Chimeric receptors and methods of use thereof

Abstract

This article provides solid tumor antigen targets for chimeric receptors and chimeric inhibitory receptors and their methods of use, such as for the treatment of cancer.

Description

Technical field

[0001] This application relates to solid tumor antigen targets for chimeric receptors and chimeric inhibitory receptors, and methods of their use, such as in the treatment of cancer. prior art

[0002] Adoptive cell therapies based on immunotherapies, such as chimeric antigen receptors (CARs), to redirect the specificity and function of immune response cells, such as T cells and natural killer (NK) cells, have been used in patients with malignant tumors. showed efficacy in patients, many of which previous studies focused on hematological malignancies (Pule et al., Nat. Med. (14):1264-1270 (2008); Maude et al., N Engl J Med. (371):1507- 17 (2014);Brentjens et al., Sci Transl Med. (5):177ra38 (2013)). For example, CAR T cells have been shown to induce complete responses in patients with CD19-expressing malignancies where chemotherapy has induced resistance and tumor progression.

[0003] Unlike T cells, natural killer (NK) cells can eliminate abnormal cells, such as cancer cells, without triggering them. NK cell activity is determined by the balance of external signals from inhibitory and activating NK cell receptors. Inhibitory receptors such as killer immunoglobulin receptors (KIRs) interact with major histocompatibility complex (MHC) class I antigens and protect normal cells from NK cell activity (see US20180057795A1).

[0004] One challenge in developing CAR therapies for solid tumors is the lack of suitable targets. The ability to identify appropriate CAR targets is important to effectively target and treat tumors without destroying normal cells expressing the same target antigen. Therefore, there is still a need for CAR-NK cell-based tumor therapies that target tumor cells but not normal cells or tissues. Contents of the invention

[0005] In one aspect, provided herein is an isolated cell comprising: (a) Inhibitory chimeric receptors comprising an extracellular antigen-binding domain that binds to an antigen, wherein the antigen is selected from the group consisting of: VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2 and SLC26A3; and (b) Chimeric receptors comprising one or more extracellular antigen-binding domains, wherein the one or more extracellular antigen-binding domains bind one or more additional antigens selected from the following The group consisting of: CEA, CEACAM1, CEACAM5 and CEACAM6.

[0006] In some aspects, the antigen-binding domain of the inhibitory chimeric receptor includes one or more single chain variable fragments (scFv), optionally wherein the one or more scFvs each include a heavy chain variable domain (VH) and A light chain variable domain (VL), optionally wherein the VH and VL are separated by a peptide linker, optionally the peptide linker comprising the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 77, and / or optionally wherein the one or more scFvs each comprise the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0007] In some aspects, the inhibitory chimeric receptor includes a transmembrane domain, and the transmembrane domain is selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain, CD25 transmembrane domain, CD7 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, LAX transmembrane domain Domain, LAT transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, TIM3 transmembrane domain, KIR3DS1 transmembrane domain, KIR3DL1 transmembrane domain, NKG2D transmembrane domain, NKG2A Transmembrane domain, TIGIT transmembrane domain, 2B4 transmembrane domain and BTLA transmembrane domain.

[0008] In some aspects, the inhibitory chimeric receptor includes a spacer between the antigen-binding domain and the transmembrane domain, optionally wherein the spacer has a polypeptide selected from the group consisting of SEQ ID NOs: 49-58 the amino acid sequence of the group; and / or

[0009] In some aspects, the inhibitory chimeric receptor comprises one or more intracellular inhibitory domains selected from the group consisting of: PD-1, CTLA4, TIGIT, LAIR1, GRB-2, Dok-1, Dok-2 , SLAP, LAG3, HAVR, BTLA, LIR1, NKG2A, KIR3DL1, GITR, PD-L1, CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22 , LAR, PTPH1, SHIP-1, RasGAP, CD94 and CD161.

[0010] In some aspects, the chimeric receptor is a CAR. In some aspects, the CAR includes one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of: CD3ζ chain intracellular signaling domain, CD3ε chain intracellular signaling domain Signaling domain, CD97 intracellular signaling domain, CD11a-CD18 intracellular signaling domain, CD2 intracellular signaling domain, ICOS intracellular signaling domain, CD27 intracellular signaling domain, CD154 intracellular signaling domain, CD8 cells Intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, ZAP40 intracellular signaling domain, CD30 intracellular signaling domain, GITR intracellular signaling domain, HVEM Intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, MyD88 intracellular signaling domain, 2B4 intracellular signaling domain, NKp46 intracellular signaling domain, NKp30 intracellular signaling domain, NKp44 cells intracellular signaling domain, NKG2D intracellular signaling domain, CD226 intracellular signaling domain and CD160 intracellular signaling domain. In some aspects, the CAR includes a transmembrane domain, and the transmembrane domain is selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain, CD25 transmembrane domain, CD7 transmembrane domain Domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, LAX transmembrane domain, LAT Transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, TIM3 transmembrane domain, KIR3DS1 transmembrane domain, KIR3DL1 transmembrane domain, NKG2D transmembrane domain, NKG2A transmembrane domain , TIGIT transmembrane domain, 2B4 transmembrane domain and BTLA transmembrane domain.

[0011] In some aspects, the CAR includes a spacer between an antigen-binding domain and a transmembrane domain, and the spacer has an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-58 .

[0012] In some aspects, the inhibitory chimeric receptor and / or the antigen-binding domain of the chimeric receptor comprises one or more single chain variable fragments (scFv), optionally wherein the one or more scFvs each comprise a heavy chain variable domain (VH) and light chain variable domain (VL), optionally wherein the VH and VL are separated by a peptide linker, optionally wherein the peptide linker comprises SEQ ID NO: 39 or SEQ ID NO: Amino acid sequence of 77.

[0013] In some aspects, the one or more scFvs each comprise the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain .

[0014] In some aspects, the one or more additional antigens are CECAM1, optionally wherein the antigen-binding domain that binds to CEACAM1 includes a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 1 and comprising The light chain variable domain (VL) of the amino acid sequence of SEQ ID NO: 2.

[0015] In some aspects, the one or more additional antigens are CECAM5, optionally wherein the antigen-binding domain that binds to CEACAM5 includes a heavy chain variable domain (VH) and a light chain variable domain selected from the group consisting of VL): (i) VH comprising the amino acid sequence of SEQ ID NO: 3 and VL comprising the amino acid sequence of SEQ. ID NO: 4; (ii) VH comprising the amino acid sequence of SEQ ID NO: 9 and VL comprising the amino acid sequence of SEQ. ID NO: 10; (iii) VH comprising the amino acid sequence of SEQ ID NO: 11 and VL comprising the amino acid sequence of SEQ. ID NO: 12; (iv) VH comprising the amino acid sequence of SEQ ID NO: 13 and VL comprising the amino acid sequence of SEQ. ID NO: 14; (v) VH comprising the amino acid sequence of SEQ ID NO: 78 and VL comprising the amino acid sequence of SEQ. ID NO: 79; and (vi) VH comprising the amino acid sequence of SEQ ID NO: 15 and VL comprising the amino acid sequence of SEQ. ID NO: 16.

[0016] In some aspects, the antigen-binding domain that binds to CEACAM5 includes a heavy chain (HC) and a light chain (LC) selected from the group consisting of: (i) HC comprising the amino acid sequence of SEQ ID NO: 5 and LC comprising the amino acid sequence of SEQ. ID NO: 6; and (ii) HC comprising the amino acid sequence of SEQ ID NO: 7 and LC comprising the amino acid sequence of SEQ. ID NO: 8; or (c) the one or more additional antigens are CECAM6, optionally wherein the antigen-binding domain binding to CEACAM6 includes a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 17 and comprising SEQ ID NO The light chain variable domain (VL) of 18 amino acid sequences.

[0017] In some aspects, the cells are immunoreactive cells. In some aspects, the inhibition of binding of the chimeric receptor to the antigen inhibits the immune response cell and / or wherein the binding of the chimeric receptor to the one or more additional antigens activates the immune response cell.

[0018] In some aspects, the chimeric receptor binds to the one or more additional antigens with low binding affinity.

[0019] In some aspects, the chimeric receptor binds to the one or more additional antigens with a lower binding affinity than the binding affinity that inhibits binding of the chimeric receptor to the antigen.

[0020] In some aspects, the inhibitory chimeric receptor binds to the antigen with low binding affinity.

[0021] In some aspects, the chimeric receptor is recombinantly expressed, optionally wherein the chimeric receptor is expressed from a vector or from a selected locus from the genome of the cell.

[0022] In some aspects, the cell line is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, natural killer T (NKT) cells, myeloid cells, Macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells and induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some aspects, the cell is autologous or the cell is allogeneic.

[0023] In some aspects, provided herein is an isolated nucleic acid encoding an inhibitory chimeric receptor as provided herein.

[0024] In some aspects, provided herein is a vector comprising a nucleic acid as provided herein.

[0025] In some aspects, provided herein is a genetically modified cell comprising a nucleic acid as provided herein or a vector as provided herein.

[0026] In some aspects, provided herein is a pharmaceutical composition comprising an effective amount of an isolated cell as provided herein, an isolated nucleic acid as provided herein, or a vector as provided herein and A pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

[0027] In some aspects, provided herein is a method of treating an individual in need thereof, the method comprising administering to the individual an effective amount of an isolated cell as provided herein, an isolated nucleic acid as provided herein , a carrier as provided herein, or a pharmaceutical composition as provided herein.

[0028] In some aspects, provided herein is a method of stimulating a cell-mediated immune response against tumor cells in an individual, the method comprising administering to an individual having a tumor an effective amount of an isolated A cell, an isolated nucleic acid as provided herein, a vector as provided herein, or a pharmaceutical composition as provided herein.

[0029] In some aspects, provided herein is a method of providing anti-tumor immunity in an individual, the method comprising administering to an individual in need thereof an effective amount of an isolated cell as provided herein, as provided herein An isolated nucleic acid, a vector as provided herein, or a pharmaceutical composition as provided herein.

[0030] In some aspects, provided herein is a method of reducing tumor burden in an individual, the method comprising administering to the individual an effective amount of an isolated cell as provided herein, an isolated cell as provided herein A nucleic acid, a vector as provided herein, or a pharmaceutical composition as provided herein. In some aspects, the method reduces tumor cell number, the method reduces tumor size, the method reduces tumor volume, and / or the method eliminates tumors in the individual.

[0031] In some aspects, provided herein is a method of treating an individual with a tumor comprising administering an effective amount of an isolated cell as provided herein, an isolated nucleic acid as provided herein, an isolated nucleic acid as provided herein, such as A carrier as provided herein or a pharmaceutical composition as provided herein.

[0032] In some aspects, provided herein is a method of treating or preventing cancer in an individual, wherein the cancer is selected from the group consisting of colorectal cancer, pancreatic cancer, lung adenocarcinoma, and gastric cancer, the method comprising treating The subject is administered an effective amount of an isolated cell as provided herein, an isolated nucleic acid as provided herein, a vector as provided herein, or a pharmaceutical composition as provided herein.

[0033] In some aspects, the method increases the time of progression-free survival of the subject; and or the method increases the time of survival of the subject.

[0034] In some aspects, provided herein is a kit for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma, and / or gastric cancer, comprising an isolated cell as provided herein, An isolated nucleic acid as provided herein, a vector as provided herein, or a pharmaceutical composition as provided herein. In some aspects, the kit further includes written instructions for using the isolated cells or pharmaceutical composition to treat and / or prevent colorectal cancer, pancreatic cancer, lung adenocarcinoma, and / or gastric cancer in an individual, or The kit further includes instructions for using the nucleic acid or vector to generate one or more antigen-specific cells for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma, and / or gastric cancer in an individual.

[0035] In some aspects, provided herein is an inhibitory chimeric receptor comprising an extracellular antigen binding domain that binds to an antigen selected from the group consisting of: VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33 , PLA2G2A, ABCA8, ATP1A2, CHP2 and SLC26A3.

[0036] In some embodiments, the antigen is VSIG2. In some embodiments, the antigen is CPM. In some embodiments, the antigen is ITM2C. In some embodiments, the antigen is SLC26A2. In some embodiments, the antigen is SLC4A4. In some embodiments, the antigen is GPA33. In some embodiments, the antigen is PLA2G2A. In some embodiments, the antigen is ABCA8. In some embodiments, the antigen is ATP1A2. In some embodiments, the antigen is CHP2. In some embodiments, the antigen is SLC26A3.

[0037] In some embodiments, when expressed on a cell, the inhibitory chimeric receptor inhibits one or more activities of the cell.

[0038] In some embodiments, the antigen is not expressed on tumor cells, or the antigen is expressed on tumor cells at a lower level than on non-tumor cells.

[0039] In some embodiments, the antigen is expressed on non-tumor cells, or the antigen is expressed on non-tumor cells at a higher level than on corresponding tumor cells.

[0040] In some embodiments, the antigen is expressed on non-neoplastic cells derived from a tissue selected from the group consisting of: lung, pancreas, gastrointestinal tract, colon, brain, neuronal tissue, endocrine, bone, bone marrow, immune System, muscle, liver, gallbladder, pancreas, kidney, bladder, male reproductive organs, female reproductive organs, fat, soft tissue and skin.

[0041] In some embodiments, the inhibitory chimeric receptor comprises one or more intracellular inhibitory domains selected from the group consisting of: PD-1, CTLA4, TIGIT, LAIR1, GRB -2, Dok-1, Dok-2, SLAP, LAG3, HAVR, BTLA, LIR1, NKG2A, KIR3DL1, GITR, PD-L1, CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST , c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, RasGAP, CD94 and CD161.

[0042] In some embodiments, the antigen binding domain comprises one or more antibodies, antigen binding fragments of antibodies, F(ab) fragments, F(ab') fragments, single chain variable fragments (scFv) or single domain antibodies ( sdAb).

[0043] In some embodiments, the antigen binding domain comprises one or more single chain variable fragments (scFv). In some embodiments, each of the one or more scFvs comprises a variable heavy domain (VH) and a variable light domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 77. In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, wherein VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain .

[0044] In another aspect, provided herein is an isolated cell comprising the inhibitory chimeric receptor of any of the above embodiments. In some embodiments, the inhibition of recombinant expression of the chimeric receptor system. In some embodiments, the suppressor chimeric receptor is expressed by a vector or a selected locus from the gene body of the cell. In some embodiments, the cell further comprises a chimeric receptor comprising one or more extracellular antigen binding domains, wherein the one or more extracellular antigen binding domains bind one or more antigens, the one or more antigens is selected from the group consisting of: CEA, CEACAM1, CEACAM5 and CEACAM6.

[0045] In another aspect, provided herein is an isolated cell comprising: (a) an inhibitory chimeric receptor comprising an extracellular antigen binding domain that binds to an antigen selected from the group consisting of: VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2 and SLC26A3; and (b) A chimeric receptor comprising one or more extracellular antigen binding domains, wherein the one or more extracellular antigen binding domains bind one or more additional antigens selected from the group consisting of Consists of groups: CEA, CEACAM1, CEACAM5 and CEACAM6.

[0046] In some embodiments, the chimeric receptor is a chimeric T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the chimeric receptor is a CAR. In some embodiments, the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of: CD3ζ chain intracellular signaling domain, CD3ε chain intracellular signaling domain, Signaling Domain, CD97 Intracellular Signaling Domain, CD11a-CD18 Intracellular Signaling Domain, CD2 Intracellular Signaling Domain, ICOS Intracellular Signaling Domain, CD27 Intracellular Signaling Domain, CD154 Intracellular Signaling Domain, CD8 Cells Intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, ZAP40 intracellular signaling domain, CD30 intracellular signaling domain, GITR intracellular signaling domain, HVEM Intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, MyD88 intracellular signaling domain, 2B4 intracellular signaling domain, NKp46 intracellular signaling domain, NKp30 intracellular signaling domain, NKp44 cellular Intracellular signaling domain, NKG2D intracellular signaling domain, CD226 intracellular signaling domain and CD160 intracellular signaling domain.

[0047] In some embodiments, the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain, CD25 transmembrane domain, CD7 transmembrane domain domain, CD3 ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, LAX transmembrane domain, LAT transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, TIM3 transmembrane domain, KIR3DS1 transmembrane domain, KIR3DL1 transmembrane domain, NKG2D transmembrane domain, NKG2A transmembrane structure domain, TIGIT transmembrane domain, 2B4 transmembrane domain and BTLA transmembrane domain.

[0048] In some embodiments, the CAR comprises a spacer between the antigen binding domain and the transmembrane domain, and the spacer has an amino acid sequence selected from the group consisting of SEQ ID NO: 49-58 .

[0049] In some embodiments, the inhibitory chimeric receptor and / or the antigen binding domain of the chimeric receptor comprises one or more antibodies, antigen binding fragments of antibodies, F(ab) fragments, F(ab') fragments, Single chain variable fragment (scFv) or single domain antibody (sdAb).

[0050] In some embodiments, the inhibitory chimeric receptor and / or the antigen binding domain of the chimeric receptor comprises one or more single chain variable fragments (scFv). In some embodiments, each of the one or more scFvs comprises a variable heavy domain (VH) and a variable light domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 77. In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, wherein VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain .

