WO2025122483A1

WO2025122483A1 - Screening and expansion of multi-target t cell populations

Info

Publication number: WO2025122483A1
Application number: PCT/US2024/058237
Authority: WO
Inventors: Farzad Haerizadeh; Masood Tayebi
Original assignee: Bio4t2 LLC
Current assignee: Bio4t2 LLC
Priority date: 2023-12-04
Filing date: 2024-12-03
Publication date: 2025-06-12
Anticipated expiration: 2026-06-04

Abstract

Disclosed herein is a platform capable of generating varying concentrations of antigens in cells. Also disclosed herein are methods for screening chimeric antigen receptors (CARs), including multitarget CARs (e.g. bi-specific, tri-specific), and CAR T cells capable of distinguishing between two cells with varying levels of the same antigen, using a highthroughput in vitro and/or in vivo approach with single domain antibodies. These methods offer a rapid way to obtain functional CAR T-cells and other modified immune cells to be used in, for example, the treatment of cancer. In some embodiments, the method/use is part of an immunotherapy treatment.

Description

SCREENING AND EXPANSION OF MULTI-TARGET T CELL POPULATIONS

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63/605991, filed December 4, 2023, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] Aspects of the present disclosure relate generally to tumor-associated antigen-specific binding polypeptides. These binding polypeptides can be incorporated into chimeric antigen receptor (CAR) constructs to be expressed in immune cells. These binding polypeptides and CARs may be used in the treatment of cancer.

Description of the Related Art

[0003] Adoptive cell therapies, such as chimeric antigen receptor (CAR) T cell therapies, have shown great promise in the treatment of cancer. CAR T cell therapies involve the use of genetically engineered T cells expressing receptors targeted to cancer-associated cell surface markers and other antigens, enabling directed killing of cancer cells while minimally affecting normal cells in a patient. For example, brexucabtagene autoleucel, tisagenlecleucel, and axicabtagene ciloleucel are FDA-approved CAR T cell therapies for CD 19+ B cell lymphoma cancers.

[0004] The CAR, which is made up of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, enables directed killing of cancer cells based on cell surface antigen expression while minimally affecting normal cells that are not expressing the targeted antigen. The extracellular antigen binding domain is often made up of an antibody or a binding fragment or derivative thereof, such as a single chain variable fragment (scFv) or single domain antibody (sdAb). SUMMARY OF THE INVENTION

[0005] Disclosed herein are high-throughput methods of identifying chimeric antigen receptor (CAR) T-cells expressing CARs specific for a cancer or one or more cancer- associated antigens with optimal architecture, composition and adjusted function. These methods may involve panning an antibody library to identify candidate antibodies specific for one or more cancer-associated antigens, assembling CAR expression constructs with different CAR components or modules combinatorically in a CAR library, and expressing the CAR library in immune cells, such as T cells, to create a pool of CAR immune cells or CAR-T libraries. This pool can be screened either in vitro or in vivo to determine actual cytotoxic efficacy and cancer-killing mediated proliferation against a cancer target expressing the one or more cancer-associated antigens, abrogating the risk of obtaining CARs that bind specifically to an antigen but are not effective in immune cell activation and/or cancer target eradication.

[0006] Also disclosed herein are methods of identifying CAR T cells expressing CARs specific for a cancer or one or more cancer-associated antigens. These methods involve creating a library of CAR T cells, with each CAR T cell expressing a different CAR expression construct binding to the one or more cancer-associated antigens, administering the library of CAR T cells to a non-human mammalian subject having a cancer, and identifying in the nonhuman mammalian subject those CAR T cell clones which are clonally amplified in the presence of the cancer.

[0007] As discussed herein, the CARs and CAR immune cells (e.g. CAR T cells) produced and identified according to the screening methods disclosed herein can be used for the treatment of a cancer in a mammal, such as a human.

[0008] Some embodiments of the present disclosure relate to a method for identifying a chimeric antigen receptor (CAR) immune cell expressing an at least one chimeric antigen receptor (CAR) specific to an at least one cancer-associated antigen. In some embodiments, the method comprises screening an antibody library for an antibody specific to the at least one cancer-associated antigen; assembling a nucleic acid CAR expression construct capable of encoding the antibody; incorporating the nucleic acid CAR expression construct into a CAR library; expressing the CAR library in a target immune cell to produce a CAR immune cells; and identifying a candidate CAR immune cell, wherein the candidate CAR immune cell has activity against the at least one cancer-associated antigen. In some embodiments, the at least one cancer-associated antigen is a transmembrane protein or an extracellular protein. In some embodiments, the antibody library is an immune antibody library, a naive antibody library, a synthetic antibody library, or semi-synthetic antibody library. In some embodiments, the antibody library comprises antibodies derived from human or antibodies that are not immunogenic in humans. In some embodiments, the antibody library is generated computationally or using machine learning processes. In some embodiments, the antibody library comprises single domain antibodies (sdAb), nanobodies, VHH fragments, VNAR fragments, single-chain variable fragments (scFv), camelid antibodies, or cartilaginous fish antibodies, or any combination thereof. In some embodiments, the antibody library comprises at least 100,000 unique antibodies. In some embodiments, screening the antibody library comprises screening by phage display, yeast display, bacterial display, ribosome display, mRNA display, or any combination thereof. In some embodiments, screening the antibody library further comprises more than one round of screening. In some embodiments, an antibody is selected under tumor microenvironment-like conditions, immunosuppressive conditions, low or high pH, low or high oxygen concentrations, low or high temperatures, low or high viscosity, or any combination thereof. In some embodiments, immunosuppressive conditions comprise high extracellular adenosine, high IL-6, IL-10, TGF-0, indoleamino-2,3- dioxygenase (IDO), VEGF, high interstitial fluid pressure (IFP), the presence of tumor- associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumor- associated neutrophils (TANs), cancer-associated fibroblasts (CAFs), other immunosuppressive cells, or any combination thereof. In some embodiments, an antibody is selected for specificity towards modified or derivative forms of the at least one cancer- associated antigen. In some embodiments, an antibody is selected for specificity to a high density of cancer-associated antigen. In some embodiments, the high density is at least 20,000 molecules of cancer-associated antigen. In some embodiments, a candidate CAR immune cell does not have activity when exposed to only to cells with less than 20,000 cancer-associated antigen molecules per cell. In some embodiments, the nucleic acid CAR expression construct comprises at least one of a signal peptide, a linker, a hinge, a transmembrane domain, a costimulatory domain, a signaling domains, a cytoplasmic domain, a functionality signal, a proliferation signal, an anti-exhaustion signal, an anti-inhibitory receptor, a tumor/cancer homing protein, a regulatory molecule, or any combination thereof. In some embodiments, at least one region of the nucleic acid CAR expression construct is computationally designed In some embodiments, the nucleic acid CAR expression construct comprises a CD8 signal peptide. In some embodiments, the nucleic acid CAR expression construct comprises a CD3^ hinge, a CD4 hinge, a CD8 hinge, and/or a CD28 hinge. In some embodiments, the nucleic acid CAR expression construct comprises a CD8a hinge. In some embodiments, the hinge is optimized for length. In some embodiments, the nucleic acid CAR expression construct comprises a CD3(^ transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct comprises a costimulatory domain. In some embodiments, the costimulatory domain is selected from a CD8 costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, a 4-1BB costimulatory domain, a 0X40 (CD 134) costimulatory domain, a CD27 costimulatory domain, a CD40 costimulatory domain, a CD40L costimulatory domain, a TLR costimulatory domain, a TNFR superfamily member costimulatory domain, an Ig superfamily member costimulatory domain, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct comprises a signaling domain. In some embodiments, the signaling domain is selected from the cytoplasmic domain of IL-2RP, IL-15R-a, MyD88, CD40, a Toll-like receptor, a IL-1 receptor signaling pathway member, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct comprises a 4- IBB costimulatory domain and/or a CD3(^ signaling domain. In some embodiments, the nucleic acid CAR expression construct further comprises a reporter sequence. In some embodiments, the reporter sequence is a fluorescent or luminescent protein. In some embodiments, the nucleic acid CAR expression construct comprises a nucleic acid sequence encoding a fluorescent eGFP+T2A self-cleaving peptide sequence, a truncated CD 19, a truncated EGFR, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct further comprises an RNAi cassette. In some embodiments, the RNAi cassette is selected from an miRNA, siRNA, piRNA, shRNA, or IncRNA cassettes, or any combination thereof. In some embodiments, the RNAi cassette downregulates a gene associated with graft-versus-host disease or immune cell function. In some embodiments, the gene associated with graft-versus-host disease or immune cell function is B2N, TCR, TRAC, p38alpha, TET2, PD-1, TIGIT, LAG3, REGNASE-1, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct further comprises a sequence encoding an at least one CAR. In some embodiments, the antibody is bicistronic, tricistronic, or polycistronic. In some embodiments, the nucleic acid CAR expression construct further comprises a sequence encoding an at least one accessory gene. In some embodiments, the at least one accessory gene enhances the anti-tumor activity and/or persistence of the CAR immune cells. In some embodiments, the at least one accessory gene is IL-18, IL-15, IL-2, IL-7, IL-15, or IL-21, or any combination thereof. In some embodiments, the CAR library comprises at least 10,000 unique CAR expression constructs. In some embodiments, the target immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof. In some embodiments, the target immune cell is human or humanized. In some embodiments, the target immune cell is derived from a human subject, a tissue, a cell, or an established cell line. In some embodiments, expressing the CAR library in the target immune cell comprises transduction. In some embodiments, the transduction is performed using a lentivirus comprising. In some embodiments, the CAR immune cells are screened for candidate CAR immune cells in vitro. In some embodiments, the CAR immune cells are screened for candidate CAR immune cells in vivo.

