks and wherein the immune effector cell experiences enhanced antitumor efficacy or resistance to mechanical suppression as compared to an immune effector cell with no mechanical priming or mechanical priming with a poly(ethylene-glycol)-based hydrogel comprising benzyl-hydrazone crosslinks.

116. The method of claim 115, wherein the poly(ethylene-glycol)-based hydrogel comprising alkyl-hydrazone crosslinks is prepared by:i) mixing poly(ethylene-glycol)-hydrazine in a buffer;182324953654vlAttorney Docket No: 243734.000233j) mixing the mixture of step a) with a collagen;k) adding a neutralization buffer to the mixture of step b);l) adding poly(ethylene-glycol)-alkyl-aldehyde to the mixture of step c);m) casting the mixture of step d) in a cell culture dish, a mold, or coverslip;n) incubating the cell culture dish, a mold, or coverslip containing the mixture of step d) until the mixture gels;o) adding buffer or culture medium to fully cover the gel of step f); andp) seeding the cell culture dish, a mold, or coverslip with the immune effector cell.

117. The method of claim 116, wherein a molar ratio of hydrazine:aldehyde in the mixture of step d) is about 2: 1 to about 1:2.

118. The method of claim 117, wherein a molar ratio of hydrazine:aldehyde in the mixture of step d) is about 1:1.

119. The method of any one of claims 116-118, wherein the buffer comprises an osmolarity of about 270-300 mOsm.

120. The method of claim 119, wherein the buffer is phosphate buffered saline.

121. The method of any one of claims 116-120, wherein the collagen is present in the mixture of step b) at a concentration of about 2.0 mg / mL to about 5 mg / mL, or about 2.5 mg / mL.

122. The method of claim 121, wherein the collagen is collagen-I.

123. The method of any one of claims 116-122, wherein the neutralization buffer is added to the mixture of step b) in an amount effective to adjust the pH of the mixture to about pH 7.0 to about pH 7.4.

124. The method of claim 123, wherein the neutralization buffer comprises sodium hydroxide.183324953654vlAttorney Docket No: 243734.000233125. The method of any one of claims 116-124, wherein the mixture of step d) comprises a total polyethylene glycol concentration of about 2.0 % w / v to about 5.0 % w / v or about 2.5% w / v.

126. The method of any one of claims 116-125, wherein the mixture of step d) is mixed by pipetting.

127. The method of any one of claims 116-126, wherein the mixture of step d) is not mixed for more than 30 seconds.

128. The method of any one of claims 116-127, wherein the incubation step of step f) is conducted at 37°C.

129. The method of any one of claims 115-128, wherein the hydrogel comprises 100% alkylhydrazone crosslinks.

130. The method of any one of claims 96-129, wherein the stiffness of the gel is adjusted by changing the wt / vol% of the PEG components.

131. The method of any one of claims 101-114 or 116-130, further comprising pre-incubating the cell culture dish of step e) with poly-L-lysine.

132. The method of any one of claims 101-114 or 116-131, wherein steps a)-e) are performed at 0°C to 5°C.

133. The method of any one of claims 101-132, wherein the immune effector cell is a T cell, NK cell, or an induced pluripotent stem cell (iPSC).

134. The method of claim 133, wherein immune effector cell expresses a chimeric antigen receptor (CAR), an antigen specific T-cell receptor, or an antibody or antigen-binding fragment thereof that binds to an antigen.184324953654vlAttorney Docket No: 243734.000233135. The method of claim 134, wherein the antigen is selected from a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, a prion antigen, and an antigen associated with an inflammation or an autoimmune disease.

136. The method of claim 135, wherein the tumor is a solid tumor.

137. The method of claim 135, wherein the tumor is breast, prostate, urinary bladder, skin, lung, ovary, sarcoma, or brain cancer.

138. The method of claim 135, wherein the tumor is a central nervous system (CNS) tumor.

139. The method of claim 138, wherein the CNS tumor is a pediatric CNS malignancy.

140. The method of claim 139, wherein the pediatric CNS malignancy is Diffuse Midline Glioma (DMG).

141. The method of claim 138, wherein the CNS tumor is medulloblastoma.

142. The method of claim 138, wherein the CNS tumor is an ependymoma.

143. The method of any one of claims 136-142, wherein the tumor is a tumor expressing B7-H3, GD2, IL13Ra2, EphA2, or HER2.

144. A biomaterials-based hydrogel platform for manufacturing immune effector cells, wherein the platform comprises a hydrogel substrate (e.g., crosslinked hydrogels) comprising tunable viscoelastic and stress relaxation properties.

145. The platform of claim 144, wherein the biomaterials-based hydrogel platform comprises poly(ethylene-glycol)-based hydrogels, alginate-based hydrogels, collagen-I based platforms, hyaluronic acid and collagen-I hydrogels, and / or elastin-like protein (ELP) based hydrogel systems.185324953654vlAttorney Docket No: 243734.000233146. The platform of claim 144, wherein the biomaterials-based hydrogel platform comprises hydrazone-based crosslinking chemistry (e.g., PEG macromers functionalized with reactive end groups (e.g., alkyl-aldehyde, benzaldehyde) to form either dynamic (alkyl-hydrazone) or stable (benzyl-hydrazone) crosslinks).186324953654vl

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