[0051] In some embodiments, the cells are immunoreactive cells. In some embodiments, binding of the chimeric inhibitory receptor to the antigen suppresses the immune response cell. In some embodiments, binding of the chimeric receptor to the one or more additional antigens activates the immune response cells.

[0052] In some embodiments, the chimeric receptor binds to the one or more additional antigens with low binding affinity.

[0053] In some embodiments, the chimeric receptor binds to the one or more additional antigens with a lower binding affinity than the binding affinity that inhibits the chimeric receptor from binding to the antigen. In some embodiments, the inhibitory chimeric receptor binds to the antigen with low binding affinity.

[0054] In some embodiments, the chimeric receptor is expressed recombinantly. In some embodiments, the chimeric receptor is expressed by a vector or a selected locus from the genome of the cell.

[0055] In some embodiments, the cell line is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, natural killer T (NKT) cells, myeloid cells, Macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells and induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0056] In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic.

[0057] In another aspect, provided herein is an isolated nucleic acid encoding an inhibitory chimeric receptor such as any of the above inhibitory chimeric receptors.

[0058] In another aspect, provided herein is a vector comprising the nucleic acid of the above aspect.

[0059] In another aspect, provided herein is a genetically modified cell comprising the nucleic acid or vector of the above aspect.

[0060] In another aspect, provided herein is a method of treating an individual in need thereof, the method comprising administering the isolated cells of any of the above embodiments.

[0061] In another aspect, provided herein is a method of stimulating a cell-mediated immune response against tumor cells in an individual, the method comprising administering to the individual having the tumor the isolated of cells.

[0062] In another aspect, provided herein is a method of providing anti-tumor immunity in an individual, the method comprising administering to an individual in need thereof an isolated cell as in any one of the above embodiments.

[0063] In another aspect, provided herein is a method of reducing tumor burden in an individual, the method comprising administering to the individual an isolated cell as in any one of the above embodiments. In some embodiments, the method reduces the number of tumor cells. In some embodiments, the method reduces tumor size. In some embodiments, the method reduces tumor volume. In some embodiments, the method eliminates tumors in the individual.

[0064] In another aspect, provided herein is a method of treating an individual with a tumor comprising administering an isolated cell as in any one of the above embodiments.

[0065] In another aspect, provided herein is a method of treating or preventing colorectal cancer in an individual, the method comprising administering to the individual an isolated cell as in any one of the above embodiments.

[0066] In another aspect, provided herein is a method of treating or preventing pancreatic cancer in an individual, the method comprising administering to the individual an isolated cell as in any one of the above embodiments.

[0067] In another aspect, provided herein is a method of treating or preventing lung adenocarcinoma in an individual, the method comprising administering to the individual an isolated cell as in any one of the above embodiments.

[0068] In another aspect, provided herein is a method of treating or preventing gastric cancer in an individual, the method comprising administering to the individual an isolated cell as in any one of the above embodiments.

[0069] In some embodiments of the above aspects, the isolated cells are administered in an effective amount. In some embodiments of the above aspects, the method increases the progression-free survival time of the individual. In some embodiments of the above aspects, the method increases the survival time of the individual.

[0070] In another aspect, provided herein is a pharmaceutical composition comprising an effective amount of isolated cells as in any one of the above embodiments and a pharmaceutically acceptable carrier, a pharmaceutically acceptable Excipients or combinations thereof. In some embodiments, the pharmaceutical composition is used for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma and / or gastric cancer.

[0071] In another aspect, provided herein is a kit for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma, and / or gastric cancer, which comprises any one of the above embodiments. An isolated cell or a pharmaceutical composition as described above. In some embodiments, the kit further includes written instructions for using the isolated cells to treat and / or prevent colorectal cancer, pancreatic cancer, lung adenocarcinoma, and / or gastric cancer in an individual.

[0072] In another aspect, provided herein is a kit for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma, and / or gastric cancer, comprising the isolated nucleic acid of the above aspect or carrier. In some embodiments, the kit further comprises use of the nucleic acid to generate one or more antigen-specific cells for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma, and / or gastric cancer in an individual written instructions. Brief description of the diagram

[0073] []

[0074] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Firm upon request and upon payment of the necessary fee.

[0075] These and other features, aspects and advantages of the present invention will be more fully understood in conjunction with the following description of the invention and the accompanying drawings.

[0076] [picture] [1] Schematic diagram depicting the members of the CEA family, their structure and function. [picture] [2] Depicted the gene expression of CEACAM5 and CEACAM6 in colorectal cancer (CRC) and normal tissues. [picture] [3A] Delineates the gene expression of CEACAM5 in colorectal cancer (CRC) and normal tissues. [picture] [3B] Delineates the gene expression of CEACAM6 in colorectal cancer (CRC) and normal tissues. [picture] [4] Depicted the protein expression of CEACAM1, CEACAM5, and CEACAM6 in patient samples (including cancer and normal tissue samples) in tissue microarrays. [picture] [5] Describes flow cytometry-based analysis of CEACAM1, CEACAM5 and CEACAM6 expression in a panel of human colon cancer cell lines obtained from ATCC. [picture] [6A] Describes the analysis of NOT targets using bulk RNAseq gene expression data from ABCA8. [picture] [6B] Describes the analysis of NOT targets using bulk RNAseq gene expression data of ATP1A2. [picture] [7A] depicts the analysis of a single cell RNAseq dataset GSE144735 after dimensionality reduction and clustering, in which different cell types are annotated and colored. [picture] [7B] Depicts the expression of ABCA8 in normal tissue (Figure 7A), with its expression in stromal cells highlighted. [picture] [8] characterized the gene expression of VSIG2 in colorectal cancer (CRC) and normal tissues. [picture] [9A] depicts gene expression analysis of VSIG2, showing expression in epithelial cells in normal and tumor tissues in the single-cell RNAseq dataset GSE144735. [picture] [9B] depicts gene expression analysis of VSIG2, showing overall gene expression in normal and tumor tissues in the single-cell RNAseq data set GSE132465. [picture] [10A] Describes comparative gene expression analysis of VSIG2 and CEACAM5. [picture] [10B] Depicts comparative gene expression analysis of VSIG2 and CEACAM5, presented as relative scores for each population. [picture] [10C] Describes comparative gene expression analysis of VSIG2 and CEACAM5 in normal gastrointestinal tissues (ileum, colon and rectum). [picture] [10D] Depicts comparative gene expression analysis of VSIG2 and CEACAM5 in normal lung epithelial tissue. [picture] [10E] Describes comparative gene expression analysis of VSIG2 and CEACAM5 in normal kidney and liver tissues. [picture]

[11] Describe the performance analysis of potential NOT target VSIG2 expression in multiple normal and cancer subtypes of the colon (dataset: GSE81861, GSE144735, GSE132465). [picture] [12A] Characterizes VSIG2 expression in healthy lung epithelial cells (EGAS00001004344). [picture] [12B] Compare the expression of VSIG2, CEACAM5 and CEACAM6 in healthy lung epithelial cells. [picture] [13A] Tissue microarray (TMA) depicting gastrointestinal tumors and healthy gastrointestinal tissues stained for VSIG2. [picture] [13B] is a representative healthy tissue sample of TMA stained for VSIG2. [picture] [13C] is a representative healthy tissue sample of TMA stained for VSIG2. [picture] [14A] depicts multiplex IHC analysis of healthy tissue stained for VSIG2 (pink) and CEACAM5 (white). Nuclei were stained with DAPI (blue). [picture] [14B] depicts multiplex IHC analysis of healthy tissue stained for VSIG2 (pink) and CEACAM5 (white). Nuclei were stained with DAPI (blue). [picture]

[15] depicts the localization analysis of VSIG2 (pink) and CEACAM5 (white) in grade 3 stage IIB colon cancer. Nuclei were stained with DAPI (blue). [picture] [16A] depicts gene expression analysis of VSIG2 and CEACAM5 in stromal cells and immune cells in colon cancer samples from two cohorts. [picture] [16B] Describes gene expression analysis of VSIG2 and CEACAM5 in stromal cells and immune cells in colon cancer samples from two cohorts. [picture] [16C] Depicts gene expression analysis of VSIG2 and CEACAM5 in stromal cells and immune cells in colon cancer samples from two cohorts. [picture] [17A] Describes gene expression analysis of CPM in colon cancer and normal tissues (dataset: GSE81861, GSE144735, GSE132465). [picture] [17B] Characterized the comparative performance of CPM and CEACAM5 and CEACAM6 in normal lung tissue (dataset EGAS00001004344). [picture] [17C] Delineate the comparative performance of CPM and CEACAM5 and CEACAM6 in healthy lung cells (dataset EGAS00001004344). [picture]

[18] describe the performance analysis of potential NOT target GPA33. [picture] [18A] Delineate GPA33 expression across multiple normal and CRC cell subtypes (dataset: GSE81861, GSE144735, GSE132465). [picture] [18B] Characterized GPA33 gene expression analysis in normal lung tissue (dataset EGAS00001004344). [picture]

[19] describe the performance analysis of potential NOT target PLA2G2A. [picture] [19A] Delineate PLA2G2A expression in multiple normal and CRC cell subtypes (dataset: GSE81861, GSE144735, GSE132465). [picture] [19B] Characterized PLA2G2A gene expression analysis in normal lung tissue (dataset EGAS00001004344). [picture]

[20] describe the performance analysis of potential NOT target ITM2C. [picture] [20A] Characterizes ITM2C manifestations in multiple normal and CRC cell subtypes (dataset: GSE81861, GSE144735, GSE132465). [picture] [20B] Delineate ITM2C gene expression analysis in normal lung tissue. [picture]

[21] describe the performance analysis of potential NOT target CHP2. [picture] [21A] Delineate CHP2 manifestations in multiple normal and CRC cell subtypes (dataset: GSE81861, GSE144735, GSE132465). [picture] [21B] Delineate CHP2 gene expression analysis in normal lung tissue. [picture]

[22] Describe the performance analysis of potential NOT target SLC26A2. [picture] [22A] Characterizes SLC26A2 manifestations in multiple normal and CRC cell subtypes (dataset: GSE81861, GSE144735, GSE132465). [picture] [22B] Delineate SLC26A2 gene expression analysis in normal lung tissue. [picture]

[23] describe the performance analysis of potential NOT target SLC4A4. [picture] [23A] Delineate SLC4A4 manifestations in multiple normal and CRC cell subtypes (dataset: GSE81861, GSE144735, GSE132465). [picture] [23B] Delineate SLC4A4 gene expression analysis in normal lung tissue. [picture]

[24] describe the performance analysis of potential NOT target SLC26A3. [picture] [24A] Delineate SLC26A3 manifestations in multiple normal and CRC cell subtypes (dataset: GSE81861, GSE144735, GSE132465). [picture] [24B] Delineate SLC26A3 gene expression analysis in normal lung tissue. [] [picture] [25A] [To the picture] [25B] Depicts the LDH activity of parental untransduced LS174t cells ( [picture] [25A]) and the LDH activity of target cells expressing mKATE ( [picture] [25B]), each after co-culture with CEA aCAR transduced T cells from the first donor ("T cell donor 1"). [] [picture] [26A] [To the picture] [26B] Depicts the LDH activity of parental untransduced LS174t cells ( [picture] [26A]) and the LDH activity of target cells expressing mKATE ( [picture] [26B]), each after co-culture with CEA aCAR-transduced T cells from a second donor (“T cell donor 2”). [] [picture] [27A] [To the picture] [27F] Delineate the interleukin activity (IL-2, IL-2, [picture] [27A];IFN-γ, [picture] [27B];TNF-α, [picture] [27C];Caspase 3, [picture] [27D]; perforin, [picture] [27E]; and granzyme B, [picture] [27F]). [] [picture]

[28] Delineate the killing activity of CEA aCAR-transduced NK cells transduced with the first set of CEA aCAR as determined by fluid-based assay. [] [picture]

[29] Delineate the killing activity of CEA aCAR-transduced NK cells transduced with a second set of CEA aCAR as determined by fluid-based assay. [] [picture]

[30] depict the killing activity of NK cells transduced with two different CEA aCARs at different effector cell to target cell (E:T) ratios as determined by fluid-based assays. [] [picture] [31A] [To the picture] [31C] Delineate surface marker activation of CEA aCAR-transduced NK cells cultured with target cells (NKp46, [picture] [31A];CD16, [picture] [31B]; and CD107a, [picture] [31C]). [] [picture]

[32] Delineated the killing activity of transduced CEA aCAR NK cells by real-time fluorescence-based analysis. [picture] [33A] [To the picture] [33B] Depicts the fold change in tumor bioluminescence after treatment of mice with NK cells expressing CEA aCAR ( [picture] [33A]) and representative tumor images from day 16 after treatment ( [picture] [33B]). [picture]

[34] depict the killing activity of NK cells transduced with different CEA aCAR constructs as measured by real-time fluorescence-based analysis. [picture]

[35] depict the killing activity of NK cells transduced with different CEA aCAR constructs as measured by real-time fluorescence-based analysis. [picture]

[36] depict representative images obtained on day 2 after NK cell treatment for mice inoculated intraperitoneally with target cells of a colorectal cancer cell line and treated with NK cells transduced with different CEA aCAR constructs. [] [picture]

[37] depict representative images obtained on day 13 after NK cell treatment for mice inoculated intraperitoneally with target cells of a colorectal cancer cell line and treated with NK cells transduced with CEA aCAR constructs. [picture] [38A] [To the picture] [38D] Provides the performance levels of different CEA-CAR lentiviral and retroviral constructs on transduced NK cells (3 days after transduction). Figure 38A, Figure 38B, and Figure 38C provide flow cytometry histograms depicting performance levels (Figure 38A, for lentiviral constructs, at 3 days post-transduction; Figure 38B, for retroviral constructs, at 3 days post-transduction 3 days after transduction; and Figure 38C, for retroviral constructs, 11 days after transduction). The dashed line represents the negative threshold (based on the performance of untransduced “virus-free” NK cells). [picture] [38D] shows the time course of expression from retroviral constructs, where the left panel provides the time course of percent expression, and the right panel provides the time course of mean fluorescence intensity (MFI). [picture] [39A] [To the picture] [39C] shows the performance of CEA CAR from different retroviral transduction systems pseudotyped with baboon endogenous viral envelope (BaEv). [picture] [39A] shows the performance of the first retroviral backbone ("Backbone 1"). [picture] [39B] shows the performance of the first retroviral vector backbone ("Backbone 1") with alternative nucleotide sequences ("New Codon Optimization"). [picture] [39C] shows the performance of the second retroviral backbone ("Backbone 2"). [picture] [40A] [To the picture] [40B] Provide the transduction efficiency of CEA CAR with different CAR structures ( [picture] [40A]) and CAR performance ( [picture] [40B]). [picture] [41A] [To the picture] [41B] shows CEA CAR NK cells (derived from two different donors, donor 7, [picture] [41A] and donor 13, [picture] [41B]) Killing at a 1:1 effector cell to target cell ratio. [] [picture] [42A] [To the picture] [42B] shows CEA CAR NK cells (derived from two different donors, donor 7, [picture] [42A] and donor 13, [picture] [42B]) Killing at a 1:2 effector cell to target cell ratio. [] [picture]

[43] showed the CEA CAR performance of NK cells on different CAR structures. [] [picture]

[44] showed NK cell activation when CEA CAR NK cells were co-cultured with target cells (colorectal cancer cell line Ls174t) as measured by granzyme B and interferon gamma. [] [picture] [45A] [To the picture] [45B] shows the fold change in tumor burden as calculated 5 days after NK cell treatment ( [picture] [45A]) and representative images ( [picture] [45B]), as measured by bioluminescence imaging in tumor-bearing mice treated with CEA CAR NK cells. [] [picture]

[46] showed the tumor response rate of mice in the study. [] [picture] [47A] [To the picture] [47B] Shows NK cells expressing CEA aCAR and off-target iCAR ( [picture] [47A]) and NK cells expressing CEA aCAR and inhibitory CAR specific for the model safety antigen HER2 ( [picture] [47B]) NK cell CAR performance level. [] [picture]

[48] ​​showed the level of NK cell performance in inhibiting CAR (specific for the safety antigen VSIG2) and activating anti-CEA CAR. [] [picture]

[49] showed killing of HER2+ Ls174t cells co-cultured with NK cells expressing off-target iCAR and CEA aCAR or HER2 iCAR and CEA aCAR. [] [picture] [50A] [To the picture] [50B] shows the phosphorylation level of NK cell activation markers after NK cells expressing aCAR / iCAR combination were co-cultured with target cells expressing safety antigens. [] [picture]

[51] shows the percentage reduction of target cells as measured by flow cytometry after overnight co-culture with NK cells expressing aCAR / iCAR combination or aCAR alone. [] Implementation

[0077] [] Cross-references to related applications

[0078] This application claims the rights and priority of U.S. Provisional Application No. 63 / 231,626 filed on August 10, 2021 and U.S. Provisional Application No. 63 / 117,861 filed on November 24, 2020; each case is based on Incorporated herein by reference in its entirety for all purposes. sequence list

[0079] This application contains a Sequence Listing that has been filed by EFS-Web and is hereby incorporated by reference in its entirety. This ASCII copy was created on January 13, 2022 and is named STB-023WO_SL, and is 255,989 bytes in size.