[0009] Some embodiments of the present disclosure relate to a method for identifying the candidate CAR immune cell of any one of the present embodiments in vitro. In some embodiments, the method comprises one or more rounds of contacting a population of the CAR immune cells with the at least one cancer-associated antigen; and isolating a subpopulation of the CAR immune cells that are activated, persist, and/or are enriched over time; wherein the subpopulation that is activated, persists, and/or is enriched over time are the candidate CAR immune cells. In some embodiments, the method further comprises sequencing the subpopulation of the CAR immune cells to identify the CAR expression constructs that are enriched and hence functionally active against the one or more cancer-associated antigens. In some embodiments, the at least one cancer-associated antigens are coated on a solid substrate or displayed on a cell, tissue, tumor cell line, or tumor cell 3D model. In some embodiments, the solid substrate is a plate or beads. In some embodiments, the cell is a cancer cell or an antigen presenting cell. In some embodiments, isolating the subpopulation of the CAR immune cells that are activated comprises sorting the CAR immune cells based on expression of CD 107a, CD69, CD71, CD25, or any combination thereof. [0010] Some embodiments of the present disclosure relate to a method for identifying the candidate CAR immune cell of any one of the present embodiments in vivo. In some embodiments, the method comprises one or more rounds of administering the CAR immune cells to a non-human mammalian subject comprising a cancer; isolating a blood or biopsy sample from the mammalian subject; and sequencing the blood or biopsy sample to identify the CAR immune cells that are amplified in the blood or biopsy sample as being functionally active against the one or more cancer-associated antigens. In some embodiments, the non-human mammalian subject is a mouse, rat, hamster, cat, dog, monkey, horse, pig, cow, sheep, or goat. In some embodiments, the non-human mammalian subject is an immunodeficient mouse or a humanized mouse. In some embodiments, the at least one cancer- associated antigen is associated with breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, brain cancer, pancreatic cancer, bladder cancer, testicular cancer, prostate cancer or gastric/stomach cancer, a hematologic malignancy, or any combination thereof.

[0011] Some embodiments of the present disclosure relate to a population of chimeric antigen receptor immune cells identified by the method of any one of the embodiments herein.

[0012] Some embodiments of the present disclosure relate to a method for identifying chimeric antigen receptor (CAR) T cells expressing CARs specific for one or more cancer-associated antigens. In some embodiments, the method comprises creating a library of CAR T cells, with each CAR T cell expressing a different CAR expression construct binding to the one or more cancer-associated antigens; administering the library of CAR T cells to a non-human mammalian subject having a cancer; and identifying in the non-human mammalian subject those CAR T cell clones which are clonally amplified and enriched in the presence of the cancer. In some embodiments, the method further comprises determining the nucleotide or protein sequence, or both, of the chimeric antigen receptors on the CAR T cell clones that were identified as being amplified in the presence of the cancer. In some embodiments, determining the nucleotide or protein sequence, or both, comprises sequencing a blood or biopsy sample from the mammalian subject comprising the clonally amplified CAR T cell clones. In some embodiments, the CAR expression construct comprises signal peptides, linkers, hinges, transmembrane domains, costimulatory domains, cytoplasmic domains, functionality signals, cancer homing proteins, or regulatory molecules, or any combination thereof. [0013] Some embodiments of the present disclosure relate to a CAR immune cell that has activity only when exposed to at least 20,000 molecules of an antigen. In some embodiments, the antigen is a cancer-associated antigen. In some embodiments, the activity is T cell activation. In some embodiments, the activity is IFN-gamma release and/or CD 107a degranulation. In some embodiments, the immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof.

[0014] Some embodiments of the present disclosure relate to a CAR immune cell that has activity only when exposed to a cell that expresses at least 20,000 molecules of an antigen. In some embodiments, the antigen is a cancer-associated antigen. In some embodiments, the activity is IFN-gamma release and/or CD 107a degranulation. In some embodiments, the immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In addition to the features described above, additional features and variations will be readily apparent from the following descriptions of the drawings and exemplary embodiments. It is to be understood that these drawings depict typical embodiments and are not intended to be limiting in scope.

[0016] FIG. 1 depicts a non-limiting example schematic of a workflow for the PrismCore™ platform used to generate CAR-T cells that recognize self-antigens only when over-expressed, as occurs on many types of solid tumors.

[0017] FIG. 2A is a bar chart which depicts the results of a non-limiting example of a plate-bound antigen response test quantifying the concentration of IFN-g released (pg/mL) in CAR T cells expressing B4T2-001 over an increasing exposure to the BT-001 antigen.

[0018] FIG. 2B is a bar chart which depicts a non-limiting example of a platebound antigen response test quantifying the concentration of IFN-g released (pg/mL) in CAR T cells expressing B4T2-001 over an increasing exposure to an antigen within the same family as BT-001.

[0019] FIG. 3 A is a set of charts which depicts a non-limiting example of CD 107a degranulation as a B4T2-001 specific antigen density dependent response in response to a variety of cell types, including Effector Only, PMA/lanomycin, BCPC3, CAP AN I, MKN45, N87 or 293 cells, as demonstrated by FACS.

[0020] FIG. 3B is a chart which depicts a non-limiting example of the surface antigen density of cells (i.e. the target antigen-PE molecules per cells) for 293, N87, MKN-45, Bxpc3, and Capan 1 cells.

[0021] FIG. 4A is a bar chart which depicts a non-limiting example of a platebound antigen response test quantifying the concentration of IFN-g released (pg/mL) of CAR T cells against normal primary human cells including epithelial, endothelial, neutrophils, and cancer cells.

[0022] FIG. 4B is a bar chart which depicts a non-limiting example of a CD 107 degranulation response test in B4T2-001 CAR T as a function of antigen density.

[0023] FIG. 5A is a bar chart which depicts a non-limiting example of a CD107a degranulation response test of CAR T cells exposed to various target cells including freshly isolated normal human hepatocytes and tumor cells. The left y axis is for the % of CD 107 degranulation response, while the right y axis marks the antigen density per cell of the target cells.

[0024] FIG. 5B is a microscopy image of normal hepatocytes exposed to CAR T cells expressing B4T2-001.

DETAILED DESCRIPTION

[0025] Disclosed herein are high-throughput methods of producing chimeric antigen receptor (CAR) libraries to be screened either in vitro or in vivo in the context of cytotoxic immune cells to identify robust CARs with optimal architecture, composition and adjusted function against desired cancer-associated antigens. By this approach, and the methods described herein, single target or multitarget CARs (e.g. bi-specific, tri-specific, or more, or CARs targeting 1, 2, 3, or more epitopes of one antigen) can be generated combinatorically and rapidly validated against tumor models.

[0026] Also disclosed herein is a platform capable of varying/expanding the number of antigens in cells. This platform allows for the generation of CAR T cells that are able to differentiate between tumors and cancer cells with high levels of antigens, and normal cells with low levels of those same antigens. In some embodiments, the CAR T cells are able to distinguish between a cell or tissue with a given level of an antigen, and another cell or tissue with a higher level of the same antigen. In some embodiments, the CAR T cells are able to distinguish between a cell or tissue with a given level of an antigen, and another cell or tissue with a lower level of the same antigen.

[0027] Some embodiments of the present disclosure relate to a method for identifying chimeric antigen receptor (CAR) immune cells expressing CARs specific for one or more cancer-associated antigens by using a high throughput approach. In this approach, an antibody library is first screened to identify candidate antibodies that are specific for one or more cancer-associated antigens. These antigens may be on a tumor, such as a solid tumor. Once the set of antibodies are determined, a set of CAR expression constructs are created from nucleic acids encoding for the candidate antibodies or their binding fragments. These expression constructs include the binding domains of the candidate antibodies along with a mixture of compatible nucleic acids encoding for different CAR-related modules to produce a CAR library.

[0028] In some embodiments, the library is then expressed within target immune cells to express the binding molecules on the surface of the immune cells. The immune cell library may be tested in vitro and in vivo to determine which of the CAR related modules are able to become activated and proliferated and provide a potential therapeutic construct for later development. In one embodiment, the immune cell library is put directly into a murine animal model having the target tumor or cancer. After an incubation period, the immune cells in the animal model can be analyzed to identify which CAR related modules within the immune cell library were clonally amplified in response to binding the target tumor and being amplified. Those CAR related modules that were identified as being amplified can then be purified and sequenced to determine the sequences of the CAR related modules that were most responsive to the target tumor. This approach allows for a rapid, high throughput approach for testing a library of CAR related modules to determine the binding receptors with the most activity in vitro or in vivo for a particular target tumor.

[0029] Some embodiments of the present disclosure relate to a method for identifying a chimeric antigen receptor (CAR) immune cell expressing an at least one chimeric antigen receptor (CAR) specific to an at least one cancer-associated antigen. In some embodiments, the method comprises screening an antibody library for an antibody specific to the at least one cancer-associated antigen; assembling a nucleic acid CAR expression construct capable of encoding the antibody; incorporating the nucleic acid CAR expression construct into a CAR library; expressing the CAR library in a target immune cell to produce a CAR immune cells; and identifying a candidate CAR immune cell, wherein the candidate CAR immune cell has activity against the at least one cancer-associated antigen. In some embodiments, the at least one cancer-associated antigen is a transmembrane protein or an extracellular protein. In some embodiments, the antibody library is an immune antibody library, a naive antibody library, a synthetic antibody library, or semi-synthetic antibody library.

[0030] In some embodiments, the antibody library comprises antibodies derived from human or antibodies that are not immunogenic in humans. In some embodiments, the antibody library is generated computationally or using machine learning processes. In some embodiments, the antibody library comprises single domain antibodies (sdAb), nanobodies, VHH fragments, VNAR fragments, single-chain variable fragments (scFv), camelid antibodies, or cartilaginous fish antibodies, or any combination thereof. In some embodiments, the antibody library comprises at least 100,000 unique antibodies. In some embodiments, screening the antibody library comprises screening by phage display, yeast display, bacterial display, ribosome display, mRNA display, or any combination thereof. In some embodiments, screening the antibody library further comprises more than one round of screening. In some embodiments, an antibody is selected under tumor microenvironment-like conditions, immunosuppressive conditions, low or high pH, low or high oxygen concentrations, low or high temperatures, low or high viscosity, or any combination thereof. In some embodiments, immunosuppressive conditions comprise high extracellular adenosine, high IL-6, IL-10, TGF- P, indoleamino-2,3-dioxygenase (IDO), VEGF, high interstitial fluid pressure (IFP), the presence of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils (TANs), cancer-associated fibroblasts (CAFs), other immunosuppressive cells, or any combination thereof. In some embodiments, an antibody is selected for specificity towards modified or derivative forms of the at least one cancer- associated antigen.

[0031] In some embodiments, an antibody is selected for specificity to a high density of cancer-associated antigen. In some embodiments, the high density is at least 20,000 molecules of cancer-associated antigen. In some embodiments, a candidate CAR immune cell does not have activity when exposed to only to cells with less than 20,000 cancer-associated antigen molecules per cell. In some embodiments, the nucleic acid CAR expression construct comprises at least one of a signal peptide, a linker, a hinge, a transmembrane domain, a costimulatory domain, a signaling domain, a cytoplasmic domain, a functionality signal, a proliferation signal, an anti-exhaustion signal, an anti-inhibitory receptor, a tumor/cancer homing protein, a regulatory molecule, or any combination thereof. In some embodiments, at least one region of the nucleic acid CAR expression construct is computationally designed. In some embodiments, the nucleic acid CAR expression construct comprises a CD8 signal peptide. In some embodiments, the nucleic acid CAR expression construct comprises a CD3(^ hinge, a CD4 hinge, a CD8 hinge, and/or a CD28 hinge. In some embodiments, the nucleic acid CAR expression construct comprises a CD8a hinge. In some embodiments, the hinge is optimized for length.