[0080] Unless otherwise indicated, the practice of the invention will employ methods of molecular biology, chemistry, biochemistry, virology and immunology that are within the ability of those skilled in the art. This technique is well documented in the literature. See, for example, Hepatitis C Viruses: Genomes and Molecular Biology (S.L. Tan, eds., Taylor & Francis, 2006); Fundamental Virology, 3rd ed., Volumes I and II (B.N. Fields and D.M. Knipe, eds.); Handbook of Experimental Immunology, Volumes I to IV (D.M. Weir and C.C. Blackwell, eds., Blackwell Scientific Publications); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Methods In Enzymology (eds. S. Colowick and N. Kaplan, Academic Press, Inc.). [Definition] []

[0081] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for the purpose of clarity and / or for timely reference, and the inclusion of such definitions herein should not necessarily be understood to mean that they are consistent with what is commonly understood in the art. difference. Techniques and procedures described or referenced herein will generally be better understood by those skilled in the art using well-known methods, such as, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Edition (2012) Cold Spring The widely used method of molecular breeding is described in Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents were generally performed according to protocols and conditions defined by the manufacturer, unless otherwise indicated.

[0082] As used herein, the singular forms "a" and "the" include plural references unless the context clearly dictates otherwise. Unless expressly indicated otherwise, the terms "include", "such as" and similar terms are intended to convey including but not limited to.

[0083] As used herein, unless expressly indicated otherwise, the term "comprising" also explicitly includes embodiments "consisting of" and "consisting essentially of" the recited elements.

[0084] The term "about" indicates and encompasses the indicated value and ranges above and below that value. In certain embodiments, the term "about" indicates ±10%, ±5%, or ±1% of the specified value. In certain embodiments, where applicable, the term "about" indicates the indicated value ± one standard deviation of that value.

[0085] As used herein, the term "activating an immune response cell" refers to inducing a change in signal transduction or protein expression in a cell, thereby triggering an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade occurs. In certain embodiments, when an endogenous TCR or an exogenous CAR binds an antigen, immune synapse formation occurs, which includes clustering of many molecules near the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc. ). This clustering of membrane-bound signaling molecules allows phosphorylation of ITAM motifs contained within the CD3 chain. This phosphorylation in turn triggers T cell activation pathways, ultimately activating transcription factors such as NF-κΒ and AP-1. These transcription factors induce the overall gene expression of T cells to increase IL-2 production, thereby performing the proliferation and expression of master regulatory T cell proteins in order to elicit T cell-mediated immune responses.

[0086] As used herein, the term "stimulate" or "stimulate an immune response" refers to the generation by one or more cell types or populations of cells of a signal that elicits an immune response. Immunostimulatory activity may include pro-inflammatory activity. In various embodiments, the immune response occurs after activation of immune cells (such as T cells or NK cells) or via receptors (including but not limited to CD28, CD137 (4-1BB), OX40, CD40, and ICOS) and their corresponding ligands. Body (including B7-1, B7-2, OX-40L and 4-1BBL) with mediation. Such polypeptides can be present in the tumor microenvironment and can activate an immune response to tumor cells. In various embodiments, promoting, stimulating or otherwise activating a pro-inflammatory polypeptide and / or its ligand enhances the immune response of the immune-responsive cells. Without being bound by a particular theory, receiving multiple stimulatory signals (e.g., co-stimulation) is essential for establishing robust and long-lasting cell-mediated immune responses (such as T cell-mediated immune responses, where T It is important that cells may be suppressed and unresponsive to antigen (also known as "T cell anergy"). In the absence of such stimulatory signals, T cells are rapidly suppressed and unresponsive to antigen. Although the roles of various co-stimulatory signals (especially when combined with each other) may differ and are still only partially understood, their co-stimulation generally increases gene expression in order to generate, for example, complete and complete expression of target cells expressing cognate antigens. and / or long-lived proliferative anti-apoptosis resistant cells, such as T cells or NK cells, that stably respond to antigen when eradicated continuously.

[0087] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain (herein also Recombinant polypeptide construct called "intracellular signaling domain").

[0088] As used herein, the term "activating CAR" or "aCAR" refers to a CAR construct / structure capable of inducing signal transduction or protein expression changes in activated CAR-expressing cells, which upon binding to a cognate aCAR ligand Initiate, activate, stimulate or increase an immune response.

[0089] As used herein, the term "inhibiting CAR" or "iCAR" refers to a CAR construct / structure capable of inducing a change in signal transduction or protein expression in a cell expressing the inhibiting CAR, which upon binding to a cognate iCAR ligand Preventing, attenuating, inhibiting, reducing, lowering, suppressing or suppressing an immune response, such as reducing the activation of immune-responsive cells that receive or have received one or more stimulatory signals, including co-stimulatory signals.

[0090] As used herein, the term "enzyme inhibitory domain" refers to a protein domain that inhibits an intracellular signal transduction cascade, such as the native T cell activation cascade. In some embodiments, the enzyme inhibitory domain of the chimeric inhibitory receptors of the invention comprises at least a portion of an extracellular domain, a transmembrane domain and / or an intracellular domain. In some embodiments, the enzyme inhibition domain comprises at least a portion of an enzyme. In some embodiments, the enzyme is selected from CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1 and RasGAP (See eg Stanford et al., Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep; 137(1): 1-19). In some embodiments, the enzyme moiety comprises an enzyme domain, an enzyme fragment, or a mutant thereof. In some embodiments, the enzyme moiety is an enzyme catalytic domain. In some embodiments, enzyme domains, enzyme fragments, or mutants thereof are selected to maximize potency and minimize basal inhibition.

[0091] As used herein, the term "intracellular signaling domain" refers to the functional portion of a protein that functions by transmitting information within the cell, by producing a second messenger, or by responding to such a messenger. Effector functions regulate cellular activity through defined signaling pathways.

[0092] As used herein, the term "extracellular antigen-binding domain" or "antigen-binding domain" (ABD) refers to a polypeptide sequence or polypeptide complex that specifically recognizes or binds to a specified antigen or epitope, such as those described herein The chimeric protein provides, for example, a polypeptide sequence or part of a polypeptide complex to which VSIG2 specifically binds. An ABD (or antibody, antigen-binding fragment and / or chimeric protein comprising the same) is said to "recognize" an epitope (or, more generally, an antigen) to which the ABD specifically binds, and that epitope is said to be The "recognition specificity" or "binding specificity" of the ABD. ABDs are said to bind to their specific antigen or epitope with specific affinity. As described herein, "affinity" refers to the strength of the non-covalent intermolecular force interaction between one molecule and another molecule. Affinity, that is, the interaction strength, can be expressed as the dissociation equilibrium constant (KD), where the lower the KD value, the stronger the intermolecular interaction. The K D value of the antibody construct is measured by methods well known in the art, including but not limited to biolayer interferometry (such as Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (such as Biacore®) and cell binding analysis (e.g., flow cytometry). Specific binding, as assessed by affinity, may refer to the binding molecule having an affinity between the ABD and its cognate antigen or epitope, where the K D value is lower than 10 -6M, 10 -7M, 10 -8M, 10 -9M or 10 -10M. Specific binding may also include recognition and binding of related biological molecules (eg, polypeptides) without specific recognition and binding of other molecules in samples (eg, biological samples) that naturally include polypeptides of the invention. In certain embodiments, specific binding refers to the binding between an ABD, an antibody, or an antigen-binding fragment and an epitope, or an antigen or an antigenic determinant that is replaceable with an identical or similar epitope, antigen, or antigenic determinant. The two preparations may be combined in a competitive manner.

[0093] ABD can be an antibody. The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0094] An ABD may be an antigen-binding fragment of an antibody. As used herein, the term "antigen-binding fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof that is sufficient to confer recognition and specific binding to a target such as an antigen or epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, single domain antibodies such as sdAb (VL or VH), camel V HH domains and those bound by antigen Multispecific antibodies formed from fragments, such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of the antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, macrobodies, microbodies, nanobodies, endobodies, diabodies, tribodies, tetrabodies, v-NARs and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0095] The number of ABDs in a binding molecule such as the chimeric proteins described herein defines the "potency" of the binding molecule. A binding molecule having a single ABD is "monovalent". A binding molecule having a plurality of ABDs is said to be "multivalent". A multivalent binding molecule having two ABDs is "bivalent". A multivalent binding molecule having three ABDs is "trivalent". A multivalent binding molecule having four ABDs is "tetravalent". In various multivalent embodiments, the plurality of ABDs all have the same recognition specificity and can be referred to as "monospecific multivalent" binding molecules. In other multivalent embodiments, at least two of the plurality of ABDs have different recognition specificities. Such binding molecules are multivalent and "multispecific". In multivalent embodiments where the ABD has a total of two recognition specificities, the binding molecule is "bispecific". In multivalent embodiments where the ABD has a total of three recognition specificities, the binding molecule is "trispecific". In multivalent embodiments where the ABD has a plurality of recognition specificities for different epitopes present on the same antigen, the binding molecule is "multiparatopic." A polyvalent example in which an ABD collectively recognizes two epitopes on the same antigen is "biparatopic".

[0096] In various multivalent embodiments, the multivalency of the binding molecule improves the affinity of the binding molecule for a specific target. As described herein, "affinity" refers to the overall strength of an interaction between two or more molecules, such as multivalent binding molecules for a specific target, where affinity is provided by the affinity of multiple ABDs Cumulative strength of interactions. Affinity can be measured by the same method as that used to determine affinity as described above. In certain embodiments, the binding molecule has an affinity for a specific target such that the interaction is a specific binding interaction, wherein the affinity between the two molecules is less than 10-6M, 10-7M, 10-8M , K D value of 10 -9M or 10 -10M. In certain embodiments, the affinity of the binding molecule for a specific target has a KD value such that the interaction is a specific binding interaction, where one or more of the individual ABD affinities need not have an affinity suitable for specific binding on its own The KD value of the respective antigen or epitope. In certain embodiments, avidity is the cumulative strength of an interaction provided by the affinities of multiple ABDs for a single antigen on a common specific target or complex, such as a single antigen found on an individual cell. In certain embodiments, affinity is the cumulative strength of an interaction provided by the affinity of multiple ABDs for individual epitopes on a shared individual antigen.

[0097] As used herein, the term "single chain variable fragment" or "scFv" refers to a fusion protein comprising at least one antigen-binding fragment containing a light chain variable region and at least one antigen-binding fragment containing a heavy chain variable region, The light chain and heavy chain variable regions are continuously connected via a short flexible polypeptide linker and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless stated otherwise, as used herein, a scFv may have the VL and VH variable regions in either order, for example, with respect to the N- and C-termini of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH- Connectome-VL.

[0098] As used herein, "variable region" refers to variable sequences resulting from recombination events, such as V, J in immunoglobulin genes in B cells or T cell receptor (TCR) genes in T cells. And / or after recombination of the D segment. In immunoglobulin genes, the variable region is usually defined by the antibody chain from which it is derived, for example, VH refers to the variable region of the antibody heavy chain, and VL refers to the variable region of the antibody light chain. Selected VH and selected VL can be associated together to form an antigen-binding domain that confers antigen specificity and binding affinity.

[0099] As used herein, the term "complementarity determining region" or "CDR" refers to the sequences within the variable regions VH and VL of an antibody that confer antigen specificity and binding affinity. For example, generally speaking, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The exact amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known protocols, including those by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Those schemes described by the Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (the "Chothia" numbering scheme), or combinations thereof. According to the Kabat numbering scheme, in some embodiments, CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and The CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to the Chothia numbering scheme, in some embodiments, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in VL Residue numbers 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in VH (e.g., mammalian VH, e.g., human VH). ); and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in VL (e.g. mammalian VL, e.g. human VL). In various embodiments, the CDRs are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CDRs are human sequences. In various embodiments, the CDRs are naturally occurring sequences.

[0100] As used herein, the term "framework region" or "FR" refers to the generally conserved sequences within the antibody variable regions VH and VL that serve as a scaffold for interspersed CDRs, typically in the form FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 arrangement (from N-terminus to C-terminus). In various embodiments, the FRs are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In specific embodiments, the FRs are human sequences. In various embodiments, the FRs are naturally occurring sequences. In various embodiments, the FRs are synthetic sequences, including but not limited to rationally designed sequences.

[0101] As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring configuration, and which generally determines the class to which the antibody belongs.

[0102] As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring configuration. Kappa (Kappa) and lambda (lambda) light chains refer to the two major antibody light chain isotypes.

[0103] As used herein, the term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, such as, for example, antibodies expressed by phage or yeast expression systems. The term should also be construed to mean an antibody produced by the synthesis of a DNA molecule encoding the antibody that expresses the antibody protein or the amino acid sequence that specifies the antibody, where the DNA or amino acid sequence has been used in this technology. Recombinant DNA or amino acid sequence technology is available and well known.

[0104] As used herein, the term "antigen" or "Ag" refers to a molecule that provokes an immune response. This immune response may involve antibody production or activation of specific immune competent cells or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can be used as an antigen.

[0105] As used herein, the term "anti-tumor effect" or "anti-tumor activity" refers to biological effects that can be manifested by various means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, and reduction in the number of metastases. , increased life expectancy, reduced tumor cell proliferation, reduced tumor cell survival rate, or improvement of various physiological symptoms related to cancer symptoms. The "anti-tumor effect" can also be first reflected by the ability of the peptides, polynucleotides, cells and antibodies of the present invention to prevent tumor occurrence, such as for preventive therapy or treatment.

[0106] As used herein, the term "autologous" refers to any material derived from the same individual that is later reintroduced into that individual.

[0107] As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more pairs of individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some embodiments, the extent to which allogeneic material from individuals of the same species differs genetically (e.g., at specific genes, such as MHC alleles) may be sufficient for antigenic interaction to occur. In some embodiments, allogeneic material from individuals of the same species may be genetically similar (eg, at specific genes such as MHC alleles) enough that no antigenic interaction occurs.

[0108] Isolated nucleic acid molecules of the invention include any nucleic acid molecule encoding a polypeptide of the invention, or fragments thereof. Such nucleic acid molecules need not be 100% homologous or identical to the endogenous nucleic acid sequence, but will typically exhibit substantial identity. Nucleic acids that have "substantial identity" or "substantial homology" to an endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. As used herein, "hybridization" refers to pairing under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (eg, genes described herein) or portions thereof. For example, the stringent salt concentration may be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. . Low stringency hybridization can be obtained in the absence of organic solvents such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Strict temperature conditions will generally include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Varying other parameters, such as hybridization time, concentration of detergent (eg, sodium dodecyl sulfate (SDS)) and inclusion or exclusion of carrier DNA, is well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as desired.

[0109] "Substantially identical" or "substantially homologous" means that a polypeptide or nucleic acid molecule exhibits the same sequence as a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the amino acid sequences described herein). any of the described nucleic acid sequences) is at least about 50% homologous or identical. Preferably, such sequence is at least about 60%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identical at the amino acid level or nucleic acid level to the sequence used for comparison. source or consistent. Sequence identity is typically measured using sequence analysis software (such as the Genetics Computer Group, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs from the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). . This software matches identical or similar sequences by specifying the degree of homology of various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, aspartic acid, glutamine Acid; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, in which a probability score between e-3 and e-100 indicates closely related sequences.

[0110] As used herein, the term "coding" refers to a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence used to serve as a template for the synthesis of other polymers and macromolecules in biological processes. The inherent properties of a specific sequence of nucleotides in a sequence of polynucleotides, such as genes, cDNA or mRNA, and the resulting biological properties. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to the gene produce a protein in a cell or other biological system. The coding strand whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing and the non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding proteins or other products of the gene or cDNA. Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are in degenerate form of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also contain introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some form.

[0111] As used herein, the term "ligand" refers to a molecule that binds to a receptor. Specifically, a ligand binds to a receptor on another cell, thereby allowing cell-cell recognition and / or interaction.

[0112] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition that is effective in achieving a particular biological outcome as described herein. In some embodiments, an "effective amount" or a "therapeutically effective amount" is an amount sufficient to arrest, ameliorate, or inhibit the continued proliferation, growth, or metastasis of a related disease or disorder (eg, a myeloid disorder).

[0113] As used herein, the term "immune response cell" refers to a cell or progenitor cell or progeny thereof that functions in an immune response (e.g., an immune effector response). Examples of immune effector cells include, but are not limited to, alpha / beta T cells, gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0114] As used herein, the term "immune effector response" or "immune effector function" refers to a function or response of an immune response cell that, for example, enhances or promotes an immune attack on a target cell. For example, an immune effector function or response may refer to the property of a T cell or NK cell that promotes the killing of a target cell or inhibits its growth or proliferation. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0115] As used herein, the term "flexible polypeptide linker" or "linker" refers to amino acids such as glycine and / or serine residues used alone or in combination and used to combine A peptide linker that links the variable heavy chain and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and includes the amino acid sequence (Gly-Gly-Gly-Gly-Ser) n (SEQ ID NO: 139) or (Gly-Gly-Gly -Ser) n (SEQ ID NO: 140), where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 or n=10. In some embodiments, flexible polypeptide linkers include, but are not limited to, Gly 4Ser (SEQ ID NO: 37) or (Gly 4Ser) 3 (SEQ ID NO: 39). In other embodiments, the linker includes a plurality of (Gly 2Ser) (SEQ ID NO: 27), (GlySer) or (Gly 3Ser) (SEQ ID NO: 32) repeating units. In some embodiments, the flexible polypeptide linker includes a Whitlow linker (e.g., GSTSGSGKPGSGEGSTKG [SEQ ID NO: 42]). Also included within the scope of the present invention are linkers such as those described in WO2012 / 138475.