[0032] In some embodiments, the nucleic acid CAR expression construct comprises a CD3^ transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct comprises a costimulatory domain. In some embodiments, the costimulatory domain is selected from a CD8 costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, a 4-1BB costimulatory domain, an 0X40 (CD 134) costimulatory domain, a CD27 costimulatory domain, a CD40 costimulatory domain, a CD40L costimulatory domain, a TLR costimulatory domain, a TNFR superfamily member costimulatory domain, an Ig superfamily member costimulatory domain, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct comprises a signaling domain. In some embodiments, the signaling domain is selected from the cytoplasmic domain of IL-2RP, IL-15R-a, MyD88, CD40, a Toll-like receptor, an IL-1 receptor signaling pathway member, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct comprises a 4- IBB costimulatory domain and/or a CD3 signaling domain. In some embodiments, the nucleic acid CAR expression construct further comprises a reporter sequence. In some embodiments, the reporter sequence is a fluorescent or luminescent protein. In some embodiments, the nucleic acid CAR expression construct comprises a nucleic acid sequence encoding a fluorescent eGFP+T2A self-cleaving peptide sequence, a truncated CD19, a truncated EGFR, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct further comprises an RNAi cassette.

[0033] In some embodiments, the RNAi cassette is selected from an miRNA, siRNA, piRNA, shRNA, or IncRNA cassettes, or any combination thereof. In some embodiments, the RNAi cassette downregulates a gene associated with graft-versus-host disease or immune cell function. In some embodiments, the gene associated with graft-versus- host disease or immune cell function is B2N, TCR, TRAC, p38alpha, TET2, PD-1, TIGIT, LAG3, REGNASE-1, or any combination thereof. In some embodiments, the nucleic acid CAR expression construct further comprises a sequence encoding at least one CAR. In some embodiments, the antibody is bicistronic, tricistronic, or polycistronic.

[0034] In some embodiments, the nucleic acid CAR expression construct further comprises a sequence encoding at least one accessory gene. In some embodiments, the accessory gene enhances the anti-tumor activity and/or persistence of the CAR immune cells. In some embodiments, the accessory gene is IL- 18, IL- 15, IL-2, IL-7, IL- 15, or IL-21, or any combination thereof. In some embodiments, the CAR library comprises at least 10,000 unique CAR expression constructs. In some embodiments, the target immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof. In some embodiments, the target immune cell is human or humanized. In some embodiments, the target immune cell is derived from a human subject, a tissue, a cell, or an established cell line. In some embodiments, expressing the CAR library in the target immune cell comprises transduction. In some embodiments, the transduction is performed using a lentivirus comprising. In some embodiments, the CAR immune cells are screened for candidate CAR immune cells in vitro. In some embodiments, the CAR immune cells are screened for candidate CAR immune cells in vivo.

[0035] Some embodiments of the present disclosure relate to a method for identifying the candidate CAR immune cell of any one of the present embodiments in vitro. In some embodiments, the method comprises one or more rounds of contacting a population of the CAR immune cells with at least one cancer-associated antigen; and isolating a subpopulation of the CAR immune cells that are activated, persist, and/or are enriched over time; wherein the subpopulation that are activated, persist, and/or enriched over time are the candidate CAR immune cells. In some embodiments, the method further comprises sequencing the subpopulation of the CAR immune cells to identify the CAR expression constructs that are enriched and hence functionally active against the one or more cancer-associated antigens. In some embodiments, the cancer-associated antigens are coated on a solid substrate or displayed on a cell, tissue, tumor cell line, or tumor cell 3D model. In some embodiments, the solid substrate is a plate or beads. In some embodiments, the cell is a cancer cell or an antigen presenting cell. In some embodiments, isolating the subpopulation of the CAR immune cells that are activated comprises sorting the CAR immune cells based on expression of CD 107a, CD69, CD71, CD25, or any combination thereof.

[0036] Some embodiments relate to a method for identifying the candidate CAR immune cell of any one of the present embodiments hi vivo. In some embodiments, the method comprises one or more rounds of administering the CAR immune cells to a non-human mammalian subject comprising a cancer; isolating a blood or biopsy sample from the mammalian subject; and sequencing the blood or biopsy sample to identify the CAR immune cells that are amplified in the blood or biopsy sample as being functionally active against the one or more cancer-associated antigens. In some embodiments, the non-human mammalian subject is an immunodeficient mouse or a humanized mouse.

[0037] Some embodiments of the present disclosure relate to a population of chimeric antigen receptor immune cells identified by the method of any one of the embodiments herein.

[0038] Some embodiments of the present disclosure relate to a method for identifying chimeric antigen receptor (CAR) T cells expressing CARs specific for one or more cancer-associated antigens. In some embodiments, the method comprises creating a library of CAR T cells, with each CAR T cell expressing a different CAR expression construct binding to the cancer-associated antigens; administering the library of CAR T cells to a non-human mammalian subject having a cancer; and identifying in the non-human mammalian subject those CAR T cell clones which are clonally amplified and enriched in the presence of the cancer. In some embodiments, the method further comprises determining the nucleotide or protein sequence, or both, of the chimeric antigen receptors on the CAR T cell clones that were identified as being amplified in the presence of the cancer. [0039] In some embodiments, determining the nucleotide or protein sequence, or both, comprises sequencing a blood or biopsy sample from the mammalian subject comprising the clonally amplified CAR T cell clones.

[0040] Some embodiments of the present disclosure relate to a CAR immune cell that has activity only when exposed to at least 20,000 molecules of an antigen. In some embodiments, the antigen is a cancer-associated antigen. In some embodiments, the activity is T cell activation. In some embodiments, the activity is IFN-gamma release and/or CD 107a degranulation. In some embodiments, the immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof.

[0041] Some embodiments of the present disclosure relate to a CAR immune cell that has activity only when exposed to a cell that expresses at least 20,000 molecules of an antigen. In some embodiments, the antigen is a cancer-associated antigen. In some embodiments, the activity is T cell activation. In some embodiments, the activity is IFN- gamma release and/or CD 107a degranulation. In some embodiments, the immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof.

PrismCore™ Platform

[0042] Chimeric antigen receptors (CARs) displayed on autologous T cells (CAR- T) have been commercially approved for targeting hematological malignancies. Their success was based on targeting self-antigens (e.g., CD19 and BCMA) on tumor cells with acceptable toxicity to normal cells. However, the translation of using CAR-T techniques on solid tumors has been stymied by a lack of suitable targets which are (1) differentially presented, (2) homogeneously expressed, and (3) for which the tumor is addicted. Some self-antigens on almost all types of invasive cancers meet these conditions. The PrismCore platform described herein was developed to produce CARs to generate CAR-T cells that selectively bind selfantigens on solid tumors. The technology tunes the CAR-T to only recognize self-antigens that are over-expressed on cancer cells and to ignore normal cells with reduced basal levels of the same antigen. As discussed in more detail below, preclinical testing in small animals and nonhuman primates of on such CAR-T, termed B4t2-001, and recognizing self-antigen BT-001 has been developed and is expected to have broad anti-tumor activity in multiple solid cancer types. [0043] There are emerging clinical data that shows administration of T cells engineered to express chimeric antigen receptors (CARs) can result in anti-tumor effects in patients with solid tumors. Expanding these observations depends on T cells recognizing antigens on the cell surface that distinguish malignant cells from normal cells. The PrismCore™ platform as exemplified in FIG. 1 was developed to identify CAR targets that are self-antigens based on three gating principles. The first principle is that putative targets homogeneously expressed on solid tumors are likely also expressed on normal cells. The second principle is that a subset of these targets on invasive cancers are over-expressed relative to lower levels representing basal expression on healthy cells. The third principle is that some over-expressed antigens confer malignant potential thus wedding the tumor phenotype to continued high target density. The platform calibrates and restricts CAR-T recognition to a range of self-antigen expression which is non-overlapping with healthy cells.

[0044] The PrismCore™ platform combines computational biology with empiric observations to generate CAR-T cells against singularly recognized and over-expressed selfantigens. Synthetic single-domain antibodies combined with structural elements and signaling components can be use to generate proto-libraries of CARs. These are computationally simulated and the subsets are synthesized as combinatorial libraries for bio-screening in T cells against putative targets. An iterative process using in vitro experimentation and in vivo models was used to generate CAR-T cells that were ranked according to functional and safety attributes. The latter included membrane proteome arrays to evaluate on target and off-tumor recognition. Lead CAR-T cells were then evaluated in non-human primates (NHP) which expressed physiologic levels of the targeted antigen. Following lymphodepletion, escalating doses of autologous T cells can be administered expressing the CARs that in the laboratory recognized as over expressed macaque homologs of the human antigen.

CAR Screening

[0045] Disclosed herein are methods for identifying chimeric antigen receptor (CAR) immune cells expressing CARs specific for one or more cancer-associated antigens. In some embodiments, these methods comprise panning an antibody library to identify candidate antibodies specific for more cancer-associated antigens, assembling CAR expression constructs from nucleic acids encoding for the candidate antibodies and a mixture of compatible nucleic acids encoding for different CAR-related modules to produce a CAR library, expressing the CAR library in target immune cells to produce CAR immune cells, and identifying candidate CAR immune cells with activity against the one or more cancer- associated antigens by screening the CAR immune cells against the cancer-associated antigens. In some embodiments, the CARs are specific for one target. In other embodiments, the CARs are specific for more than one target. In some embodiments, the CARs are specific for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targets. In some embodiments, the CARs are bi-specific or tri- specific.

[0046] In some embodiments of any of the methods disclosed herein, the antibody library is an immune antibody library, a naive antibody library, a synthetic antibody library, or a semi-synthetic antibody library. In some embodiments, the antibody library comprises antibodies derived from human, or antibodies that are not immunogenic in humans, or both. In some embodiments, the antibody library comprises single domain antibodies (sdAb), nanobodies, VHH fragments, VNAR fragments, single-chain variable fragments (scFv), camelid antibodies, or cartilaginous fish antibodies, or any combination thereof. One exemplary library that can be used is a fully humanized, synthetic, sdAb library. However, any other antibody library that can be prepared or is available can be used for the methods disclosed herein. In some embodiments, the antibody library comprises single domain antibodies. In some embodiments, the antibody library comprises at least 50, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 500000, or 1000000 unique antibodies, or any number of antibodies within a range defined by any two of the aforementioned number of antibodies.

[0047] In some embodiments, the antibody library is generated computationally or using machine learning processes for diversity and or affinity. An exemplary method of generating the antibody library computationally includes modifying a universal VHH framework with synthetic diversity in one or more complementary determining regions (CDRs), such as CDR1, CDR2, or CDR3, or any combination thereof. The diversity of the CDRs are introduced by randomizing the library of sequences encoding for the antibodies with degenerate codons. For example, an NNK codon library can be employed, where the NNK codon comprises N (25% mix of A/T/C/G) and K (50% mix of T/G). In some embodiments, the NNK codon library is constructed with all possible amino acids, or with some amino acids (e.g. cysteine) and stop codon combinations excluded. Other degenerate codon mixes can be substituted for the NNK codon library with minimal experimentation. In other embodiments, the antibody library can be generated using a trimer codon mix [trinucleotide-directed mutagenesis (TRIM)], which improves balanced representation of sense codons while reducing the chance of stop codons, improving efficiency of antibody generation and testing. In some embodiments, artificial intelligence-based prediction can be used to randomize specific binding pockets of the antibodies using available binding models or structure data.

[0048] In some embodiments of any of the methods disclosed herein, panning the antibody library comprises screening for the candidate antibodies by phage display, yeast display, bacterial display, ribosome display, or mRNA display, or any combination thereof. In some embodiments, panning the antibody library comprises one or more rounds of selection, wherein the candidate antibodies are selected for specificity towards the one or more cancer- associated antigens or cells or tissues displaying the one or more cancer-associated antigens. In some embodiments, the candidate antibodies are selected under conditions including but not limited to tumor microenvironment-like conditions, immunosuppressive conditions, low or high pH, low or high oxygen concentrations, low or high temperatures, low or high viscosity, or any combination thereof, or for specificity towards modified or derivative forms of the one or more cancer-associated antigens. In some embodiments, the immunosuppressive conditions may comprise high extracellular adenosine, high IL-6, IL-10, TGF-[3, indoleamino-2,3- dioxygenase (IDO), VEGF, high interstitial fluid pressure (IFP), the presence of tumor- associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumor- associated neutrophils (TANs), cancer-associated fibroblasts (CAFs), or other immunosuppressive cells, or any combination thereof.

[0049] In some embodiments of any of the methods disclosed herein, the nucleic acids encoding for the antibodies identified by panning of the antibody library are assembled into CAR expression constructs with other CAR related modules. In some embodiments, the CAR expression constructs are assembled using multi-fragment assembly reactions known in the art. One exemplary method of assembling CAR expression constructs involves using Type IIS restriction enzymes to generate nucleic acid fragments with compatible overhang sequences and ligating the nucleic acid fragments with a ligase. As Type IIS restriction enzymes cleave outside of their recognition sites, multiple compatible nucleic acid fragments may be prepared simultaneously. In other embodiments, the CAR expression constructs can be assembled by overlap extension PCR or any other method of assembling nucleic acid constructs from more than one nucleic acid fragment can be employed. In some embodiments, the different CAR related modules comprise signal peptides, linkers with various lengths and compositions, hinges, transmembrane domains, costimulatory domains, activation domains, signaling domains, cytoplasmic domains, functionality signals, proliferation signals, antiexhaustion signals, anti-inhibitor receptors, cancer homing proteins, or regulatory molecules, or any combination thereof. Some exemplary hinges comprise CD3(^ hinge, CD4 hinge, CD8 hinge, CD28 hinge, IgGl hinge, or IgG4 hinge, or the like, or computationally designed synthetic hinges with various lengths. Some exemplary transmembrane domains comprise CD3^ transmembrane domain, CD8a transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, or ICOS transmembrane domain, or computationally designed transmembrane domains. Some exemplary costimulatory domains comprise a CD8 costimulatory domain, CD28 costimulatory domain, 4- IBB costimulatory domain, 0X40 (CD 134) costimulatory domain, ICOS costimulatory domain, CD27 costimulatory domain, CD40 costimulatory domain, CD40L costimulatory domain, TLR costimulatory domain, or costimulatory domains of other TNFR superfamily members or Ig superfamily members, MYD88-CD40 costimulatory domain, KIR2DS2 costimulatory domain, or other signaling via cytoplasmic domains of IL-2RP, IL-15R-U, MyD88, CD40, or any other Toll-like receptor or IL-1 receptor signaling pathway members. In some embodiments, the different CAR related modules are derived from CD8, CD28, 4-1BB, CD3(^, or any combination thereof. The CAR may also be modified with various additions, including but not limited to cytokines, chemokines, cytokine receptors, chemokine receptors, antigen receptors or ligands, antibodies, or enzymes.

[0050] In some embodiments of any of the methods disclosed herein, the CAR expression constructs comprise RNAi cassettes, including but not limited to miRNA, siRNA, piRNA, shRNA, or IncRNA cassettes, or any combination thereof. In some embodiments, the RNAi cassettes are used to downregulate one or more genes which may interfere with the desired or proper activity of the CAR immune cell. In some embodiments, the RNAi cassettes downregulate genes associated with graft-versus-host disease or immune cell function, including but not limited to B2N, TCR, TRAC, p38alpha, TET2, PD-1, TIGIT, LAG3, REGNASE-1, or any combination thereof. In some embodiments, the CAR expression constructs each comprise at least one CAR cassette comprising a CAR gene sequence. In some embodiments, the CAR expression constructs are bicistronic, tricistronic, or polycistronic constructs. In some embodiments, the CAR expression constructs are expressed with accessory genes that enhance the anti-tumor activity and/or persistence of the CAR immune cells, including but not limited to IL- 18, IL- 15, IL-2, IL-7, IL- 15, or IL-21, or any combination thereof. In some embodiments, the CAR expression constructs comprise a CAR expression reporter sequence. One category of a CAR expression reporter sequence may be a fluorescent or luminescent reporter, such as GFP, eGFP, RFP, or other conventional fluorescent protein, for example, expressed in the CAR immune cell as either a separate cassette, or added to the CAR cassette with or without a self-cleaving peptide sequence (e.g. P2A, T2A, E2A, F2A). In other embodiments, the CAR expression reporter sequence encodes for another cell surface marker that can be detected, e.g. with an antibody specific for the protein encoded by the reporter sequence. For example, the CAR expression reporter sequence may comprise a truncated CD 19 cassette or a truncated EGFR cassette, or both. In some embodiments, these truncated variants only comprise extracellular and transmembrane domains and lack intracellular domains such that they do not actuate any signaling pathways. In some embodiments, the CAR library comprises at least 10, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 unique CAR expression constructs, or any number of constructs within a range defined by any two of the aforementioned number of constructs.

[0051] In some embodiments of any of the methods disclosed herein, the target immune cells comprise T cells, NK cells, NK T cells, dendritic cells, macrophages, or any combination thereof. In some embodiments, the target immune cells are derived from a patient or from a cell line. For example, the target immune cells may be Jurkat cells. In some embodiments, expressing the CAR library in the target immune cells comprises transducing the target immune cells with the CAR library. In some embodiments, the target immune cells are transduced with lentivirus, gamma retrovirus, or other retroviral vector, comprising the CAR library.

[0052] In some embodiments of any of the methods disclosed herein, the CAR immune cells are screened in vitro. In some embodiments, screening in vitro comprises one or more rounds of a) contacting the CAR immune cells with the one or more cancer-associated antigens, isolating a subpopulation of the CAR immune cells that are activated, persist, and/or are enriched over time, and b) sequencing the subpopulation of the CAR immune cells to identify the CAR expression constructs that are enriched and hence functionally active against the one or more cancer-associated antigens. In some embodiments, the subpopulation of the CAR immune cells are isolated by pull down or cell sorting, or the like. In some embodiments, the one or more cancer-associated antigens are coated on a solid substrate or displayed on a cell, tissue, tumor cell line, or tumor cell 3D model. In some embodiments, the solid substrate is a plate or set of beads. In some embodiments, the cell is a cancer cell or an antigen presenting cell. In some embodiments, the antigen presenting cell comprises the one or more cancer- associated antigens. In some embodiments, isolating the subpopulation of the CAR immune cells that are activated comprises sorting the CAR immune cells. In some embodiments, the CAR immune cells are sorted based on expression of cell markers associated with activation, including but not limited to CD 107a, CD69, CD71, CD25, or any combination thereof.

[0053] In some embodiments of any of the methods disclosed herein, the CAR immune cells are screened in vivo. In some embodiments, screening the CAR immune cells in vivo comprises administering the CAR immune cells to a mammalian subject comprising a cancer or tumor, isolating a blood or biopsy sample from the mammalian subject, and sequencing the immune cells in the blood or biopsy sample to identify the CAR immune cells that are amplified in the blood or biopsy and being enriched and hence functionally active against the one or more cancer-associated antigens. In some embodiments, the biopsy sample is spleen, heart, or tumor tissue, or any other tissue containing the CAR immune cells. In some embodiments, the mammalian subject is a non-human mammalian subject. In some embodiments, the cancer is xenogeneic, such as a patient derived xenograft (PDX), or syngeneic. In some embodiments, the cancer has been isolated from a human patient with the cancer.

[0054] Disclosed herein are the antibodies, CAR expression constructs, CAR library, or populations of CAR immune cells identified by any one of the methods disclosed herein.

[0055] Also disclosed herein are methods for identifying CAR T cells expressing CARs specific for one or more cancer-associated antigens. In some embodiments, the methods comprise creating a library of CAR T cells, with each CAR T cell expressing a different CAR expression construct binding to the one or more cancer-associated antigens, administering the library of CAR T cells to a mammalian subject having a cancer, and identifying in the mammalian subject those CAR T cell clones which are clonally amplified in the presence of the cancer. In some embodiments, the methods further comprise determining the nucleotide or protein sequence, or both, of the CARs on the CAR T cell clones that were identified as being amplified in the presence of the cancer. In some embodiments, determining the nucleotide or protein sequence, or both, comprises sequencing a blood or biopsy sample from the mammalian subject comprising the clonally amplified CAR T cell clones. In some embodiments, the CARs are specific for one target, or one or more different epitopes of one target. In other embodiments, the CARs are specific for more than one target. In some embodiments, the CARs are specific for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targets. In some embodiments, the CARs are specific for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different epitopes of one target. In some embodiments, the CARs are bi-specific or tri-specific. In some embodiments, the mammalian subject is a non-human mammalian subject.