[0116] As used herein, the term "treatment" refers to the reduction or amelioration of the progression, severity, or severity of a proliferative disorder (e.g., cancer) by the administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). degree and / or duration, or improve one or more symptoms of the proliferative disease (preferably, one or more identifiable symptoms). In some embodiments, alleviating or ameliorating refers to improving at least one measurable physical parameter of a proliferative disorder that is not necessarily discernible to the patient, such as tumor growth. In other embodiments, the term "treating" or "treating" refers to inhibiting the progression of a proliferative disorder physically (by, for example, stabilizing discernible symptoms), physiologically (by, for example, stabilizing physical parameters), or both. In some embodiments, alleviation or improvement includes reducing or stabilizing tumor size or cancer cell count.

[0117] As used herein, the term "individual" is intended to include living organisms (e.g., mammals, humans) that can elicit an immune response. [Other Interpretation Statutes] []

[0118] Ranges recited herein should be understood to be a simplification of all values ​​within the range, including the recited end points. For example, the range 1 to 50 should be understood to include from 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 and 50 any value, combination of values ​​or sub-range.

[0119] Unless otherwise indicated, reference to a compound having one or more stereocenters means each of its individual stereoisomers and all combinations of stereoisomers. [Solid Tumor Antigens] []

[0120] Certain aspects of the invention relate to chimeric receptors and cells genetically modified to express one or more chimeric receptors that bind to relevant antigens, such as immune response cells, and the use of such receptors and cells in therapy and / or prevent solid malignant tumors (such as lung cancer, pancreatic cancer, gastrointestinal cancer, colon cancer, brain cancer, neuronal tissue cancer, endocrine tumors, bone cancer, bone marrow cancer, immune system cancer, muscle cancer, liver cancer, gallbladder cancer, Kidney cancer, bladder cancer, male reproductive organ cancer, female reproductive organ cancer, fat cancer, soft tissue cancer and skin cancer) and other lesions that require antigen-specific immune responses. Malignant cells have developed a series of mechanisms to protect themselves from immune recognition and elimination. The present invention provides immunogenicity within the tumor microenvironment for the treatment of such malignant cells.

[0121] Certain aspects of the present invention relate to chimeric receptors that specifically bind one or more antigens expressed on myeloid cells and are genetically modified to express immunity of such chimeric receptors useful in the treatment of solid tumor malignancies. Response cells. Solid cancers are reproductive diseases caused by genetic and epigenetic alterations that disrupt key processes such as cell proliferation and differentiation. Solid Tumor Malignancies can be chronic or acute.

[0122] In certain embodiments, the present invention relates to solid tumor antigens and solid tumor antigens suitable for use in chimeric receptors (e.g., chimeric TCRs or CARs) to increase efficacy and / or reduce off-tumor toxicity in the treatment of solid tumors. combination. In certain embodiments, the solid tumor antigen is a member of the CEA family. In certain embodiments, the solid tumor antigen is a member of the CEA family selected from the group consisting of: CEA, CEACAM1, CEACAM5, and CEACAM6. As used herein, "CEA" refers to the highly related protein (CD66 protein) family, including but not limited to CEACAM1 (CD66a), CEACAM5 (CD66e), and CEACAM6 (CD66c). In certain embodiments, an antibody or antigen-binding fragment that binds CEA binds more than one CD66 protein.

[0123] [surface] [1] Provide CEA family antigens suitable for use in the chimeric receptors described in the methods and compositions provided herein. [surface] [1] [Solid Tumor Antigens] [Antigen] [UniProt] [Login ID] [name] [short description] CEA - carcinoembryonic antigen-related cell adhesion molecule Highly relevant cell surface glycoproteins that function in cell adhesion CEACAM1 P13688 carcinoembryonic antigen-related cell adhesion molecule 1 Cell surface glycoprotein that functions as a co-inhibitory receptor in the immune response, insulin action and function, and as an activator during angiogenesis CEACAM5 P06731 carcinoembryonic antigen-related cell adhesion molecule 5 Cell surface glycoproteins with roles in cell adhesion, intracellular signaling, and tumor progression CEACAM6 P40199 carcinoembryonic antigen-related cell adhesion molecule 6 Cell surface glycoproteins that play a role in cell adhesion and tumor progression

[0124] In some embodiments, the solid tumor antigen is CEACAM1 antigen. CEACAM1 is also known in the art as BGP, BGP1, BGPI or CD66a. In some embodiments, the solid tumor antigen is CEACAM5 antigen. CEACAM5 was previously known in the art as CEA. Currently, CEACAM5 is also known as meconium antigen 100, carcinoembryonic antigen or CD66e. In some embodiments, the solid tumor antigen is CEACAM6 antigen. CEACAM6 is also known in the art as CEAL, NCA, normal cross-reactive antigen, non-specific cross-reactive antigen, or CD66c. [Chimeric receptor] []

[0125] Certain aspects of the invention relate to chimeric receptors and nucleic acids encoding such chimeric receptors that bind to relevant antigens. Antibodies and antigen-binding fragments

[0126] In some embodiments, the chimeric receptors comprise compounds capable of binding solid tumor antigens, such as CEA family antigens such as [surface] One or more of the antigen-binding domains listed in [1]. The antigen binding domain of the chimeric receptor may comprise [surface] Antibody sequences of representative anti-CEA antibodies or antigen-binding fragments thereof are provided in [2]. [Table 2] [Exemplary solid tumor antigen binding agents] [Solid tumor antigen] [Antibody pure line] [pure line sequence] CEACAM1 MRG1 VH: QVQLQQSGAELVRPGTSVKVSCKASGYAFTNNLIEWVKQRPGQGLEWIGVINPGSGDTNYNEKFKGKATLTADKSSNTAYMQLSSLTSDDSAVYFCARGDYYGGFAVDYWGQGTSVTVSS (SEQ ID NO: 1) VL: DIQMTQTTSSLSASLGDRVTISCRTSQDIGNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKSLPRTFGGGTKLEI (SEQ ID NO: 2) CEACAM5 Labetuzumab (aka, hMN14) VH: EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS (SEQ ID NO: 3) VL: DIQLTQSPSSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK (SEQ ID NO: 4) CEACAM5 Cibisatamab HC: QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSSTAYMELRRSSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS (SEQ ID NO: 5) LC: DIQMTQSPSSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYAEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6) CEACAM5 Tusamitamab HC: EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO: 7) LC: DIQMTQSPASLSSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8) CEACAM5 BW431 / 26 VH: QLQESGPGLVRPSQTLSLTCTVSGFTISSGYSWHWVRQPPGRGLEWIGYIQYSGITNYNPSLKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREDYDYHWYFDVWGQGSLVTVSS (SEQ ID NO: 9) VL: GVHSDIQMTQSPSSSLSASVGDRVTITCSTSSSSVSYMHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQWSSYPTFGQGTKVEIKR (SEQ ID NO: 10) CEACAM5 A5B7 VH: MDFQVQIFSFLLISASVIMSRGQTVLSQSPAILSASPGEKVTMTCRASSSVTYIHWYQQKPGSSPKSWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQHWSSKPPTFGGGTKLEIKR (SEQ ID NO: 11) VL: MDFQVQIFSFLLISASVIMSRGQTVLSQSPAILSASPGEKVTMTCRASSSVTYIHWYQQKPGSSPARFSGSGTSYSLTISRVEAEDAATYYCQHWSSKPPTFGGGTKLEIKR (SEQ ID NO: 12) CEACAM5 MFE23 VH: ETVIKYLLPTAAAGLLLLAAQPAMAQVKLQQSGAELVRSGTSVKLSCTASGFNIKDSYMHWLRQGPEQGLEWIGWIDPENGDTEYAPKFQGKATFTTDTSSNTAYLQLSSLTSEDTAVYYCNEGTPTGPYYFDYWGQGTTVTVSS (SEQ ID NO: 13) VL: ENVLTQSPAIMSASPGKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPLTFGAGTKLELKRAA (SEQ ID NO: 14) CEACAM5 hMFE23 VH: QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSLSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSS (SEQ ID NO: 78) VL: ENVLTQSPSSMSVSVGDRVTIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIK (SEQ ID NO: 79) Glycosylated CEACAM5 FM4 (also known as "MG7") VH: EVKLVESGGGLVQPGGSLRLSCSISGFTFTDYYMNWVRQSPGKALEWLGFIRNKVNGDTTEYSASVKGRFTISRDISQSILYLQMNTLRTEDSATYYCARDKGIAYYFDYWGQGTTLTVSS (SEQ ID NO: 15) VL: QIVLSQSPAILFASPGEKVTMTCRASSSVSYIHWYQQKPGSSPKPWIHGTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSNLSTFGGGTKLEIK (SEQ ID NO: 16) CEACAM6 Tinurilimab HC: QVTLRESGPALVKPTQTLTLTCTFSGFSLSTYGIGVGWIRQPPGKALEWLAHIWWNDNKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARISLPYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17) LC: DIQLTQSPSFLSASVGDRVTITCKASQNVGTAVAWYQQKPGKAPKLLIYSASNRYTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18)

[0127] Certain aspects of the invention relate to chimeric receptors (eg, CARs or chimeric TCRs) comprising extracellular antigen-binding domains that bind to one or more antigens of the invention. In some embodiments, the antigen-binding domain is derived from an antibody or antigen-binding fragment thereof. In some embodiments, the antigen binding domain comprises [surface] [2] The CDR sequence of the antibody or its antigen-binding fragment. CDR sequences and known systems for defining them, such as Kabat, are discussed in detail above.

[0128] Suitable antibodies of the invention include any antibody, whether natural or synthetic, full length or fragments thereof, monoclonal or polyclonal, that binds strongly and specifically to a solid tumor antigen, such as CEA, CEACAM1, CEACAM5 or CEACAM6. In some embodiments, the antibody can have at most about 10-6M, at most about 10-7M, at most about 10-8M, at most about 10-9M, at most about 10-10M, at most about 10-11M, or at most about 10 -12M of K D.

[0129] In some embodiments, antibodies and derivatives thereof that can be used include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, primatized (CDR grafted) antibodies, mosaic antibodies, Single chain antibodies, phage-produced antibodies (eg from phage display libraries) and functional binding fragments of antibodies. For example, antibody fragments capable of binding to solid tumor antigens or portions thereof include, but are not limited to, Fv, Fab, Fab' and F(ab')2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. By way of example and without limitation, papain or pepsin cleavage can produce Fab or F(ab')2 fragments, respectively. Other proteases with the necessary substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies in various truncated forms can also be produced using antibody genes that have introduced one or more stop codons upstream of the natural stop site. For example, a chimeric gene encoding the F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH domain and hinge region of the heavy chain.

[0130] Methods of generating antibodies targeting specific antigens are generally known in the art. Synthetic and engineered antibodies are described, for example, in US4816567, EP0125023Bl, US4816397, EP0120694Bl, WO 86 / 01533, EP0194276Bl, US5225539, EP0239400Bl, EP0451216Bl, EP0519596Al and US4946778.

[0131] In some embodiments, commercially available antibodies can be used to bind to solid tumor antigens. The CDRs of commercially available antibodies are readily obtained by those skilled in the art using conventional sequencing techniques. Furthermore, those skilled in the art are able to construct nucleic acids encoding scFv and chimeric receptors such as CAR and TCR based on the CDRs of such commercially available antibodies.

[0132] In some embodiments, the chimeric receptor comprises an antigen binding domain that specifically binds CEA.

[0133] In some embodiments, the chimeric receptor comprises an antigen binding domain that specifically binds CEACAM1. In some embodiments, the CEACAM1-specific antigen binding domain is derived from an anti-CEACAM1 antibody, such as an MRG1 antibody or an antigen-binding fragment thereof. In certain embodiments, the CEACAM1-specific antigen-binding domain comprises at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, SEQ ID NO: 1, A heavy chain variable domain (VH) that is at least 97%, at least 98%, at least 99% or 100%) identical to an amino acid sequence and contains at least 90% (e.g., at least 91%, at least 92%) the same amino acid sequence as SEQ ID NO:2 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences of light chain variable domains (VL). In certain embodiments, the second antigen binding site comprises VH and VL sequences belonging to SEQ ID NO: 1 and 2, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen binding domain may be a scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor can have a multispecific antigen binding domain. For example, the chimeric receptor can be specific for CEACAM1 and one or more additional antigens. In some embodiments, the chimeric receptor can be specific for CEACAM1 and CEACAM5. In some embodiments, the chimeric receptor can be specific for CEACAM1 and CEACAM6. In some embodiments, the chimeric receptor can be specific for CEACAM5 and CEACAM6.

[0134] In some embodiments, the chimeric receptor comprises an antigen binding domain that specifically binds CEACAM5. In some embodiments, the CEACAM5-specific antigen binding domain is derived from an anti-CEACAM5 antibody, such as labetuzumab (ie, hMN14) or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5-specific antigen-binding domain comprises at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, SEQ ID NO: 3, A heavy chain variable domain (VH) that is at least 97%, at least 98%, at least 99% or 100%) identical to an amino acid sequence and contains at least 90% (e.g., at least 91%, at least 92%) the same amino acid sequence as SEQ ID NO:4 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences of light chain variable domains (VL). In certain embodiments, the second antigen binding site comprises the VH and VL sequences belonging to SEQ ID NO: 3 and 4, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen binding domain may be a scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor can have a multispecific antigen binding domain. For example, the chimeric receptor can be specific for CEACAM5 and one or more additional antigens.

[0135] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as cerebrumumab or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a protein that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%) identical to SEQ ID NO: 5. A heavy chain (HC) that is at least 97%, at least 98%, at least 99% or 100%) identical to an amino acid sequence and contains at least 90% (e.g., at least 91%, at least 92%, at least 93) identical to SEQ ID NO: 6 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical light chain (LC) amino acid sequence. In certain embodiments, the second antigen binding site comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) belonging to the HC and LC sequences of SEQ ID NO: 5 and 6, respectively. In certain embodiments, the second antigen binding site comprises the HC and LC sequences belonging to SEQ ID NO: 5 and 6, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen-binding domain may be a scFv comprising a light chain variable domain and a heavy chain variable domain. In some embodiments, the chimeric receptor can have multispecific antigen binding domains. For example, the chimeric receptor can be specific for CEACAM5 and one or more additional antigens.

[0136] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as tercertuzumab or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a protein that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%) identical to SEQ ID NO: 7. A heavy chain (HC) that is at least 97%, at least 98%, at least 99% or 100%) identical to an amino acid sequence and contains at least 90% (e.g., at least 91%, at least 92%, at least 93) identical to SEQ ID NO: 8 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical light chain (LC) amino acid sequence. In certain embodiments, the second antigen binding site comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) belonging to the HC and LC sequences of SEQ ID NO: 7 and 8, respectively. In certain embodiments, the second antigen binding site comprises the HC and LC sequences belonging to SEQ ID NO: 7 and 8, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen-binding domain may be a scFv comprising a light chain variable domain and a heavy chain variable domain. In some embodiments, the chimeric receptor can have multispecific antigen binding domains. For example, the chimeric receptor can be specific for CEACAM5 and one or more additional antigens.

[0137] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as BW431 / 26 or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a protein that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%) identical to SEQ ID NO: 9. A heavy chain variable domain (VH) that is at least 97%, at least 98%, at least 99% or 100%) identical to an amino acid sequence and includes at least 90% (e.g., at least 91%, at least 92%) identical to SEQ ID NO: 10 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) of the light chain variable domain (VL) identical amino acid sequence. In certain embodiments, the second antigen binding site comprises the VH and VL sequences belonging to SEQ ID NO: 9 and 10, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen-binding domain may be a scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor can have multispecific antigen binding domains. For example, the chimeric receptor can be specific for CEACAM5 and one or more additional antigens.

[0138] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as A5B7 or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5-specific antigen-binding domain comprises at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, SEQ ID NO: 11, A heavy chain variable domain (VH) that is at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence and includes at least 90% (e.g., at least 91%, at least 92%) identical to SEQ ID NO: 12 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) of the light chain variable domain (VL) identical amino acid sequence. In certain embodiments, the second antigen binding site comprises the VH and VL sequences belonging to SEQ ID NO: 11 and 12, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen-binding domain may be a scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor can have multispecific antigen binding domains. For example, the chimeric receptor can be specific for CEACAM5 and one or more additional antigens.