Definitions

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

[0057] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0058] The articles “a” and “an” are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0059] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. [0060] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0061] The term “% w/w” or “% wt/wt” means a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100.

Sequences

[0062] The terms “nucleic acid” or “nucleic acid molecule” as used herein refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g. plasmid, virus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used for amplification and/or expression of the nucleic acid or nucleic acids in various biological systems. Typically, the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0063] A nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a nucleic acid as used herein refers to a sequence being after the 3 ’-end of a previous sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “upstream” on a nucleic acid as used herein refers to a sequence being before the 5 ’-end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “grouped” on a nucleic acid as used herein refers to two or more sequences that occur in proximity either directly or with extra nucleic acids in between, e.g. linkers, repeats, or restriction enzyme sites, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.

[0064] The term “codon optimized” regarding a nucleic acid as used herein refers to the substitution of codons of the nucleic acid to enhance or maximize translation in a host of a particular species without changing the polypeptide sequence based on species-specific codon usage biases and relative availability of each aminoacyl -tRN A in the target cell cytoplasm. Codon optimization and techniques to perform such optimization is known in the art. Those skilled in the art will appreciate that gene expression levels are dependent on many factors, such as promoter sequences and regulatory elements. In this aspect, many synthetic genes can be designed to increase their protein expression level. [0065] The terms “peptide”, “polypeptide”, and “protein” as used herein refers to macromolecules comprised of amino acids linked by peptide bonds. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available. By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g. linkers, repeats, epitopes, or tags, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a polypeptide as used herein refers to a sequence being after the C-terminus of a previous sequence. The term “upstream” on a polypeptide as used herein refers to a sequence being before the N-terminus of a subsequent sequence.

[0066] In some embodiments, the nucleic acid or peptide sequences presented herein and used in the examples are functional in various biological systems including but not limited to humans, mice, rats, monkeys, primates, cats, dogs, rabbits, E. coli, yeast, and mammalian cells. In other embodiments, nucleic acid or peptide sequences sharing at least or lower than 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, or any percentage within a range defined by any two of the aforementioned percentages similarity to the nucleic acid or peptide sequences presented herein and used in the examples can also be used with no effect on the function of the sequences in biological systems. As used herein, the term “similarity” refers to a nucleic acid or peptide sequence having the same overall order of nucleotide or amino acids, respectively, as a template nucleic acid or peptide sequence with specific changes such as substitutions, deletions, repetitions, or insertions within the sequence. In some embodiments, two nucleic acid sequences sharing as low as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity can encode for the same polypeptide by comprising different codons that encode for the same amino acid during translation.

[0067] As disclosed herein, sequences having a percent homology to any of the sequences disclosed herein are envisioned and may be used. The term “% homology” refers to the degree of conservation between two sequences when considering their three-dimensional structure. For example, homology between two protein sequences may be dependent on structural motifs, such as beta strands, alpha helices, and other folds, as well as their distribution throughout the sequence. Homology may be determined through structural determination, either empirically or in silico. In some embodiments, any sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to any of the sequences disclosed herein may be used. In some embodiments, any sequence having at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 substitutions, deletions, or additions relative to any of the sequences disclosed herein, which may or may not affect the overall percent homology, may be used.

[0068] As applied herein, sequences having a certain “percent similarity” or “percent identity” to any of the sequence disclosed herein are envisioned and may be used. In some embodiments, these sequences may include peptide sequences, nucleic acid sequences, CDR sequences, variable region sequences, or heavy or light chain sequences. As understood in the art with respect to peptide sequences, “similarity” refers to the comparison of amino acids based on their properties, including but not limited to size, polarity, charge, pK, aromaticity, hydrogen bonding properties, or presence of functional groups (e.g. hydroxyl, thiol, amine, carboxyl, and the like). The term “% similarity” refers to the percentage of units (i.e. amino acids) that are the same between two or more sequences relative to the length of the sequence. When the two or more sequences being compared are the same length, the percent similarity will be respective that length. When two or more sequences being compared are different lengths, deletions and/or insertions may be introduced to obtain the best alignment. The similarity of two amino acids may dictate whether a certain substitution is conservative or non-conservative. Methods of determining the conservativeness of an amino acid substitution are generally known in the art and may involve substitution matrices. Commonly used substitution matrices include BLOSUM45, BLOSUM62, BLOSUM80, PAM100, PAM120, PAM160, PAM200, PAM250, but other substitution matrices or approaches may be used as considered appropriate by the skilled person. A certain substitution matrix may be preferential over the others when considering aspects such as stringency, conservation and/or divergence of related sequences (e g. within the same species or broader), and length of the sequences in question. As used herein, a peptide sequence having a certain percent similarity to another sequence will have up to that percent of amino acids that are either identical or an acceptable substitution as governed by the method of similarity determination used. In some embodiments, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to any of the sequences disclosed herein may be used. In some embodiments, any sequence having at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 similar substitutions relative to any of the sequences disclosed herein may be used. As applied to antibody sequences, these similar substitutions may apply to antigen-binding regions (i.e. CDRs) or regions that do not bind to antigens or are only secondary to antigen binding (i.e. framework regions).

[0069] As applied herein, sequences having a certain “percent identity” to any of the sequence disclosed herein are envisioned and may be used. The term to “percent identity” refers to the percent similarity between two or more sequences. In some embodiments, any sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 99%, 100%, or any integer that is between 60 and 100% identity, to any of the sequences disclosed herein may be used.

[0070] The term “consensus sequence” as used herein with regard to sequences refers to the generalized sequence representing all of the different combinations of permissible amino acids at each location of a group of sequences. A consensus sequence may provide insight into the conserved regions of related sequences where the unit (e.g. amino acid or nucleotide) is the same in most or all of the sequences, and regions that exhibit divergence between sequences. In the case of antibodies, the consensus sequence of a CDR may indicate amino acids that are important or dispensable for antigen binding. It is envisioned that consensus sequences may be prepared with any of the sequences provided herein, and the resultant various sequences derived from the consensus sequence can be validated to have similar effects as the template sequences.

Antigen Binding Molecules and Antibodies

[0071] As used herein, the term "antibody" denotes the meaning ascribed to it by one of skill in the art, and further it is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.

[0072] The term "antibody library" refers to a collection of antibodies and/or antibody fragments displayed for screening and/or combination into full antibodies. The antibodies and/or antibody fragments may be displayed on a ribosome; on a phage; or on a cell surface, in particular a yeast cell surface.

[0073] The term "compete," as used herein with regard to an antibody or binding polypeptide, means that a first antibody or binding polypeptide, or an antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody or binding polypeptide, or an antigen-binding portion thereof, such that the result of binding of the first antibody or binding polypeptide with its cognate epitope is detectably decreased in the presence of the second antibody or binding polypeptide compared to the binding of the first antibody or binding polypeptide in the absence of the second antibody or binding polypeptide. The alternative, where the binding of the second antibody or binding polypeptide to its epitope is also detectably decreased in the presence of the first antibody or binding polypeptide, can, but need not be the case. Regardless of the mechanism by which such competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing antibodies or binding polypeptides are encompassed and can be useful for the methods disclosed herein.

[0074] An antibody or binding polypeptide that "preferentially binds" or "specifically binds" (used interchangeably herein) to an epitope is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, and/or more rapidly, and/or with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody or binding polypeptide "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, and/or avidity, and/or more readily, and/or with greater duration than it binds to other substances.

[0075] The term “humanized” as applies to a non-human (e.g. rodent or primate) antibodies are hybrid immunoglobulins, immunoglobulin chains or fragments thereof which contain minimal sequence derived from non-human immunoglobulin.

[0076] The term “single domain binding polypeptide” or “single domain antibody” (sdAb) as used herein refers to a single peptide strand (e.g. not bound to another peptide strand with disulfide bonds) comprising an intact immunoglobulin domain or other protein fold which can recognize antigens. Single domain binding polypeptides or sdAbs may be derived from typical heavy or light immunoglobulin chains, such as from human, or from alternative sources such as dromedaries (e.g. VHH) and cartilaginous fish (e.g. VNAR). In some embodiments, the single domain binding polypeptide or sdAb comprises one, two, or three complementarity determining regions (CDRs). In some embodiments, the single domain binding polypeptide or sdAb comprises one, two, or three of a CDR1, CDR2, and CDR3.

[0077] Unless otherwise specified, the complementarity determining regions (CDRs) disclosed herein follow the IMGT definition. However, the CDRs, either separately or within the context of the variable domains, can also be interpreted by Kabat, Chothia, or other definitions as understood by those of skill in the art.

[0078] The term "single-chain variable fragment" (scFv) as used herein is a fusion protein comprising the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, in which the VH and VL are covalently linked to form a VH: VL heterodimer. The VH and VL are either joined directly or joined by a peptide-encoding linker, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid including VH- and VL-encoding sequences. In some embodiments, the VH and VL of the scFv each comprises one, two, or three CDRs. In some embodiments, the VH and VL of the scFv each comprises one, two, or three of a CDR1, CDR2, and CDR3.

[0079] In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody or binding polypeptide is accomplished by solving the structure of the antibody or binding polypeptide and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the IMGT approach (Lefranc et al., 2003) Dev Comp Immunol. 27:55-77), computational programs such as Paratome (Kunik et al., 2012, Nucl Acids Res. W521-4), the AbM definition, and the conformational definition.

[0080] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM.TM., A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198. The contact definition is based on an analysis of the available complex crystal structures. See, e g., MacCallum et al., 1996, J. Mol. Biol., 5:732- 45. In another approach, referred to herein as the "conformational definition" of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156- 1166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, IMGT, Paratome, AbM, and/or conformational definitions, or a combination of any of the foregoing.

Antigen binding polypeptides

[0081] In addition to entire immunoglobulins (or their recombinant counterparts), immunoglobulin fragments or “binding fragments” comprising the epitope binding site (e.g., Fab', F(ab')2, single-chain variable fragment (scFv), diabody, minibody, nanobody, singledomain antibody (sdAb), VHH fragments, VNAR fragments, or other fragments) are useful as antibody moieties in the present invention. Such antibody fragments may be generated from whole immunoglobulins by ricin, pepsin, papain, or other protease cleavage. Minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques. For instance "Fv" immunoglobulins for use in the present invention may be produced by linking a variable light chain region to a variable heavy chain region via a peptide linker (e.g., polyglycine or another sequence which does not form an alpha helix or beta sheet motif). Nanobodies or single-domain antibodies can also be derived from alternative organisms, such as dromedaries, camels, llamas, alpacas, sharks, or cartilaginous fish. In some embodiments, antibodies can be conjugates, e.g. pegylated antibodies, drug, radioisotope, or toxin conjugates. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the targeting and/or depletion of cellular populations expressing the marker.

[0082] The term “single-domain antibody” (sdAb) as used herein refers to a single peptide strand (e.g. not bound to another peptide strand with disulfide bonds) comprising an intact immunoglobulin domain or other protein fold which can recognize antigens. sdAbs may be derived from typical heavy or light immunoglobulin chains, such as from human, or from alternative sources such as dromedaries (e.g. VHH) and cartilaginous fish (e.g. VNAR). Chimeric Antigen Receptors (CARs)

[0083] The term “chimeric antigen receptor (CAR)” as used herein refers to engineered biological receptors that confers an artificial specificity in an immune cell towards a certain antigen, such as a tumor-associated antigen. An exemplary immune cell in which CARs can be used are T cells, but it is envisioned that CARs can be engineered into any amenable cytotoxic immune cell, including but not limited to T cells, Natural Killer (NK) cells, Natural Killer T (NKT) cells, dendritic cells, or macrophages. In this aspect, any disclosure pertaining to CAR T cells can also be applied to other immune cells comprising CARs. At their core, CARs comprise an extracellular antigen-recognizing domain (e.g. tumor receptor ligand, or antibody), hinge, transmembrane, and intracellular signaling domain (endodomain). Different combinations of these CAR components may result in different specificities and efficacy against certain cancer antigens.

[0084] Also disclosed herein are chimeric antigen receptors (CARs) comprising: any one of the CEA5 single domain binding polypeptides disclosed herein, any one of the CEA6 single domain binding polypeptides disclosed herein, any one of the MSLN single domain binding polypeptides disclosed herein, any one of the MUC1 single domain binding polypeptides disclosed herein, any one of the EPCAM single domain binding polypeptides disclosed herein, any one of the GPC3 single domain binding polypeptides disclosed herein, or any one of the FAP single domain binding polypeptides disclosed herein, or any combination thereof, including two or more of any of the single domain binding polypeptides disclosed herein.

[0085] In some embodiments, the CAR comprises at least two single domain binding polypeptides and the CAR is a multivalent CAR. In some embodiments, the CAR comprises two single domain binding polypeptides and the CAR is a bivalent CAR. In some embodiments, the CAR comprises three single domain binding polypeptides and the CAR is a trivalent CAR.

[0086] In some embodiments, the CAR further comprises one or more signal peptides, linkers with various lengths and composition, hinges, transmembrane domains, costimulatory domains, signaling domains, cytoplasmic domains, functionality signals, proliferation signals, anti-exhaustion signals, anti-inhibitory receptors, tumor/cancer homing proteins, or regulatory molecules, or any combination thereof. In some embodiments, the costimulatory domains comprise CD8, CD28, ICOS, 4-1BB, 0X40 (CD134), CD27, CD40, CD40L, TLR or other TNFR superfamily member or Ig superfamily member costimulatory domains, or other signaling via cytoplasmic domains of IL-2RP, IL-15R-a, MyD88, or CD40 or any other Toll-like receptor or IL-1 receptor signaling pathway members.

[0087] In some embodiments, the CARs disclosed herein are constructed by assembling CAR expression constructs from nucleic acids encoding for any one of the single domain binding polypeptides disclosed herein and a mixture of compatible nucleic acids encoding for different CAR modules. In some embodiments, different combinations of CARs are produced for use in a CAR library for screening for CAR efficacy (in vitro or in vivo). In some embodiments, unique CARs are produced separately. In some embodiments, the CARs are specific for one target. In some embodiments, the CARs are specific for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targets. In some embodiments, the CARs are bi-specific or tri-specific.

[0088] To construct any one of CARs disclosed herein, the nucleic acids encoding for the single domain binding polypeptides identified by panning of the antibody library are assembled into CAR expression constructs with other CAR modules. In some embodiments, the CAR expression constructs are assembled using multi-fragment assembly reactions known in the art. One exemplary method of assembling CAR expression constructs involves using Type IIS restriction enzymes to generate nucleic acid fragments with compatible overhang sequences and ligating the nucleic acid fragments with a ligase. As Type IIS restriction enzymes cleave outside of their recognition sites, multiple compatible nucleic acid fragments may be prepared simultaneously. In other embodiments, the CAR expression constructs can be assembled by overlap extension PCR. It is envisioned that any other method of assembling nucleic acid constructs from more than one nucleic acid fragment can be employed. In some embodiments, the different CAR modules comprise signal peptides, linkers, hinges, transmembrane domains, costimulatory domains, activation domains, signaling domains, cytoplasmic domains, functionality signals, proliferation signals, anti -exhaustion signals, antiinhibitor receptors, cancer homing proteins, or regulatory molecules, or any combination thereof. Nucleic Acids

[0089] Also disclosed herein are nucleic acids that encode for a polypeptide. In some embodiments, the polypeptide is a binding polypeptide. In some embodiments, the polypeptide is a single domain binding polypeptide. In some embodiments, the polypeptide is any one of the single domain binding polypeptides disclosed herein. In some embodiments, the polypeptide comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to: any one of the CEA5 single domain binding polypeptides disclosed herein, any one of the CEA6 single domain binding polypeptides disclosed herein, any one of the MSLN single domain binding polypeptides disclosed herein, any one of the EPCAM single domain binding polypeptides disclosed herein, any one of the GPC3 single domain binding polypeptides disclosed herein, or any one of the FAP single domain binding polypeptides disclosed herein, or any combination thereof, including two or more of the binding polypeptides disclosed herein.

[0090] Any one of the nucleic acids that encode for a binding polypeptide can be prepared by recombinant DNA technology, synthetic chemistry techniques, or a combination thereof. For example, sequences of nucleic acids encoding for the binding polypeptide may be cloned into an expression vector using standard molecular techniques known in the art. Sequences can be obtained from other vectors encoding the desired protein sequence, from PCR-generated fragments using respective template nucleic acids, or by assembly of synthetic oligonucleotides encoding the desired sequences. In some embodiments, the expression vector may be a CAR expression vector, in which it is provided to an immune cell so that it expressed the CAR. In some embodiments, the expression vector may be an expression vector suited for large scale antibody or binding polypeptide production, from which the peptide products can be isolated for further use.

[0091] Expression of binding polypeptides or CARs may be confirmed by nucleic acid or protein assays known in the art. For example, the presence of transcribed mRNA of binding polypeptides or CARs can be detected and/or quantified by conventional hybridization assays (e.g. Northern blot analysis), amplification procedures (e.g. RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based technologies (see e.g. U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934), using probes complementary to any region of a polynucleotide that encodes for the binding polypeptides or CARs. Expression of the binding polypeptides or CARs can also be determined by examining the expressed peptide. A variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), “sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays, and SDS- PAGE.

Methods of Use or Treatment

[0092] As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker).

[0093] As used herein, the terms “treating” or “treatment” (and as well understood in the art) means an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. “Treating” and “treatment” as used herein also include prophylactic treatment. Treatment methods comprise administering to a subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may comprise a series of administrations. The compositions are administered to the subject in an amount and for a duration sufficient to treat the subject. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.

[0094] The terms “effective amount” or “effective dose” as used herein refers to that amount of a recited composition or compound that results in an observable designated effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the designated response for a particular subject and/or application. The selected dosage level can vary based upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of doselimiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0095] The term “administering” includes enteral, oral administration, topical contact, administration as a suppository, parenteral, intra-arteriole, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, intraventricular, intradermal, intracranial, parenteral, subdermal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “coadminister” it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein.

[0096] As used herein, the term "therapeutic target" refers to a gene or gene product that, upon modulation of its activity (e.g., by modulation of expression, biological activity, and the like), can provide for modulation of the disease phenotype. As used throughout, "modulation" is meant to refer to an increase or a decrease in the indicated phenomenon (e.g., modulation of a biological activity refers to an increase in a biological activity or a decrease in a biological activity).

[0097] As used herein, the term “standard of care”, “best practice” and “standard therapy” refers to the treatment that is accepted by medical practitioners to be an appropriate, proper, effective, and/or widely used treatment for a certain disease. The standard of care of a certain disease depends on many different factors, including the biological effect of treatment, region or location within the body, patient status (e.g. age, weight, gender, hereditary risks, other disabilities, secondary conditions), toxicity, metabolism, bioaccumulation, therapeutic index, dosage, and other factors known in the art. Determining a standard of care for a disease is also dependent on establishing safety and efficacy in clinical trials as standardized by regulatory bodies such as the US Food and Drug Administration, International Council for Harmonisation, Health Canada, European Medicines Agency, Therapeutics Goods Administration, Central Drugs Standard Control Organization, National Medical Products Administration, Pharmaceuticals and Medical Devices Agency, Ministry of Food and Drug Safety, and the World Health Organization. The standard of care for a disease may include but is not limited to surgery, radiation, chemotherapy, targeted therapy, or immunotherapy.

[0098] Also disclosed herein are methods of treating a cancer in a subject in need thereof. In some embodiments, the methods comprise administering a chimeric antigen receptor cell to the subject. In some embodiments, the methods comprise administering any one of the chimeric antigen receptor cells disclosed herein. In some embodiments, the chimeric antigen receptor cell expresses and/or comprises: any one of the CEA5 single domain binding polypeptides disclosed herein, any one of the CEA6 single domain binding polypeptides disclosed herein, any one of the MSLN single domain binding polypeptides disclosed herein, any one of the MUC1 single domain binding polypeptides disclosed herein, any one of the EPCAM single domain binding polypeptides disclosed herein, any one of the GPC3 single domain binding polypeptides disclosed herein, or any one of the FAP single domain binding polypeptides disclosed herein, or any combination thereof, including two or more of the single domain binding polypeptides disclosed herein. In some embodiments, the chimeric antigen receptor cell is a CAR-T cell. In some embodiments, the chimeric antigen receptor cell is derived from the subject and is autologous to the subject. In some embodiments, the chimeric antigen receptor cell is allogeneic to the subject. In some embodiments, the chimeric antigen receptor cell is from a cell line (e.g. Jurkat). In some embodiments, the subject is a human.

[0099] In some embodiments, the chimeric antigen receptor cell is administered once per day, twice per day, three times per day or more. In some embodiments, the chimeric antigen receptor cell is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more. In some embodiments, the immune cell is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[0100] In some embodiments, the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub -doses per day.

[0101] The ranges for administration are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages is altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0102] In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.

EXAMPLES

[0103] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the invention, as it is described herein above and in the claims.