[0139] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as MFE23 or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5-specific antigen-binding domain includes a protein that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%) identical to SEQ ID NO: 13. A heavy chain variable domain (VH) that is at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence and includes at least 90% (e.g., at least 91%, at least 92%) identical to SEQ ID NO: 14 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) of the light chain variable domain (VL) identical amino acid sequence. In certain embodiments, the second antigen binding site comprises the VH and VL sequences belonging to SEQ ID NO: 13 and 14, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen-binding domain may be a scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor can have multispecific antigen binding domains. For example, the chimeric receptor can be specific for CEACAM5 and one or more additional antigens.

[0140] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody, such as hMFE23 or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5-specific antigen-binding domain comprises at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, SEQ ID NO: 78, A heavy chain variable domain (VH) that is at least 97%, at least 98%, at least 99% or 100%) identical in amino acid sequence and comprises at least 90% (e.g., at least 91%, at least 92%) of SEQ ID NO: 79 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) of the light chain variable domain (VL) identical amino acid sequence. In certain embodiments, the second antigen binding site comprises VH and VL sequences belonging to SEQ ID NO: 78 and 79, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen-binding domain may be a scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor can have multispecific antigen binding domains. For example, the chimeric receptor can be specific for CEACAM5 and one or more additional antigens.

[0141] In some embodiments, the CEACAM5-specific antigen-binding domain is derived from an anti-CEACAM5 antibody capable of specifically binding glycosylated CEACAM5. In some embodiments, the glycosylated CEACAM5-specific antigen binding domain is derived from an anti-glycosylated CEACAM5 antibody, such as FM4 (also referred to herein as "MG7") or an antigen-binding fragment thereof. In certain embodiments, the CEACAM5-specific antigen binding domain comprises at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A heavy chain variable domain (VH) that is at least 97%, at least 98%, at least 99% or 100%) identical in amino acid sequence and comprises at least 90% (e.g., at least 91%, at least 92%) of SEQ ID NO: 16 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) of the light chain variable domain (VL) identical amino acid sequence. In certain embodiments, the second antigen binding site comprises VH and VL sequences belonging to SEQ ID NO: 15 and 16, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen-binding domain may be a scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor can have multispecific antigen binding domains. For example, the chimeric receptor can be specific for CEACAM5 and one or more additional antigens.

[0142] In some embodiments, the chimeric receptor comprises an antigen-binding domain that specifically binds CEACAM6. In some embodiments, the CEACAM6-specific antigen-binding domain is derived from an anti-CEACAM6 antibody, such as tenucrelimab or an antigen-binding fragment thereof. In certain embodiments, the CEACAM6-specific antigen binding domain comprises at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%) of SEQ ID NO: 17 , at least 97%, at least 98%, at least 99% or 100%) of the heavy chain (HC) of an amino acid sequence identical to SEQ ID NO: 18 and comprising at least 90% (eg, at least 91%, at least 92%, at least A light chain (LC) with an amino acid sequence that is 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the second antigen binding site comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) belonging to the HC and LC sequences of SEQ ID NO: 17 and 18, respectively. In certain embodiments, the second antigen binding site comprises HC and LC sequences belonging to SEQ ID NO: 17 and 18, respectively, determined according to Kabat, Chothia, MacCallum, or any other CDR determination method known in the art. Heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3. The antigen-binding domain may be a scFv comprising a light chain variable domain and a heavy chain variable domain. In some embodiments, the chimeric receptor can have multispecific antigen binding domains. For example, the chimeric receptor can be specific for CEACAM6 and one or more other antigens. [T] [cell receptor] [(TCR)]

[0143] Certain aspects of the invention relate to chimeric receptors that specifically bind to antigens expressed on solid tumor cells. In some embodiments, the chimeric receptor is a chimeric T cell receptor (TCR). The TCRs of the present invention are disulfide-bridged heterodimeric proteins containing two variable chains that appear as part of a complex with an invariant CD3 chain molecule. TCRs are found on the surface of T cells and are responsible for recognizing antigens in the form of peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, a TCR of the invention comprises an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0144] Each chain of a TCR is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane, followed by the transmembrane region and short cytoplasmic tail. The variable domains bind to the peptide / MHC complex. Each variable region has three complementarity determining regions (CDRs).

[0145] In certain embodiments, the TCR can form a receptor complex with the three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or CD247ζ / η. T cells expressing TCR complexes are activated when the TCR complex engages its antigen and MHC (peptide / MHC).

[0146] In some embodiments, the TCR of the present invention is a recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, the TCR differs from a naturally occurring TCR by at least one amino acid residue. In some embodiments, the TCR differs from a naturally occurring TCR by at least 2 amino acid residues, at least 3 amino acid residues, at least 4 amino acid residues, at least 5 amino acid residues, At least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues or more amino acid residues. In certain embodiments, the TCR is modified with at least one amino acid residue relative to a naturally occurring TCR. In some embodiments, the TCR is modified by at least 2 amino acid residues, at least 3 amino acid residues, at least 4 amino acid residues, at least 5 amino acid residues relative to a naturally occurring TCR. , at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amines amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, At least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues Acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, or more amino acid residue modifications. chimeric TCR

[0147] In some embodiments, the TCR of the present invention comprises one or more antigen-binding domains, and the one or more antigen-binding domains can be grafted to a TCR chain, such as one or more constant domains of TCR α chain or TCR β chain, To generate a chimeric TCR that specifically binds to a target antigen of the invention (eg, a solid tumor antigen). Without wishing to be bound by theory, it is believed that following antigen binding, the chimeric TCR can signal via the TCR complex. For example, antibodies or antibody fragments (e.g. scFv) can be grafted to the constant domains of a TCR chain (such as TCR alpha chain and / or TCR beta chain), e.g. extracellular constant domain, transmembrane domain and at least the cytoplasmic domain part. As another example, the CDRs of an antibody or antibody fragment can be grafted into the TCR alpha chain and / or beta chain to generate a chimeric TCR that specifically binds to an antigen of the invention (eg, a solid tumor antigen). Such chimeric TCR can be produced by methods known in the art (eg Willemsen RA et al., Gene Therapy 2000; 7:1369-1377; Zhang T et al., Cancer Gene Ther 2004 11: 487-496; and Aggen et al., Gene Ther. 2012 Apr; 19(4): 365-74). [Chimeric Antigen Receptor] [(CAR)]

[0148] Certain aspects of the invention pertain to specific binding to solid tumors (e.g., lung, pancreas, gastrointestinal tract, colon, brain, neuronal tissue, endocrine, bone, bone marrow, immune system, muscle, liver, gallbladder, kidney , bladder, male reproductive organs, female reproductive organs, adipose, soft tissue or skin tumors) chimeric receptors for antigens expressed. In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In some embodiments, a CAR (or an immune response cell genetically engineered to contain one or more CARs, see below) specifically binds a CEA family member tumor antigen, such as [surface] Any of the CEA antigens of [1]. In some embodiments, a CAR (or an immune response cell genetically engineered to contain one or more CARs) may comprise [surface] Antibody sequences or antigen-binding fragments of representative anti-CEA antibodies provided in [2]. In some embodiments, a CAR (or an immune response cell genetically engineered to contain one or more CARs) may comprise a scFv derived from an antibody capable of binding to a solid tumor antigen, such as [surface] Representative anti-CEA scFv provided in [3].

[0149] In some embodiments, CARs are engineered receptors that graft or confer relevant specificities on immune effector cells. In certain embodiments, CARs can be used to graft the specificity of antibodies onto immune response cells such as T cells. In some embodiments, a CAR of the invention comprises an extracellular antigen binding domain (eg, scFv) fused to a transmembrane domain, fused to one or more intracellular signaling domains.

[0150] In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of an immune response cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce immune response cell stimulation. In some embodiments, the immune response cells are activated to kill target cells. In some embodiments, activation of the immune response cells results in the expression and / or secretion of cytokines or chemokines by the immune response cells. In some embodiments, stimulation of the immune response cells results in expression and / or secretion of cytokines or chemokines by the immune response cells. In some embodiments, stimulation of the immune response cells induces differentiation of the immune response cells. In some embodiments, stimulation of the immune-responsive cells induces proliferation of the immune-responsive cells.

[0151] The CAR of the present invention can be a first-generation, second-generation or third-generation CAR. "First-generation" CARs contain a single intracellular signaling domain, typically derived from the T-cell receptor chain. "First generation" CARs typically have an intracellular signaling domain derived from the CD3-ζ (CD3ζ) chain, which is the primary transmitter of signals from endogenous TCRs. "First-generation" CARs provide de novo antigen recognition and activate CD4+ and CD8+ T cells through the CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second-generation" CARs add a second intracellular signaling domain from one of several costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide T cells with additional signal. "Second generation" CARs provide co-stimulation (eg, CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have indicated that "second generation" CARs can improve the antitumor activity of immune response cells such as T cells. "Third generation" CARs have multiple intracellular co-stimulatory signaling domains (such as CD28 and 4-1BB) and an intracellular activation signaling domain (CD3ζ).

[0152] In some embodiments, the extracellular antigen-binding domain of the CAR of the invention binds to one or more antigens expressed on cells, such as solid tumor cells, with a dissociation constant (KD) of about 2×10 −7 M or less , about 1×10 -7M or less, about 9×10 -8M or less, about 1×10 -8M or less, about 9×10 -9M or less, about 5×10 -9M or less , about 4×10 -9M or less, about 3×10 -9M or less, about 2×10 -9M or less, or about 1×10 -9M or less. In some embodiments, the KD is in the range of about 2×10 −7 M to about 1×10 −9 M.

[0153] The binding of the extracellular antigen-binding domain of the CAR of the present invention can be performed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, biological analysis (such as growth inhibition) or Western Blot analysis. assay) to measure. Each of these assays typically detects the presence of a particularly relevant protein-antibody complex by employing a labeling reagent (such as an antibody or scFv) specific for the complex of interest. For example, scFv can be radiolabeled and used in RIA analysis. Radioisotopes can be detected by means such as the use of gamma counters or scintillation counters or by automated radiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent label. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent proteins (such as EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent proteins (such as ECFP, Cerulean, and CyPet), and yellow fluorescent proteins Proteins (eg YFP, Citrine, Venus and YPet).

[0154] In some embodiments, the CARs of the invention comprise binding to solid tumors (e.g., lung, pancreas, gastrointestinal tract, colon, brain, neuronal tissue, endocrine, bone, bone marrow, immune system, muscle, liver, gallbladder, kidney The extracellular antigen-binding domain, the transmembrane domain and one or more intracellular signaling domains of one or more antigens expressed on cells such as bladder, male reproductive organ, female reproductive organ, fat, soft tissue or skin tumor). In some embodiments, the extracellular antigen binding domain comprises a scFv. In some embodiments, the extracellular antigen binding domain includes Fab fragments that may be cross-linked. In certain embodiments, the extracellular binding domain is an F(ab)2 fragment. extracellular antigen binding domain

[0155] In some embodiments, the extracellular antigen-binding domain of the CAR of the invention specifically binds to tissues such as lung, pancreas, gastrointestinal tract, colon, brain, neuronal tissue, endocrine, bone, bone marrow, immune system, muscle, liver One or more antigens expressed on solid tumor cells of gallbladder, kidney, bladder, male reproductive organs, female reproductive organs, fat, soft tissue or skin tumor cells. In certain embodiments, the extracellular antigen binding domain binds to one or more antigens expressed on solid tumor cells (solid tumor antigens). In some embodiments, the one or more solid tumor antigens are human polypeptides.

[0156] The antigen-binding domain of the present invention may include any domain that binds to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, conjugated antibodies, human antibodies, humanized antibodies and functional fragments thereof, Including but not limited to single domain antibodies (sdAb), such as the heavy chain variable domain (VH), light chain variable domain (VL) and variable domain (VHH) of camel-derived nanobodies, and this technology Alternative scaffolds known to function as antigen-binding domains, such as recombinant fibronectin domains, T cell receptors (TCRs), recombinant TCRs with increased affinity or fragments thereof, such as single chain TCRs, and their analogs. In some cases, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be beneficial for the antigen-binding domain of a CAR to comprise human or humanized residues of the antigen-binding domain of an antibody or antibody fragment.

[0157] In some embodiments, the extracellular antigen binding domain comprises an antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a chimeric antibody. In some embodiments, the extracellular antigen binding domain comprises an antigen binding fragment of an antibody.

[0158] In some embodiments, the extracellular antigen binding domain comprises an F(ab) fragment. In certain embodiments, the extracellular antigen binding domain comprises an F(ab') fragment.

[0159] In some embodiments, the extracellular antigen binding domain comprises a scFv.

[0160] Various scFvs derived from antibodies capable of binding to solid tumor antigens are provided at [surface] [3] in. In some embodiments, the extracellular antigen binding domain comprises such as [surface] scFv provided in [3]. [table 3] [scFv] [Amino acid sequence] [SEQ ID NO] hMN14 scFv (VL-VH) DIQLTQSPSSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKGGSGSGGSGSGGSGSEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLK DRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS

[19] hMFE23 scFv (VH-VL) QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVTIACSASSSVPYMHWLQQKPGKSPK LLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIK

[20] MG7 ("FM4") scFv (VL-VH) EVKLVESGGGLVQPGGSLRLSCSISGFTFTDYYMNWVRQSPGKALEWLGFIRNKVNGDTTEYSASVKGRFTISRDISQSILYLQMNTLRTEDSATYYCARDKGIAYYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLSQSPAILFASPGEKVTMTCRASSSVSYIHWYQQKPG SSPKPWIHGTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSNLSTFGGGTKLEIK [twenty one] MRG1 scFv (VH-VL) QVQLQQSGAELVRPGTSVKVSCKASGYAFTNNLIEWVKQRPGQGLEWIGVINPGSGDTNYNEKFKGKATLTADKSSNTAYMQLSSLTSDDSAVYFCARGDYYGGFAVDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRTSQDIGNYLNWYQQKPDG TVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKSLPRTFGGGTKLEI [twenty two] Terceltuzumab scFv (VH-VL) DIQMTQSPASLSSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRGGGGSGGGGSGGGGSEVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPST VKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSSA [twenty three] Terceltuzumab scFv (VL-VH) EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSSAGGGGSGGGGSGGGGSDIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVY NTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKR [twenty four] A5B7 scFv (VH-VL) EVQLLESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANGYTTEYSASVKGRFTISRDKSKSTLYLQMNTLQAEDSAIYYCTRDRGLRFYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVLYLQMNTLQAEDSAIYYCTR DRGLRFYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVLTQSPSSLSVSVGDRVTITCRASSSVTYIHWYQQKPGLAPKSLIYATSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQHWSSKPPTFGQGTKVEVKRTV

[25] A5B7 scFv (VL-VH) Question TTEYSASVKGRFTISRDKSKSTLYLQMNTLQAEDSAIYYCTRDRGLRFYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVLYLQMNTLQAEDSAIYYCTRDRGLRFYFDYWGQGTLVTVSS

[26]

[0161] In some embodiments, the extracellular antigen binding domain comprises two single chain variable fragments (scFv). In some embodiments, the two scFvs each bind to a different epitope on the same antigen. In some embodiments, the extracellular antigen binding domain includes a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to different epitopes on the same antigen. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a chimeric scFv. In certain embodiments, the scFv includes a heavy chain variable domain (VH) and a light chain variable domain (VL). In certain embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises such as [surface] The amino acid sequence shown in [4]. [surface] [4] [Applicable peptide linkers] [Connector] [Amino acid sequence] [SEQ ID NO:] (G 2S) 1scFv linker GGS 27 (G 2S) 2scFv linker GGSGGS 28 (G 2S) 3scFv linker GGSGGSGGS 29 (G 2S) 4scFv linker GGSGGSGGSGGS 30 (G 2S) 5scFv linker GGSGGSGGSGGSGGS 31 (G 3S) 1scFv linker GGGS 32 (G 3S) 2scFv linker GGGSGGGS 33 (G 3S) 3scFv linker GGGSGGGSGGGS 34 (G 3S) 4scFv linker GGGSGGGSGGGSGGGS 35 (G 3S) 5scFv linker GGGSGGGSGGGSGGGSGGGS 36 (G 4S) 1 linker GGGGS 37 (G 4S) 2 linker GGGGSGGGGS 38 (G 4S) 3 linker GGGGSGGGGSGGGGS 39 (G 4S) 4 linker GGGGSGGGGSGGGGSGGGGS 40 (G 4S) 5 linker GGGGSGGGGSGGGGSGGGGSGGGGS 41 Whitlow connector GSTSGSGKPGSGEGSTKG 42 scFv linker 2 EAAAKEAAAKEAAAKEAAAK 43 (GGSGS) 3scFv linker GGSGSGGSGSGGSGS 77

[0162] In certain embodiments, the peptide linkage system is encoded by a nucleic acid comprising the sequence GGCGGAGGCGGATCAGGTGCGGAGGAAGTGGCGGCGGAGGATCT (SEQ ID NO: 44).

[0163] In certain embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0164] In some embodiments, the one or more scFvs each comprise the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When two or more scFv are linked together, each scFv can be linked to the next scFv using a peptide linker. In some embodiments, the one or more scFvs are each separated by a peptide linker. In some embodiments, the peptide linker separating each scFv includes, e.g. [surface] The amino acid sequence shown in [4].