Example 1: Generating Multi-Target CAR T Cells

Plasmid preparation and quality check (QC)

[0104] The Maxi Plasmid Purification kit (Zymo, D4203) was used for CAR plasmid preparation. Plasmid concentration and quality was analyzed by Nanodrop (260/280 ratio and 260/230 ratio) and the ToxinSensor Chromogenic LAL Endotoxin Assay (Genescript, L00350). Good-quality plasmid DNA will have an A26/A280 ratio of 1.8-2.0, an A260/A230 ratio greater than 2.0, and less than 0.1 EU/pg of endotoxin.

Plasmid assembly and QC

[0105] Synthetic genes were cloned into the pLenti vector and the construct was confirmed using Sanger sequencing. The CAR constructs contained a signal peptide (e.g. CD8 signal peptide), hinge (e.g. CD8a stalk), transmembrane domain (e g. CD8 transmembrane domain or CD28 transmembrane domain), and signaling domains (e.g. 4- IBB costimulatory domain or CD3(^ signaling domain).

[0106] To monitor CAR expression levels, the expressed cassette of the CAR plasmids can also be engineered to express fluorescent eGFP+T2A self-cleaving peptide sequences. Alternatively, truncated CD 19 or truncated EGFR cassettes can be used to monitor by antibody detection. Human sequences (e.g. CD8, 4- IBB, CD3Q are accessible on GenBank. Virus production and titration

[0107] To produce lentivirus, human embryonic kidney 293T (HEK293T) cells were co-transfected with CAR transgene-encoding pLenti transfer plasmid and one or more necessary packaging plasmids (i.e. encoding for Gag, Pol, Rev, VSVG, and optionally Tat). The supernatants of the HEK293T cultures were collected at either 48 or 72 hours after transfection, centrifuged, and filtered with a 0.45 pm filter.

[0108] Virus titration was done in Jurkat cells transduced with diluted lentivirus collections. After 48 hours, transduced Jurkat cells were stained with biotinylated recombinant protein L and phycoerythrin (PE)-conjugated streptavidin, and CAR abundance was measured by flow cytometry.

T cell transduction and expansion

[0109] Human PBMCs were isolated from peripheral blood of healthy human donors by density gradient centrifugation with the Lymphoprep reagent (StemCell Technologies). PBMCs were resuspended at IxlO⁶ cells/mL in X-VIVO 15 serum-free hematopoietic medium (Lonza, 04-418QCN) with 10 ng/mL IL-2 (Novoprotein, GMP-CD66) and 10 ng/mL IL-7 (Novoprotein, GMP-CD47). PBMCs were stimulated with 50 ng/mL anti- CD3 antibody (Novoprotein, GMP-A018) for 24 hours. Then, PBMCS were transduced with CAR lentivirus. CAR surface levels, CD3, and CD4/CD8 ratios (using antibodies from Biolegend, 317344, 301012, and 317412) were measured 12 or 14 days after initial stimulation of PBMCs with the anti-CD3 antibody.

T cell cryopreservation and thawing

[0110] After 14 days of CAR-T expansion, CAR-T cells were centrifuged and the supernatant was discarded. The cell pellet was resuspended in chilled CryoStor CS10 (StemCell Technologies, 07930) at a viable cell density of 5 x 107 cells/mL. Aliquots of cell suspension were dispensed into cryovials. The cryovials were cooled l°C/minute. The frozen cells were transferred to liquid nitrogen.

[oni] To thaw, the cells were quickly thawed in a 37°C water bath with gentle agitation. The thawed cells were transferred to a 50 mL conical tube and washed by adding 20 mL of fresh growth medium dropwise. The cells were centrifuged and resuspended in X-VIVO 15 medium at a cell density of 1 x 106 cells/mL.

Cytotoxicity assay

[0112] The cytotoxicity of the CAR T-cells was determined by standard luciferasebased assays. Briefly, target cells expressing firefly luciferase were co-cultured with CAR-T cells in triplicate at the indicated effectortarget ratios using white-walled 96-well plates with 2 x 104 target cells in a total volume of 100 pL per well in X-VIVO 15 medium. Target cells alone as a control were plated at the same cell density. After 48 hours of co-culture, 100 pL of luciferase substrate (ONE-Glo, Promega) was directly added to each well. Emitted light was detected with a luminescence plate reader.

IFN-y evaluation

[0113] 1 x 10⁶ per well of CAR T-cells were stimulated with target cells at indicated effector: target cell ratios in 6-well tissue culture plates for 24 hours. Interferon gamma (ZFN-y) and IL-2 secretion was quantitated through enzyme-linked immunosorbent assay (ELISA) using the human IL-2 ELISA Kit II (BD, 550611) and human IFN-y ELISA Kit II (BD, 550612).

CD25/CD69 assay

[0114] 1 x IO⁶ per well of CAR T-cells were stimulated with target cells at indicated effectortarget cell ratios in 6-well tissue culture plates for 24 hours. Cells were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher) to label dead cells and with anti-CD3, and CAR-T cells were identified as CD3+ GFP+ cells by flow cytometry. CD69 and CD25 on CAR-T cells were stained with Alexa Fluor 700 anti-human CD69 antibody (Biolegend) and PE anti-CD25 antibody (Biolegend).

Sequencing of CAR T cell libraries

[0115] Cells were collected from CAR-T library screens. Genomic DNA (gDNA) was extracted using the Blood/Cell Genome DNA Mini kit (Tiangen, DP304-03). 75 ng of gDNA was used for PCT to amplify the CAR region of the library. The amplified region was TA cloned with the TA/Blunt-Zero Cloning kit (Vazyme, C601) and sequence with conventional Sanger sequencing or by any next generation sequencing platforms or methods such as Illumina HiSEQ, or in general using any short read or long read next generation sequencing platforms.

Example 2: Generating cells with varying antigen concentrations using the PrismCore Platform

[0116] This platform has identified CAR-T cells to three targetsas described herein. Target BT-001 is a self-antigen that was selected based on over-expression on the cell surface of multiple solid tumors and that it participates in pathobiology. Libraries with various diversities from 1E10 to 1E11 camelid antibodies were designed, synthesized, and screened followed by binding analysis.

Example 3: Generating CAR T cells using the PrismCore Platform

[0117] 1E3 to 1E4 antibody variants were combinatorically assembled into multiple CAR libraries. CAR-T libraries were evaluated in a battery of laboratory tests including assessing effector functions in response to varying densities of BT-001 and overall fitness. Selected CAR-T candidates were then advanced to in vivo evaluation in mice to evaluate their anti-tumor effect, including sustained biological activity. The lead CAR-T, termed B4T2-001, was then infused in NHP and despite circulating engineered T cells being measurable there was no toxicity found with this lead.

Example 4: CAR T cells are selective for varying antigen concentrations

[0118] Selectivity of B4T2-001 CAR T cells was tested using a plate-bound antigen-response test. As shown in FIG. 2A, there was a robust IFN-gamma release in cells as a response to increasing doses of the specific antigen BT-001. However, this same CAR T cell population did not respond to the presence of a non-specific antigen from the same family of proteins (FIG. 2B). The CAR T cells therefore display a high degree of safety and selectivity.

[0119] CD107a degranulation in response to antigen exposure was also assessed. CAR T cells were exposed to a variety of cell types at an effector : target ratio of 1 :1, and degranulation was quantified using FACS (FIG. 3A). The antigen density across varying cell surfaces are as shown in FIG. 3B. Compared to control, the B4T2-001 cells demonstrated specific antigen density-dependent responses, whereby the degranulation increased in proportion to the antigen density.

[0120] B4T2-001 CART cells were also tested for their response to human primary cells. There was no significant IGN-gamma release when exposed to normal human cells, including normal lung and intestinal epithelial cells, which express BT-001 at low levels (FIG. 4A). In contrast, there was a significant response of CAR T cells to tumor/cancer cell lines. Similarly, CD 107a degranulation was observed in CAR T cells exposed to high antigen density tumor cells, and was not observed in CAR T cells exposed to low antigen density normal cells (FIG. 4B).

[0121] B4T2-001 CAR-T cells were incubated with various target cells including freshly isolated normal human hepatocytes (FIG. 5B) at a 1: 1 effector to target ratio for 24 hours, before CD 107a degranulation response was evaluated by FACS (FIG. 5 A). Once again, B4T2-001 CAR-T cells exhibited no response to normal hepatocytes.

[0122] Together, this data indicates that B4T2-001 CAR T cells are only activated when in the presence of target antigens at a density that passes a threshold only seen in tumor and metastatic cells.

[0123] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0124] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

[0125] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0126] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0127] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0128] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0129] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0130] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0131] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

WHA T IS CLAIMED IS:

1. A method for identifying a chimeric antigen receptor (CAR) immune cell expressing at least one chimeric antigen receptor (CAR) specific to at least one cancer- associated antigen, comprising: screening an antibody library for an antibody specific to the at least one cancer- associated antigen; assembling a nucleic acid CAR expression construct capable of encoding the antibody; incorporating the nucleic acid CAR expression construct into a CAR library; expressing the CAR library in a target immune cell to produce CAR immune cells; and identifying a candidate CAR immune cell, wherein the candidate CAR immune cell has activity against the at least one cancer-associated antigen.

2. The method of claim 1, wherein the at least one cancer-associated antigen is a transmembrane protein or an extracellular protein.

3. The method of claim 1 or 2, wherein the antibody library is an immune antibody library, a naive antibody library, a synthetic antibody library, or semi-synthetic antibody library.

4. The method of any one of claims 1-3, wherein the antibody library comprises antibodies derived from human or antibodies that are not immunogenic in humans.

5. The method of any one of claims 1-4, wherein the antibody library is generated computationally or using machine learning processes.

6. The method of any one of claims 1-5, wherein the antibody library comprises single domain antibodies (sdAb), nanobodies, VHH fragments, VNAR fragments, single-chain variable fragments (scFv), camelid antibodies, or cartilaginous fish antibodies, or any combination thereof.

7. The method of any one of claims 1-6, wherein the antibody library comprises at least 100,000 unique antibodies.

8. The method of any one of claims 1-7, wherein screening the antibody library comprises screening by phage display, yeast display, bacterial display, ribosome display, mRNA display, or any combination thereof.

9. The method of any one of claims 1 -8, wherein screening the antibody library further comprises more than one round of screening.

10. The method of any one of claims 1-9, wherein an antibody is selected under tumor microenvironment-like conditions, immunosuppressive conditions, low or high pH, low or high oxygen concentrations, low or high temperatures, low or high viscosity, or any combination thereof.

11. The method of claim 10, wherein immunosuppressive conditions comprise high extracellular adenosine, high IL-6, IL-10, TGF-P, indoleamino-2,3-dioxygenase (IDO), VEGF, high interstitial fluid pressure (IFP), the presence of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils (TANs), cancer-associated fibroblasts (CAFs), other immunosuppressive cells, or any combination thereof.