[0165] In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 43.

[0166] In some embodiments, the cell includes a first chimeric receptor and a second chimeric receptor. The antigen binding domain of the first chimeric receptor and the antigen binding domain of the second chimeric receptor may be suitable antigen binding domains described herein or known in the art. For example, the first or second antigen-binding domain may be one or more antibodies, antigen-binding fragments of antibodies, F(ab) fragments, F(ab') fragments, single-chain variable fragments (scFv), or single-chain variable fragments. Domain antibody (sdAb). In some embodiments, the antigen-binding domain of the first chimeric receptor and / or the second chimeric receptor includes two single chain variable fragments (scFv). In some embodiments, the two scFvs each bind to a different epitope on the same antigen.

[0167] In some embodiments, the extracellular antigen binding domain comprises a single domain antibody (sdAb). In certain embodiments, the sdAb is a humanized sdAb. In certain embodiments, the sdAb is a chimeric sdAb.

[0168] In some embodiments, the CAR of the present invention may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains. domain, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, or ten or more antigens binding domain. In some embodiments, the two or more antigen binding domains each bind the same antigen. In some embodiments, the two or more antigen binding domains each bind to a different epitope of the same antigen. In some embodiments, the two or more antigen binding domains each bind a different antigen. In some embodiments, the two or more antigen binding domains provide a CAR with logic gating, such as OR logic gating.

[0169] In some embodiments, the CAR contains two antigen binding domains. In some embodiments, the two antigen binding domains are connected to each other via a flexible linker. In some embodiments, each of the two antigen-binding domains can be independently selected from the group consisting of an antibody, an antigen-binding fragment of an antibody, a scFv, an sdAb, a recombinant fibronectin domain, a T cell receptor (TCR), a recombinant protein with increased affinity TCR and single-chain TCR. In some embodiments, the CAR comprising two antigen-binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0170] In certain embodiments, the bispecific CAR or tanCAR contains an antigen binding domain comprising a bispecific antibody or antibody fragment (eg scFv). In some embodiments, the VH can be upstream or downstream of the VL within each antibody or antibody fragment (eg, scFv) of the bispecific antibody molecule. In some embodiments, the upstream antibody or antibody fragment (e.g. scFv) is arranged with its VH (VH 1 ) upstream of its VL (VL 1 ), and the downstream antibody or antibody fragment (e.g. scFv) is arranged with its VL (VL 2) upstream of its VH (VH 2), such that the overall bispecific antibody molecule has the arrangement VH 1 -VL 1 -VL 2 -VH 2. In other embodiments, the upstream antibody or antibody fragment (e.g. scFv) is arranged with its VL (VL 1 ) upstream of its VH (VH 1 ), and the downstream antibody or antibody fragment (e.g. scFv) is arranged with its VH (VH 2) upstream of its VL (VL 2), such that the overall bispecific antibody molecule has the arrangement VL 1-VH 1-VH 2-VL 2. In some embodiments, a linker is placed between two antibodies or antibody fragments (eg, scFv), eg, between VL1 and VL2 if the construct is arranged as VH1-VL1-VL2-VH2 , or between VH 1 and VH 2 if the structure arrangement is VL 1-VH 1-VH 2-VL 2. The linker can be a linker as described herein, such as a (Gly 4-Ser) n linker (SEQ ID NO: 138), where n is 1, 2, 3, 4, 5, or 6. Generally speaking, the length of the linker between two scFvs should be sufficient to avoid mismatching between the domains of the two scFvs. In some embodiments, the linker is disposed between VL and VH of the first scFv. In some embodiments, the linker is disposed between VL and VH of the second scFv. In constructs with multiple linkers, any two or more of the linkers may be the same or different. Thus, in some embodiments, a bispecific CAR or tanCAR comprises VL, VH, and may further comprise one or more linkers in an arrangement as described herein.

[0171] In some embodiments, the bivalent receptor comprises a CEA CAR and a CEACAM1 CAR. In some embodiments, the bivalent receptor comprises a CEA CAR and a CEACAM5 CAR. In some embodiments, the bivalent receptor comprises a CEA CAR and a CEACAM6 CAR. In some embodiments, the bivalent receptor comprises CEACAM1 CAR and CEACAM5 CAR. In some embodiments, the bivalent receptor comprises CEACAM1 CAR and CEACAM6 CAR. In some embodiments, the bivalent receptor comprises a CEACAM5 CAR and a CEACAM6 CAR.

[0172] In some embodiments, the bivalent receptor comprises a CEA CAR and a VSIG2 CAR. In some embodiments, the bivalent receptor comprises a CEA CAR and a CPM CAR. In some embodiments, the bivalent receptor includes CEA CAR and ITM2C CAR. In some embodiments, the bivalent receptor includes CEA CAR and SLC26A2 CAR. In some embodiments, the bivalent receptor includes CEA CAR and SLC4A4 CAR. In some embodiments, the bivalent receptor includes CEA CAR and GPA33 CAR. In some embodiments, the bivalent receptor includes CEA CAR and PLA2G2A CAR. In some embodiments, the bivalent receptor includes CEA CAR and ABCA8 CAR. In some embodiments, the bivalent receptor includes CEA CAR and ATP1A2 CAR. In some embodiments, the bivalent receptor includes CEA CAR and CHP2 CAR. In some embodiments, the bivalent receptor includes CEA CAR and SLC26A3 CAR.

[0173] In some embodiments, the bivalent receptor comprises CEACAM1 CAR and VSIG2 CAR. In some embodiments, the bivalent receptor comprises CEACAM1 CAR and CPM CAR. In some embodiments, the bivalent receptor comprises a CEACAM1 CAR and an ITM2C CAR. In some embodiments, the bivalent receptor comprises CEACAM1 CAR and SLC26A2 CAR. In some embodiments, the bivalent receptor comprises CEACAM1 CAR and SLC4A4 CAR. In some embodiments, the bivalent receptor comprises CEACAM1 CAR and GPA33 CAR. In some embodiments, the bivalent receptor comprises a CEACAM1 CAR and a PLA2G2A CAR. In some embodiments, the bivalent receptor comprises CEACAM1 CAR and ABCA8 CAR. In some embodiments, the bivalent receptor comprises CEACAM1 CAR and ATP1A2 CAR. In some embodiments, the bivalent receptor includes CEACAM1 CAR and CHP2 CAR. In some embodiments, the bivalent receptor includes CEACAM1 CAR and SLC26A3 CAR.

[0174] In some embodiments, the bivalent receptor includes CEACAM5 CAR and VSIG2 CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and CPM CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and ITM2C CAR. In some embodiments, the bivalent chimeric antigen receptor includes CEACAM5 CAR and SLC26A2 CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and SLC4A4 CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and GPA33 CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and PLA2G2A CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and ABCA8 CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and ATP1A2 CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and CHP2 CAR. In some embodiments, the bivalent receptor includes CEACAM5 CAR and SLC26A3 CAR.

[0175] In some embodiments, the bivalent receptor includes CEACAM6 CAR and VSIG2 CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and CPM CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and ITM2C CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and SLC26A2 CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and SLC4A4 CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and GPA33 CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and PLA2G2A CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and ABCA8 CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and ATP1A2 CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and CHP2 CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and CHP2 CAR. In some embodiments, the bivalent receptor includes CEACAM6 CAR and SLC26A3 CAR.

[0176] In some embodiments, the bivalent chimeric receptor comprises a CAR having an antigen-binding domain targeting any of the antigens provided in Table 1. In some embodiments, the bivalent chimeric receptors comprise compounds derived from, e.g. [surface] The CAR of the antigen-binding domain of the antibody provided in [2]. In some embodiments, the bivalent chimeric receptor comprises [surface] CAR of the antigen binding domain of the scFv provided in [2]. In some embodiments, the bivalent chimeric receptor comprises a targeting [surface] A CAR of two or more antigen binding domains of any antigen pairing provided in [5]. In some embodiments, the bivalent chimeric antigen receptor comprises a CAR with any combination of two or more antigen binding domains as described herein.

[0177] In some embodiments, the chimeric receptor comprises a bicistronic chimeric antigen receptor system, eg, a chimeric antigen receptor system comprising an activating CAR and an inhibitory CAR. In some embodiments, the bicistronic chimeric antigen receptor system comprises a targeting [surface] The CAR of the antigen binding domain of any antigen provided in [1] and, for example, has a targeting [surface] An inhibitory CAR of the antigen binding domain of any of the antigens provided in [8]. In some embodiments, the bicistronic chimeric antigen receptor system comprises [surface] The CAR of the antigen-binding domain of the antibody provided in [2] and, for example, has a targeting [surface] An inhibitory CAR of the antigen binding domain of any of the antigens provided in [7]. In some embodiments, the bicistronic chimeric antigen receptor system comprises a CAR with two or more antigen binding domains targeting any of the antigen pairings provided in Table 5, wherein at least one antigen binding domain is An activating CAR, and at least one antigen-binding domain is an inhibiting CAR. In some embodiments, the bicistronic chimeric antigen receptor system comprises a CAR with two or more antigen binding domains as described herein, wherein at least one antigen binding domain is an activating CAR, and at least one The antigen-binding domain is an inhibitory CAR. transmembrane domain

[0178] In some embodiments, the transmembrane domain of the CAR of the invention comprises a hydrophobic alpha-helix spanning at least a portion of the cell membrane. Different transmembrane domains have been shown to result in different receptor stabilities. Following antigen recognition, receptor clustering occurs and signals are transmitted to the cell. In some embodiments, the transmembrane domain of the CAR of the present invention may comprise CD8 polypeptide, CD28 polypeptide, CD25 polypeptide, CD7 polypeptide, CD3-ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA- 4 polypeptides, LAX polypeptides, LAT polypeptides, PD-1 polypeptides, LAG-3 polypeptides, TIM3 polypeptides, KIR3DS1 polypeptides, KIR3DL1 polypeptides, NKG2D polypeptides, NKG2A polypeptides, TIGIT polypeptides, 2B4 polypeptides, BTLA polypeptides, LIR-1 (LILRB1) polypeptides The transmembrane domain may be a synthetic peptide, or any combination thereof.

[0179] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide can be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide can be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-zeta polypeptide. Any suitable CD3-ζ polypeptide can be used. Exemplary CD3-ζ polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide can be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide can be used. Exemplary 4-1BB polypeptides include, but are not limited to, NCBI reference numbers NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an OX40 polypeptide. Any suitable OX40 polypeptide can be used. Exemplary OX40 polypeptides include, but are not limited to, NCBI reference numbers NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide can be used. Exemplary ICOS polypeptides include, but are not limited to, NCBI reference numbers NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide can be used. Exemplary CTLA-4 polypeptides include, but are not limited to, NCBI reference numbers NP_005205.2 and NP_033973.2. In some embodiments, the transmembrane domain is derived from a PD-1 polypeptide. Any suitable PD-1 polypeptide can be used. Exemplary PD-1 polypeptides include, but are not limited to, NCBI reference numbers NP_005009 and NP_032824. In some embodiments, the transmembrane domain is derived from a LAG-3 polypeptide. Any suitable LAG-3 polypeptide can be used. Exemplary LAG-3 polypeptides include, but are not limited to, NCBI reference numbers NP_002277.4 and NP_032505.1. In some embodiments, the transmembrane domain is derived from a 2B4 polypeptide. Any suitable 2B4 polypeptide can be used. Exemplary 2B4 polypeptides include, but are not limited to, NCBI reference numbers NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide. Any suitable BTLA polypeptide can be used. Exemplary BTLA polypeptides include, but are not limited to, NCBI reference numbers NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide can be used. Exemplary LIR-1 (LILRB1) polypeptides include, but are not limited to, NCBI reference numbers NP_001075106.2 and NP_001075107.2.

[0180] In some embodiments, the transmembrane domain contains a polypeptide containing the same compound as NCBI reference numbers NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2 , NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_05 7466.1, NP_061199.2, NP_861445.4 or NP_001032808 .2 sequence or fragment thereof at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or a polypeptide with 100% homologous amino acid sequence. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide may comprise one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide may have a reference number as NCBI Reference No. 070977. 1. NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_0611 99.2, NP_861445.4 or NP_001032808.2 At least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180 , an amino acid sequence of a contiguous portion of at least 190, at least 200, at least 210, at least 220, at least 230 or at least 240 amino acids in length.

[0181] Other examples of suitable polypeptides from which the transmembrane domain may be derived include, but are not limited to, the T cell receptors CD27, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CD11a, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1 , VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18 , LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D and the transmembrane region of the α, β or ζ chain of NG2C. In some embodiments, the transmembrane domain may comprise [surface] Any of the amino acid sequences listed in [5], or with [surface] At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% of any of the amino acid sequences listed in [5] % or at least 99% identical amino acid sequence. [surface] [5] [Amino acid sequence] [SEQ ID NO:] [describe] IYIWAPLAGTCGVLLLSLVIT 45 CD8 transmembrane domain FWVLVVVGGVLACYSLLVTVAFIIFWV 46 CD28 transmembrane domain VAAILGLGLVLGLLGPLAILL 47 OX40 transmembrane domain FLVIIVILSALFLGTLACFCV 48 2B4 transmembrane domain spacer

[0182] In some embodiments, the CAR of the present invention may also include a spacer connecting the extracellular antigen-binding domain and the transmembrane domain. The spacer may be flexible enough to allow the antigen binding domain to be oriented in different directions to facilitate antigen recognition. In some embodiments, the spacer can be a hinge from a human protein. For example, a spacer (also referred to herein as a "hinge") may be a human Ig (immunoglobulin) hinge, including, but not limited to, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer can comprise an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, a LNGFR hinge, or a PDGFR-beta extracellular linker. In some embodiments, the spacer is localized between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region may include [surface] Any of the amino acid sequences listed in [6], or with [surface] At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% of any of the amino acid sequences listed in [6] % or at least 99% identical amino acid sequence. In some embodiments, a nucleic acid encoding any of the spacers of the invention may comprise [surface] Any of the nucleic acid sequences listed in [7], or [surface] Any one of the nucleic acid sequences listed in [7] is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or A nucleic acid sequence that is at least 99% identical. [surface] [6] [Amino acid sequence] [SEQ ID NO:] [describe] AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP 49 CD28 hinge TTTPAPRPPTPAPTIALQPLSLRPEACRPAAGGAVHTRGLDFACD 50 CD8 hinge ESKYGPPCPSCP 51 IgG4 minimal hinge ESKYGPPAPSAP 52 IgG4 minimal hinge, no disulfide bridge ESKYGPPCPPCP 53 IgG4 S228P minimal hinge, enhanced disulfide bridge formation EPKSCDKTHTCP 54 IgG1 minimal hinge AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN 55 Extended CD8a hinge ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEADAEC 56 LNGFR hinge ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC 57 Truncated LNGFR hinge (TNFR-Cys1) AVGQDTQEVIVVPHSLPFKV 58 PDGFR-β extracellular linker [surface] [7] [nucleic acid sequence] [SEQ ID NO:] [describe] GCAGCAGCTATCGAGGTGATGTATCCTCCGCCCTACCTGGATAATGAAAAGAGTAATGGGACTATCATTCATGTAAAAGGGAAGCATCTTTGTCCTTCTCCCTTTTCCCCGGTCCGTCTAAACCT 59 CD28 hinge ACCACAAACACCAGCTCCTAGACCTCCAACTCCTGCTCCTACAATCGCCCTGCAGCCACTGTCTCTGAGGCCTGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACCAGAGGACTGGATTTCGCCTGCGAC 60 CD8 hinge GAAAGCAAGTACGGTCCACCTTGCCCTAGCTGTCCG 61 IgG4 minimal hinge GAATCCAAGTACGGCCCCCCAGCGCCTAGTGCCCCA 62 IgG4 minimal hinge, no disulfide bridge GAATCTAAATATGGCCCGCCATGCCCGCCTTGCCCA 63 IgG4 S228P minimal hinge, enhanced disulfide bridge formation GAACCGAAGTCTTGTGATAAAACTCATACGTGCCCG 64 IgG1 minimal hinge GCTGCTGCTTTCGTACCCGTGTTCCTCCCTGCTAAGCCTACGACTACCCTCGCACCGAGACCACCCACGCCAGCACCCACGATTGCTAGCCAGCCCCTTAGTTTGCGACCAGAAGCTTGTCGGCCTGCTGCTGGTGGCGCGGTACATACCCGCGGCCTTGATTTTGCTTGCGATATATATCTGGGCGCCTCTGGCCGGAACATGCGGGGTCCTCCTCCT TTCTCTGGTTATTACTCTCTACTGTAATCACAGGAAT 65 Extended CD8a hinge GCCTGCCCGACCGGGCTCTACACTCATAGCGGGGAATGTTGTAAGGCATGTAACTTGGGTGAGGGCGTCGCACAGCCCTGCGGAGCTAACCAAACAGTGTGCGAACCCTGCCTCGATAGTGTGTGACGTTCTCTGATGTTGTATCAGCTACAGCCTTGCAAACCATGTACTGAGTGCGTTGGACTTCAGTCAATGAGCGCTCCATGTGTGGAG GCAGATGATGCGGTCTGTCGATGTGCTTACGGATACTACCAAGACGAGACAACAGGGCGGTGCGAGGCCTGTAGAGTTTGTGAGGCGGGCTCCGGGCTGGTGTTTTCATGTCAAGACAAGCAAAATACGGTCTGTGAAGAGTGCCCTGATGGCACCTACTCAGACGAAGCAGATGCAGAATGC 66 LNGFR hinge GCCTGCCCCTACAGGACTCTACACGCATAGCGGTGAGTGTTGTAAAGCATGCAACCTCGGGGAAGGTGTAGCCCAGCCATGCGGGGCTAACCAAACCGTTTGC 67 Truncated LNGFR hinge (TNFR-Cys1) GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTG 68 PDGFR-β extracellular linker

[0183] In some embodiments, the spacer comprises the sequence shown in SEQ ID NO: 49. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 50. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 51. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 52. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 53. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 54. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 55. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 58.

[0184] In some embodiments, the CAR of the present invention may further include a short oligopeptide or polypeptide linker, the length of which is between 2 amino acid residues and 10 amino acid residues, and can be located at the transmembrane of the CAR. A bond is formed between the domain and the cytoplasmic region. A non-limiting example of a suitable linker is a glycine-serine dyad. In some embodiments, the linker comprises the amino acid sequence of GGCKJSGGGKJS (SEQ ID NO: 69). intracellular signaling domain

[0185] In some embodiments, a CAR of the invention includes one or more cytoplasmic domains or regions. The cytoplasmic domain or region of the CAR may include an intracellular signaling domain. The intracellular signaling domain is typically responsible for activating one or more effector functions of immune cells (eg, T cells or NK cells) engineered to express a CAR of the invention. For example, the effector function of a T cell may be cytolytic activity or auxiliary activity, such as interleukin secretion. Thus, in some embodiments, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. In embodiments where a truncated portion of an intracellular signaling domain is used, such truncated portion can be used in place of the corresponding intact chain, so long as the truncated portion transduces an effector function signal.

[0186] Examples of suitable intracellular signaling domains that may be used in the CARs of the invention include, but are not limited to, the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that cooperate to initiate signal transduction upon antigen receptor engagement, and any derivatives or variants of such sequences and any recombinant sequences having the same functional capabilities.

[0187] Without wishing to be bound by theory, it is believed that the signal generated by the TCR alone is not sufficient to fully activate T cells, and thus full activation also requires secondary and / or co-stimulatory signals. Thus, T cell activation can be initiated by two distinct classes of cytoplasmic signaling sequences, namely those that trigger antigen-dependent primary activation via the TCR (primary intracellular signaling domain) and those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domain) The antigen-dependent manner is mediated by those cytoplasmic signaling sequences (secondary cytoplasmic domains, such as costimulatory domains) that provide secondary or costimulatory signals.

[0188] In some embodiments, the primary signaling domain regulates primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of suitable ITAM-containing primary intracellular signaling domains that may be used in the CARs of the invention include, but are not limited to, CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also Referred to as "ICOS"), FcεRI, DAP10, DAP12 and CD66d.

[0189] In some embodiments, a CAR of the invention includes an intracellular signaling domain, such as a primary signaling domain of a CD3-ζ polypeptide. The CD3-ζ polypeptide of the present invention may have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% with the sequence of NCBI reference number NP_932170 or NP_001106864.2 or a fragment thereof , amino acid sequences that are at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous. In some embodiments, the CD3-ζ polypeptide can comprise one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide may have an NCBI reference number of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, An amino acid sequence of a contiguous portion of at least 120, at least 130, at least 140, at least 150, or at least 160, at least 170, or at least 180 amino acids in length.

[0190] In other embodiments, the primary signaling domain includes a modified ITAM domain, such as a mutant ITAM domain that has altered (eg, increased or decreased) activity compared to a native ITAM domain. In one embodiment, the primary signaling domain includes a primary intracellular signaling domain containing a modified ITAM, for example, a primary intracellular signaling domain containing an optimized and / or truncated ITAM. In one embodiment, the primary signaling domain contains one, two, three, four or more ITAM primitives.

[0191] In some embodiments, the intracellular signaling domain of a CAR of the invention may comprise a CD3-ζ signaling domain itself or it may be combined with any other desired intracellular signaling domain that may be used in the context of a CAR of the invention. For example, the intracellular signaling domain of a CAR can include a CD3-ζ chain portion and a costimulatory signaling domain. The costimulatory signaling domain may refer to the portion of the CAR that includes the intracellular domain of the costimulatory molecule. Costimulatory molecules of the present invention are cell surface molecules other than antigen receptors or their ligands that may be required for lymphocytes to respond effectively to antigens. Examples of suitable costimulatory molecules include, but are not limited to, CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD- 1. Lymphocyte function-associated antigen 1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3 and ligands that specifically bind CD83, class I MHC molecules, TNF receptor proteins, immunoglobulin-like proteins, cell mediators receptor, integrin, signaling lymphocyte-activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRD3S1, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103 , ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG ( CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and their analogs.

[0192] Non-limiting examples of intracellular signaling domains (ICDs) are provided in [surface] [8] in. [surface] [8] [Amino acid sequence] [SEQ ID NO:] [describe] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 70 4-1BB ICD RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 71 CD28ICD ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 72 OX40ICD WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS 73 2B4ICD RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 74 CD3zICD

[0193] In some embodiments, intracellular signaling sequences within the cytoplasmic portion of the CAR of the invention can be linked to each other in random or prescribed order. In some embodiments, for example, the length is between 2 amino acids and 10 amino acids (e.g., 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, Short oligopeptides or polypeptide linkers of 7 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids) can form bonds between intracellular signaling sequences. In one embodiment, a glycine-serine dyad may be used as a suitable linker. In one embodiment, a single amino acid (such as alanine or glycine) can be used as a suitable linker.

[0194] In some embodiments, the intracellular signaling domain includes two or more costimulatory signaling domains, such as two costimulatory signaling domains, three costimulatory signaling domains, four costimulatory signaling domains, Five costimulatory signaling domains, six costimulatory signaling domains, seven costimulatory signaling domains, eight costimulatory signaling domains, nine costimulatory signaling domains, 10 costimulatory signaling domains or more Costimulatory signaling domain. In one embodiment, the intracellular signaling domain includes two costimulatory signaling domains. In some embodiments, the two or more costimulatory signaling domains are separated by a linker of the invention. In one embodiment, the linker is a glycine residue. In another embodiment, the linker is an alanine residue.

[0195] In some embodiments, the CAR of the invention further includes an epitope tag. An epitope tag is a polypeptide sequence included within a polypeptide in the form of a label that can be detected, for example, by a monoclonal antibody. Examples of epitope tags include FLAG tag, strep tag, HA tag, V5 tag and myc tag. An exemplary epitope tag is the myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 75).

[0196] In some embodiments, cellular expressions of the invention include CARs that bind an antigen-binding domain of a target antigen of the invention, a transmembrane domain of the invention, a primary signaling domain, and one or more costimulatory signaling domains. Natural Killer CAR (NK CAR)

[0197] In some embodiments, the CAR of the invention includes one or more components of natural killer (NK) cells, thereby forming an NK-CAR. The NK component can be a transmembrane domain, hinge domain or cytoplasmic domain from any suitable natural killer cell receptor, including but not limited to killer cell immunoglobulin-like receptors (KIR), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4 , KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIRS DPI; natural cytotoxicity receptors (NCR), such as NKp30, NKp44, NKp46; immune cell receptor signaling The conductive lymphocyte-activating molecule (SLAM) family, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; Fc receptors (FcR), such as CD16 and CD64; and Ly49 receptors, such as LY49A and LY49C. In some embodiments, NK-CAR can interact with an adapter molecule or intracellular signaling domain (such as DAP12). The structural components of CAR as described above are also applicable to the structure of NK CAR.

[0198] Exemplary configurations and sequences of CARs containing NK receptor components are described in International Patent Publication WO2014 / 145252, published on September 18, 2014.

[0199] One advantage of CAR NK cell therapy over CAR T therapy is that it significantly reduces the risk of inducing graft-versus-host disease (GvHD). Therefore, since no serious toxicity to CAR NK cells is observed or expected, treatment can be administered without hospitalization, thereby significantly reducing the substantial indirect costs associated with CAR-T cell-based therapies due to post-treatment hospitalization (Oberschmidt et al. (2017), Front Immunol, 8:654). [Chimeric inhibitory receptor] []

[0200] Certain aspects of the invention relate to chimeric inhibitory receptors. Chimeric inhibitory receptors can be used, for example, as NOT logic gates for controlling cellular activity, such as immune cell activity. In some embodiments, chimeric inhibitory receptors of the invention specifically bind to one or more antigens expressed on normal cells but not on tumor cells.

[0201] In some embodiments, the chimeric inhibitory receptor comprises an antigen binding domain, a transmembrane domain of the invention (e.g., any suitable transmembrane domain used in combination with a chimeric receptor of the invention), and an intracellular structure area. In some embodiments, the chimeric inhibitory receptor inhibits one or more activities of cells, such as immunoreactive cells.

[0202] In some embodiments, the chimeric inhibitory receptor may comprise an enzyme inhibitory domain. When the chimeric inhibitory receptor is located proximal to a receptor in the cell membrane, such as an immunoreceptor, binding of a cognate antigen to the antigen binding domain will activate the enzyme inhibitory domain to inhibit activation of the receptor. As used herein, the term "enzyme inhibitory domain" refers to a protein domain that inhibits an intracellular signal transduction cascade, such as the native T cell activation cascade. Thus, the disclosed chimeric inhibitory receptors can be engineered to contain appropriate antigen binding domains that will reduce immune responses, for example, in the presence of cognate antigens. Uses of the chimeric inhibitory receptors of the present invention include, but are not limited to, reducing immune responses, controlling T cell activation, and controlling CAR-NK or CAR-T responses.

[0203] In some embodiments, the enzyme inhibitory domain of the chimeric inhibitory receptors of the invention comprises at least a portion of an extracellular domain, a transmembrane domain and / or an intracellular domain. In some embodiments, the enzyme inhibition domain comprises at least a portion of an enzyme. In some embodiments, the enzyme is selected from CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1 and RasGAP (See eg Stanford et al., Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep; 137(1): 1-19). In some embodiments, the enzyme moiety comprises an enzyme domain, an enzyme fragment, or a mutant thereof. In some embodiments, the enzyme moiety is an enzyme catalytic domain. In some embodiments, enzyme domains, enzyme fragments, or mutants thereof are selected to maximize potency and minimize basal inhibition.

[0204] In some embodiments, the enzyme inhibitory domain comprises one or more modifications that regulate basal inhibition. Examples of modifications include, but are not limited to, truncation mutations, amino acid substitutions, introduction of positions for post-translational modifications (examples of which are known to those skilled in the art), and addition of new functional groups. In some embodiments, enzyme domains, enzyme fragments, or mutants thereof are selected to maximize potency and minimize basal inhibition. In some embodiments, the one or more modifications reduce basal inhibition. In other embodiments, the one or more modifications increase basal inhibition.

[0205] In some embodiments, the enzyme inhibitory domain inhibits, for example, immune receptor activation upon recruitment of a chimeric inhibitory receptor of the invention proximal to the immune receptor. In some embodiments, the immune receptor is a naturally occurring immune receptor. In some embodiments, the immune receptor is a naturally occurring antigen receptor. In some embodiments, the immune receptor is selected from T cell receptors, pattern recognition receptors (PRR), NOD-like receptors (NLR), Toll-like receptors (TLR), killer-activated receptors (KAR), killer Inhibitory receptors (KIR), complement receptors, Fc receptors, B cell receptors and interleukin receptors. In some embodiments, the immune receptor is a T cell receptor. In some embodiments, the immune receptor is a chimeric immune receptor. In some embodiments, the chimeric immune receptor is a chimeric TCR or CAR.

[0206] In some embodiments, chimeric inhibitory receptors of the invention may also comprise one or more intracellular inhibitory helper signaling domains. In some embodiments, the intracellular inhibitory helper signaling domain comprises an inhibitory domain. In some embodiments, the one or more intracellular inhibitory helper signaling domains comprise one or more ITIM-containing proteins or fragments thereof. ITIM is a conserved amino acid sequence found in the cytoplasmic tail of many inhibitory immunoreceptors. In some embodiments, the one or more ITIM-containing proteins or fragments thereof are selected from PD-1, CTLA4, TIGIT, and LAIR1. In some embodiments, the one or more intracellular inhibitory accessory signaling domains comprise one or more non-ITIM scaffold proteins or fragments thereof. In some embodiments, the one or more non-ITIM scaffold proteins or fragments thereof are selected from GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, BTLA, GITR, and PD-L1. Other examples of suitable intracellular inhibitory helper signaling domains include, but are not limited to, PD-L1, TIM3, LIR1, NKG2A, VISTA, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, KIR3DL1, A2aR, MHC class I, MHC class II, GAL9, adenosine monophosphate, and TGFβ.

[0207] In some embodiments, the inhibitory chimeric receptor binds an antigen expressed on a non-tumor cell. Exemplary antigens for use in chimeric inhibitory receptors are described in [surface] [9]. [Table 9] [Antigen] [UniProt] [Login ID] [name] [short description] VSIG2 Q96IQ7 Contains V-set and immunoglobulin domain protein 2 Contains an immunoglobulin domain and is highly expressed in gastrointestinal gland cells CPM P14384 carboxypeptidase M Removes C-terminal basic residues (Arg or Lys) from peptides and proteins; believed to play a role in controlling cell surface peptide hormone and growth factor activity ITM2C Q9NQX7 Intrinsic membrane protein 2C Negative regulator of amyloid-beta peptide production SLC26A2 P50443 sulfate transporter A sulfate transporter that functions in endochondral bone formation SLC4A4 Q9Y6R1 Electrogenic sodium bicarbonate cotransporter 1 An electrogenic sodium / bicarbonate cotransporter that regulates bicarbonate influx / efflux at the basolateral membrane and regulates intracellular pH GPA33 Q99795 cell surface A33 antigen Functions in cell-cell recognition and signaling PLA2G2A P14555 Membrane-associated phospholipase A2 Secreted calcium-dependent phospholipase A2 mainly targeting extracellular phospholipids ABCA8 O94911 ATP-binding cassette family A member 8 ATP-dependent lipophilic drug transporter ATP1A2 P50993 Sodium / potassium transporting ATPase subunit α2 The catalytic component of an active enzyme that catalyzes the hydrolysis of ATP plus the exchange of sodium and potassium ions across the cell membrane CHP2 O43745 calcineurin B homolog 2 Cofactor that regulates cellular pH by controlling plasma membrane-type Na+ / H+ exchange activity. SLC26A3 P40879 Chloride anion exchanger Chlorine Particles / Bicarbonate Exchanger

[0208] In some embodiments, the chimeric inhibitory receptor binds a VSIG2 antigen. In some embodiments, the chimeric inhibitory receptor binds a CPM antigen. In some embodiments, the chimeric inhibitory receptor binds an ITM2C antigen. In some embodiments, the chimeric inhibitory receptor binds the SLC26A2 antigen. In some embodiments, the chimeric inhibitory receptor binds the SLC4A4 antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen. In some embodiments, the chimeric inhibitory receptor binds the ATP1A2 antigen. In some embodiments, the chimeric inhibitory receptor binds a CHP2 antigen. In some embodiments, the chimeric inhibitory receptor binds the SLC26A3 antigen.

[0209] In some embodiments, the chimeric inhibitory receptor is a multispecific receptor comprising two or more antigen binding domains such that the chimeric inhibitory receptor can bind two or more antigens. Alternatively, cells can be edited to express two or more chimeric inhibitory receptors that bind to different antigens. Exemplary antigenic pairings for chimeric inhibitory receptors are found in [surface]

[10] . In some embodiments, the bicistronic chimeric receptor system comprises two or more targeting [surface] A CAR of the antigen binding domain of any antigen pair provided in

[10] , wherein the antigen pair comprises a CEA family member and an inhibitory antigen. [immune response cells] []

[0210] Certain aspects of the invention relate to cells, such as immunoreactive cells, genetically engineered to contain one or more chimeric receptors of the invention, or one or more nucleic acids encoding such chimeric receptors, and the use of such A cell therapy method for solid tumors.

[0211] In some embodiments, the immune response cells are genetically engineered to contain one or more tumor antigens that specifically bind to CEA family members, such as [surface] CAR (or nucleic acid encoding the same) of any one of the CEA antigens of [1]. In some embodiments, the immune response cells are genetically engineered to contain one or more [surface] CAR (or nucleic acid encoding it) of the antibody sequence of the representative anti-CEA antibody provided in [2] or an antigen-binding fragment thereof. In some embodiments, the immune-responsive cells are genetically engineered to contain one or more scFvs comprising antibodies derived from antibodies capable of binding to solid tumor antigens, such as [surface] Representative anti-CEA scFv CAR (or nucleic acid encoding it) provided in [3]. In some embodiments, a CAR that specifically binds a CEA family member tumor antigen may be considered an activated CAR ("aCAR") as described herein.