12. The method of any one of claims 1-11, wherein an antibody is selected for specificity towards modified or derivative forms of the at least one cancer-associated antigen.

13. The method of any one of claims 1-12, wherein an antibody is selected for specificity to a high density of cancer-associated antigen.

14. The method of claim 13, wherein the high density is at least 20,000 molecules of cancer-associated antigen.

15. The method of any one of claims 13 or 14, wherein a candidate CAR immune cell does not have activity when exposed to only to cells with less than 20,000 cancer- associated antigen molecules per cell.

16. The method of any one of claims 1-15, wherein the nucleic acid CAR expression construct comprises at least one of a signal peptide, a linker, a hinge, a transmembrane domain, a costimulatory domain, a signaling domains, a cytoplasmic domain, a functionality signal, a proliferation signal, an anti-exhaustion signal, an anti-inhibitory receptor, a tumor/cancer homing protein, a regulatory molecule, or any combination thereof.

17. The method of claim 16, wherein at least one region of the nucleic acid CAR expression construct is computationally designed.

18. The method of any one of claims 1-17, wherein the nucleic acid CAR expression construct comprises a CD8 signal peptide.

19. The method of any one of claims 1-18, wherein the nucleic acid CAR expression construct comprises a CD3(^ hinge, a CD4 hinge, a CD8 hinge, and/or a CD28 hinge.

20. The method of any one of claims 1-19, wherein the nucleic acid CAR expression construct comprises a CD8a hinge.

21. The method of any one of claims 16-20, wherein the hinge is optimized for length.

22. The method of any one of claims 1-21, wherein the nucleic acid CAR expression construct comprises a CD3(^ transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or any combination thereof.

23. The method of any one of claims 1-22, wherein the nucleic acid CAR expression construct comprises a costimulatory domain.

24. The method of claim 23, wherein the costimulatory domain is selected from a CD8 costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, a 4-1BB costimulatory domain, a 0X40 (CD134) costimulatory domain, a CD27 costimulatory domain, a CD40 costimulatory domain, a CD40L costimulatory domain, a TLR costimulatory domain, a TNFR superfamily member costimulatory domain, an Ig superfamily member costimulatory domain, or any combination thereof.

25. The method of any one of claims 1-24, wherein the nucleic acid CAR expression construct comprises a signaling domain.

26. The method of any one of claims 1-25, wherein the signaling domain is selected from the cytoplasmic domain of IL-2RP, IL-15R-a, MyD88, CD40, a Toll-like receptor, a IL- 1 receptor signaling pathway member, or any combination thereof.

27. The method of any one of claims 1-26, wherein the nucleic acid CAR expression construct comprises a 4- IBB costimulatory domain and/or a CD3(^ signaling domain.

28. The method of any one of claims 1-27, wherein the nucleic acid CAR expression construct further comprises a reporter sequence.

29. The method of claim 28, wherein the reporter sequence is a fluorescent or luminescent protein.

30. The method of any one of claims 1-29, wherein the nucleic acid CAR expression construct comprises a nucleic acid sequence encoding a fluorescent eGFP+T2A self-cleaving peptide sequence, a truncated CD 19, a truncated EGFR, or any combination thereof.

31. The method of any one of claims 1-30, wherein the nucleic acid CAR expression construct further comprises an RNAi cassette.

32. The method of claim 31, wherein the RNAi cassette is selected from an miRNA, siRNA, piRNA, shRNA, or IncRNA cassettes, or any combination thereof.

33. The method of claim 31 or 32, wherein the RNAi cassette downregulates a gene associated with graft-versus-host disease or immune cell function.

34. The method of claim 33, wherein the gene associated with graft-versus-host disease or immune cell function is B2N, TCR, TRAC, p38alpha, TET2, PD-1, TIGIT, LAG3, REGNASE-1, or any combination thereof.

35. The method of any one of claims 1-34, wherein the nucleic acid CAR expression construct further comprises a sequence encoding an at least one CAR.

36. The method of any one of claims 1-35, wherein the antibody is bicistronic, tricistronic, or polycistronic.

37. The method of any one of claims 1-36, wherein the nucleic acid CAR expression construct further comprises a sequence encoding an at least one accessory gene.

38. The method of claim 37, wherein the at least one accessory gene enhances the anti-tumor activity and/or persistence of the CAR immune cells.

39. The method of claim 37 or 38, wherein the at least one accessory gene is IL-18, IL- 15, IL-2, IL-7, IL- 15, or IL-21, or any combination thereof.

40. The method of any one of claims 1-39, wherein the CAR library comprises at least 10,000 unique CAR expression constructs.

41. The method of any one of claims 1-40, wherein the target immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof.

42. The method of any one of claims 1-41, wherein the target immune cell is human or humanized.

43. The method of any one of claims 1-42, wherein the target immune cell is derived from a human subject, a tissue, a cell, or an established cell line.

44. The method of any one of claims 1 -43, wherein the expressing the CAR library in the target immune cell comprises transduction.

45. The method of claim 44, wherein the transduction is performed using a lentivirus comprising.

46. The method of any one of claims 1-45, wherein the CAR immune cells are screened for candidate CAR immune cells in vitro.

47. A method for identifying the candidate CAR immune cell of any one of claims 1-46 in vitro, wherein the method comprises one or more rounds of: a) contacting a population of the CAR immune cells with the at least one cancer-associated antigen; and b) isolating a subpopulation of the CAR immune cells that are activated, persist, and/or are enriched over time; c) wherein the subpopulation that are activated, persist, and/or enriched over time are the candidate CAR immune cells.

48. The method of claim 47, wherein the method further comprises sequencing the subpopulation of the CAR immune cells to identify the CAR expression constructs that are enriched and hence functionally active against the one or more cancer-associated antigens.

49. The method of claim 47 or 48, wherein the at least one cancer-associated antigens are coated on a solid substrate or displayed on a cell, tissue, tumor cell line, or tumor cell 3D model.

50. The method of claim 49, wherein the solid substrate is a plate or beads.

51. The method of claim 49 or 50, wherein the cell is a cancer cell or an antigen presenting cell.

52. The method of any one of claims 47-51, wherein isolating the subpopulation of the CAR immune cells that are activated comprises sorting the CAR immune cells based on expression of CD 107a, CD69, CD71, CD25, or any combination thereof.

53. The method of any one of claims 1-45, wherein the CAR immune cells are screened candidate CAR immune cell in vivo.

54. A method for identifying the candidate CAR immune cell of any one of claims 1-46 in vivo, wherein the method comprises one or more rounds of:

-SO- a) administering the CAR immune cells to a non-human mammalian subject comprising a cancer; b) isolating a blood or biopsy sample from the mammalian subject; and c) sequencing the blood or biopsy sample to identify the CAR immune cells that are amplified in the blood or biopsy sample as being functionally active against the one or more cancer-associated antigens.

55. The method of claim 54, wherein the non-human mammalian subject is a mouse, rat, hamster, cat, dog, monkey, horse, pig, cow, sheep, or goat.

56. The method of claim 55, wherein the non-human mammalian subject is an immunodeficient mouse or a humanized mouse.

57. The method of any one of claims 1-56, wherein the at least one cancer- associated antigen is associated with breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, brain cancer, pancreatic cancer, bladder cancer, testicular cancer, prostate cancer or gastric/stomach cancer, a hematologic malignancy, or any combination thereof.

58. A population of chimeric antigen receptor immune cells identified by the method of any one of claims 1-57.

59. A method for identifying chimeric antigen receptor (CAR) T cells expressing CARs specific for one or more cancer-associated antigens, comprising: creating a library of CAR T cells, with each CAR T cell expressing a different CAR expression construct binding to the one or more cancer-associated antigens; administering the library of CAR T cells to a non-human mammalian subject having a cancer; and identifying in the non-human mammalian subject those CAR T cell clones which are clonally amplified and enriched in the presence of the cancer.

60. The method of claim 59, further comprising determining the nucleotide or protein sequence, or both, of the chimeric antigen receptors on the CAR T cell clones that were identified as being amplified in the presence of the cancer.

61. The method of claim 60, wherein determining the nucleotide or protein sequence, or both, comprises sequencing a blood or biopsy sample from the mammalian subject comprising the clonally amplified CAR T cell clones.

62. The method of any one of claims 59-61 , wherein the CAR expression construct comprises signal peptides, linkers, hinges, transmembrane domains, costimulatory domains, cytoplasmic domains, functionality signals, cancer homing proteins, or regulatory molecules, or any combination thereof.

63. The method of any one of claims 59-62, wherein the mammalian subject is a mouse, rat, hamster, cat, dog, monkey, horse, donkey, pig, cow, sheep, or goat.

64. The method of claim 63, wherein the mammalian subject is an immunodeficient mouse or a humanized mouse.

65. The method of any one of claims 59-64, wherein the cancer is breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, brain cancer, pancreatic cancer, bladder cancer, testicular cancer, prostate cancer or gastric/stomach cancer, a hematologic malignancy, or any combination thereof.

66. A CAR immune cell that has activity only when exposed to at least 20,000 molecules of an antigen.

67. The CAR immune cell of claim 66, wherein the antigen is a cancer-associated antigen.

68. The CAR immune cell of claim 67, wherein the cancer is breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, brain cancer, pancreatic cancer, bladder cancer, testicular cancer, prostate cancer or gastric/stomach cancer, a hematologic malignancy, or any combination thereof.

69. The CAR immune cell of any one of claims 66-68, wherein the activity is T cell activation.

70. The CAR immune cell of any one of claims 66-69, wherein the activity is IFN- gamma release and/or CD 107a degranulation.

71. The CAR immune cell of any one of claims 66-70, wherein the immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof.

72. A CAR immune cell that has activity only when exposed to a cell that expresses at least 20,000 molecules of an antigen.

73. The CAR immune cell of claim 72, wherein the antigen is a cancer-associated antigen.

74. The CAR immune cell of claim 73, wherein the cancer is breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, brain cancer, pancreatic cancer, bladder cancer, testicular cancer, prostate cancer or gastric/stomach cancer, a hematologic malignancy, or any combination thereof.

75. The CAR immune cell of any one of claims 72-75, wherein the activity is T cell activation.

76. The CAR immune cell of any one of claims 72-75, wherein the activity is IFN- gamma release and/or CD 107a degranulation.

77. The CAR immune cell of any one of claims 72-76, wherein the immune cell is a T cell, NK cell, NK T cell, dendritic cell, macrophage, or any combination thereof.