[0212] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cells are primary cells. In some embodiments, the mammalian cell is a cell strain. In some embodiments, the mammalian cell is a bone marrow cell, blood cell, skin cell, bone cell, muscle cell, lung cell, gastrointestinal tract cell, brain cell, neuronal cell, fat cell, liver cell, or heart cell. In some embodiments, the cells are stem cells. Exemplary stem cells include, but are not limited to, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mature stem cells, and tissue-specific stem cells, such as hematopoietic stem cells (blood stem cells), mesenchymal stem cells (MSCs), neural stem cells, epithelial stem cells, or Skin stem cells. In some embodiments, the cells are cells derived or differentiated from the stem cells of the invention. In some embodiments, the cells are immune cells. Immune cells of the invention can be isolated or differentiated from stem cells of the invention (eg, from ESCs or iPSCs). Exemplary immune cells include, but are not limited to, T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, autologous killer T cells, αβ T cells, and γδ T cells), B cells, natural killer (NK) cells , dendritic cells, bone marrow cells, macrophages and monocytes. In some embodiments, the cell is a neuronal cell. Neuronal cells of the present invention can be isolated or differentiated from stem cells of the present invention (eg, from ESCs or iPSCs). Exemplary neuronal cells include, but are not limited to, neural progenitor cells, neurons (e.g., sensory neurons, motor neurons, choline-induced neurons, GABA-induced neurons, glutamate-induced neurons, dopamine Induced neurons or serotonin-inducible neurons), astrocytes, oligodendritic glial cells, and microglia.

[0213] In some embodiments, the cells are immune responsive cells. The immune response cells of the present invention can be isolated or differentiated from the stem cells of the present invention (eg, from ESCs or iPSCs). Exemplary immune response cells of the invention include, but are not limited to, lymphoid lineage cells. The lymphoid lineage (including B cells, T cells, and natural killer (NK) cells) provides antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, detection of foreign cells to the host, and similar functions. Examples of immunoreactive cells of the lymphoid lineage include, but are not limited to, T cells, natural killer (NK) cells, embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells (eg, those stem cells from which lymphoid cells are derived or differentiated). T cells may be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, the T cells of the present invention can be any type of T cells, including but not limited to T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem cell) T-like memory T cells) and two types of effector memory T cells: such as T EM cells and T EMRA cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. Cells Toxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic cells or tumor cells. The patient's own T cells can be activated by introducing one or more chimeric receptors, such as chimeric TCR or CARs are genetically modified to target specific antigens.

[0214] Natural killer (NK) cells may be lymphocytes that are part of cell-mediated immunity and function during the innate immune response. NK cells do not require prior activation to perform their cytotoxic effects on target cells.

[0215] In some embodiments, the immune response cells of the invention are T cells. The T cells of the present invention can be autologous, allogeneic, or derived from engineered progenitor cells or stem cells in vitro.

[0216] In some embodiments, the immune response cells of the invention are universal T cells lacking TCR-αβ. In this technology, methods for developing universal T cells are described, for example, in Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119:5697-5705.

[0217] In some embodiments, an immune-responsive cell of the invention is an isolated immune-responsive cell comprising one or more chimeric receptors of the invention. In some embodiments, the immune response cells comprise one or more, two or more, three or more, four or more, five or more, six or more , seven or more, eight or more, nine or more, or ten or more chimeric receptors of the invention.

[0218] In some embodiments, the immune response cells are T cells. In some embodiments, the immune response cells are natural killer (NK) cells. Cells expressing multiple chimeric receptors

[0219] In some embodiments, cells of the invention (eg, immune response cells) comprise two or more chimeric receptors of the invention. In some embodiments, the cell comprises two or more chimeric receptors, wherein one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell contains three or more chimeric receptors, wherein one of the three or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises four or more chimeric receptors, wherein one of the four or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises five or more chimeric receptors, wherein one of the five or more chimeric receptors is a chimeric inhibitory receptor.

[0220] In some embodiments, the two or more chimeric receptors each comprise a different antigen binding domain, eg, that bind to the same antigen or to different antigens. In some embodiments, each antigen bound by the two or more chimeric receptors is expressed on the same cell type (eg, the same solid tumor cell type). In one embodiment, the cell comprises a first chimeric receptor that targets a first antigen and includes an intracellular signaling domain with a co-stimulatory signaling domain instead of a primary signaling domain, and a second, different antigen that targets and includes a second chimeric receptor having an intracellular signaling domain with a primary signaling domain instead of a costimulatory signaling domain. Without wishing to be bound by theory, it is believed that co-stimulatory signaling domains (such as 4-1BB, CD28 or OX-40) are placed on the first chimeric receptor and primary signaling domains (such as CD3-zeta chain) are placed On the second chimeric receptor, the activity of the chimeric receptor can be restricted to cells expressing both targets. Thus, in some embodiments, a cell (e.g., an immune response cell) of the invention comprises: (a) a first chimeric receptor comprising an antigen binding domain that binds a first antigen, a transmembrane domain, and co-stimulatory signaling domain; and (b) a second chimeric receptor comprising an antigen-binding domain that binds the second antigen, a transmembrane domain and a primary signaling domain. In some embodiments, a cell (e.g., an immunoreactive cell) of the invention comprises: (a) a first chimeric receptor comprising an antigen binding domain that binds a first antigen, a transmembrane domain, and a primary signaling domain; and (b) a second chimeric receptor comprising an antigen-binding domain that binds a second antigen, a transmembrane domain, and a costimulatory signaling domain. In some embodiments, cells (e.g., immunoreactive cells) of the invention include: (a) a first chimeric receptor comprising an antigen binding domain that binds a first antigen, a transmembrane domain, a primary signaling domain, and a costimulatory domain; and (b) a second chimeric receptor comprising an antigen binding domain that binds a second antigen, a transmembrane domain, a primary signaling domain, and a costimulatory domain. In embodiments where both the first chimeric receptor and the second chimeric receptor each comprise a costimulatory signaling domain, the costimulatory signaling domain of the first chimeric receptor and the costimulatory signaling domain of the second chimeric receptor The signaling domain can be derived from the same protein, such as from 4-1BB, CD28 or OX40. Alternatively, the costimulatory signaling domain of the first chimeric receptor can be derived from a different protein than the costimulatory signaling domain of the second chimeric receptor.

[0221] In embodiments where a cell of the invention (e.g., an immunoreactive cell) expresses two or more distinct chimeric receptors, the antigen binding domain of each of the different chimeric receptors can be designed such that The antigen binding domains do not interact with each other. For example, a cell (e.g., an immunoreactive cell) of the invention expressing a first chimeric receptor and a second chimeric receptor can contain an antigen-binding domain comprising an antigen-binding domain that does not form an association with the antigen-binding domain of the second chimeric receptor The first chimeric receptor. For example, the antigen-binding domain of a first chimeric receptor can comprise an antibody fragment, such as a scFv, while the antigen-binding domain of a second chimeric receptor can comprise a VHH.

[0222] Without wishing to be bound by theory, it is believed that in cells with a plurality of chimeric membrane-embedded receptors, each comprising an antigen-binding domain, interactions between the antigen-binding domains of the individual receptors may be undesirable due to such Interactions may inhibit the ability of one or more of these antigen-binding domains to bind its cognate antigen. Thus, in embodiments in which a cell of the invention (eg, an immunoreactive cell) expresses two or more chimeric receptors, the chimeric receptors comprise antigen binding domains that minimize such inhibitory interactions. In one embodiment, the antigen binding domain of one chimeric receptor comprises a scFv and the antigen binding domain of a second chimeric receptor comprises a single VH domain, such as a camel, shark or lamprey single VH domain, or a source of A single VH domain based on human or mouse sequences.

[0223] In some embodiments, the binding of the antigen binding domain of the first chimeric receptor to its cognate antigen when present on the cell surface is not substantially reduced by the presence of the second chimeric receptor. In some embodiments, the binding of the antigen binding domain of the first chimeric receptor to its cognate antigen in the presence of the second chimeric receptor is such that the antigen binding domain of the first chimeric receptor and its cognate antigen bind in the absence of the second chimeric receptor. The binding of two chimeric receptors is 85%, 90%, 95%, 96%, 97%, 98% or 99%. In some embodiments, when present on the surface of a cell, the antigen binding domains of the first chimeric receptor and the second chimeric receptor associate with each other less than when both are scFv antigen binding domains. In some embodiments, the antigen binding domains of the first chimeric receptor and the second chimeric receptor associate with each other 85%, 90%, 95%, 96%, 96% less than if both were scFv antigen binding domains 97%, 98% or 99%.

[0224] In embodiments where the cells of the invention (eg, immune response cells) comprise two or more distinct chimeric receptors of the invention that bind to different antigens, the two or more chimeric receptors provide the cell with Logic gates, such as OR logic gates, AND logic gates, NOT logic gates, or any combination of such logic gates. Thus, in certain embodiments, a cell (eg, an immune response cell) of the invention contains two or more chimeric receptors, and binding of the first chimeric receptor to the first antigen is capable of activating the cell. In some embodiments, a cell of the invention (eg, an immune response cell) contains two or more chimeric receptors, and binding of the second chimeric receptor to a second antigen is capable of stimulating the cell. In some embodiments, cells of the invention (e.g., immune response cells) comprise two or more chimeric receptors, and a first chimeric receptor binds to a first antigen and a second chimeric receptor binds to a second Antigen binding is required to activate the cell. In some embodiments, cells of the invention (e.g., immune response cells) comprise two or more chimeric receptors, and a first chimeric receptor binds to a first antigen and a second chimeric receptor binds to a second Antigen binding is required to stimulate the cell. In some embodiments, a cell (eg, an immune-responsive cell) of the invention includes two or more chimeric receptors, and the cell exhibits a response to cells that are positive for both the first antigen and the second antigen. The lytic activity is to a greater extent than the cytolytic activity against cells positive only for the first antigen or only for the second antigen. In some embodiments, cells of the invention (e.g., immune response cells) comprise two or more chimeric receptors, and the first chimeric receptor binds to a first antigen or the second chimeric receptor binds to a second Antigen binding activates the immune response cells.

[0225] In some embodiments, cells of the invention (eg, immunoreactive cells) comprise a cleaved chimeric receptor system, such as a cleaved CAR system. Exemplary cleavage chimeric receptor systems are described in WO2014 / 055442 and WO2014 / 055657. In some embodiments, the cleaved chimeric receptor system comprises a first chimeric receptor having a first antigen binding domain and a costimulatory domain (e.g., 4-1BB) and having a second antigen binding domain and an intracellular signaling domain. (e.g., CD3-ζ). In such embodiments, when the cell encounters the first antigen, the costimulatory domain is activated and the cell proliferates. Additionally, when the cell encounters the second antigen, the intracellular signaling domain is activated and cell-killing activity is induced. Thus, in some embodiments, cells of the invention (eg, immune response cells) are fully activated only in the presence of the two antigens.

[0226] In some embodiments, cells of the invention (eg, immunoreactive cells) exhibit cytolytic activity against cells positive for both the first antigen and the second antigen and against cells positive for the first antigen alone. The cytolytic activity of the cells is greater than that of In certain embodiments, the first chimeric receptor binds to the first antigen with low binding affinity or low binding affinity. In certain embodiments, the first chimeric receptor binds to a low-accessibility epitope of the first antigen. In certain embodiments, the first chimeric receptor binds to the first antigen with a lower binding affinity than the second chimeric receptor binds to the second antigen. In some embodiments, the first chimeric receptor binds to the first antigen with a binding affinity that is at least 5-fold lower than the binding affinity of the second chimeric receptor to the second antigen. In some embodiments, the binding affinity of the first chimeric receptor to the first antigen is at least 10-fold, 20-fold, 30-fold, 40-fold lower than the binding affinity of the second chimeric receptor to the second antigen , 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 5000 times, 1000 times, 5000 times, or 10000 times.

[0227] In some embodiments, pair selection should favor redundant representation of the two target antigens in the tumor in order to minimize the risk of antigen escape. Thus, in some embodiments, cells (e.g., immune response cells) of the invention comprise (i) a first chimeric receptor that binds to a first antigen and (ii) a second chimeric receptor that binds to a second antigen. , wherein the combination of the two chimeric receptors binding to their target antigens produces a therapeutic effect. In many embodiments, binding to only one target antigen does not achieve a therapeutic effect.

[0228] In some embodiments, a cell (eg, an immunoreactive cell) of the invention comprises one or more chimeric receptors that bind a CEA family antigen, and optionally the cell also comprises an inhibitory chimeric receptor. In some embodiments, the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric receptor binds the CEACAM6 antigen. In some embodiments, the chimeric receptor binds to [surface] CEA family antigens described in [1]. In some embodiments, the chimeric receptor that binds to a CEA family antigen comprises an antigen binding domain derived from an antibody selected from the group consisting of: MRG1, labetuzumab (i.e., hMN14), cerbituzumab, tet Certolumab, BW431 / 26, A5B7, MFE23, hMFE23, FM4 (also known as "MG7"), Tenurelimab. In some embodiments, the antigen binding domain of a chimeric receptor that binds to a CEA family antigen comprises a [surface] scFv with the amino acid sequence provided in [3]. In some embodiments, chimeric receptors that bind to CEA family antigens comprise such as [surface] The amino acid sequence shown in

[28] and / or is represented by [surface] The polynucleotide sequence encoding provided in

[28] . In some embodiments, the inhibitory chimeric receptor binds a VSIG2 antigen. In some embodiments, the inhibitory chimeric receptor binds a CPM antigen. In some embodiments, the inhibitory chimeric receptor binds an ITM2C antigen. In some embodiments, the inhibitory chimeric receptor binds the SLC26A2 antigen. In some embodiments, the inhibitory chimeric receptor binds the SLC4A4 antigen. In some embodiments, the inhibitory chimeric receptor binds to the GPA33 antigen. In some embodiments, the inhibitory chimeric receptor binds to the PLA2G2A antigen. In some embodiments, the inhibits the chimeric receptor from binding to the ABCA8 antigen. In some embodiments, the inhibitory chimeric receptor binds ATP1A2 antigen. In some embodiments, the chimeric receptor inhibits binding to CHP2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the SLC26A3 antigen. In some embodiments, the chimeric receptor is a multispecific receptor comprising two or more antigen binding domains such that the chimeric receptor can bind two or more antigens.

[0229] In some embodiments, the immune response cells may comprise one or more tumor-targeting chimeric receptors and one or more inhibitory chimeric receptors targeting antigens not expressed on the tumor. Combinations of tumor-targeting chimeric receptors and inhibitory chimeric receptors in the same immune response cells can be used to reduce on-target and off-tumor toxicity. For example, if a healthy cell expresses both an antigen recognized by a tumor-targeting chimeric receptor and an antigen recognized by an inhibitory chimeric receptor, an immune reactive cell expressing the tumor antigen can bind to the healthy cell. In such cases, inhibiting the chimeric antigen will also bind its cognate ligand on healthy cells, and inhibiting the inhibitory function of the chimeric receptor will reduce, reduce, prevent or inhibit immune response cells through the tumor-targeting chimeric ligand. Activated by binding to receptors.

[0230] In some embodiments, the inhibits chimeric receptor binding to VSIG2 antigen. In some embodiments, the chimeric receptor inhibits binding to CPM antigen. In some embodiments, the inhibits the chimeric receptor from binding to the ITM2C antigen. In some embodiments, the inhibitory chimeric receptor binds to SLC26A2 antigen. In some embodiments, the inhibitory chimeric receptor binds to SLC4A4 antigen. In some embodiments, the inhibitory chimeric receptor binds to the GPA33 antigen. In some embodiments, the inhibitory chimeric receptor binds to the PLA2G2A antigen. In some embodiments, the inhibits the chimeric receptor from binding to the ABCA8 antigen. In some embodiments, the inhibitory chimeric receptor binds ATP1A2 antigen. In some embodiments, the inhibitory chimeric receptor binds a CHP2 antigen. In some embodiments, the inhibitory chimeric receptor binds the SLC26A3 antigen.

[0231] Alternatively, cells may express two or more chimeric receptors that bind to different antigens. Exemplary antigen pairings are shown in [surface]

[10] . [surface]

[10] [Antigen] [1] [Antigen] [2] [] [Antigen] [1] [Antigen] [2] CEACAM1 CEACAM5 CEACAM6 SLC4A4 CEACAM1 CEACAM6 CEACAM6 GPA33 CEACAM1 VSIG2 CEACAM6 PLA2G2A CEACAM1 CPM CEACAM6 ABCA8 CEACAM1 ITM2C CEACAM6 ATP1A2 CEACAM1 SLC26A2 CEACAM5 SLC26A2 CEACAM1 SLC4A4 CEACAM5 SLC4A4 CEACAM1 GPA33 CEACAM5 GPA33 CEACAM1 PLA2G2A CEACAM5 PLA2G2A CEACAM1 CHP2 CEACAM5 ABCA8 CEACAM1 SLC26A3 CEACAM5 ATP1A2 CEACAM1 ABCA8 CEACAM5 CHP2 CEACAM1 ATP1A2 CEACAM5 SLC26A3 CEACAM5 CEACAM6 CEACAM6 VSIG2 CEACAM5 VSIG2 CEACAM6 CPM CEACAM5 CPM CEACAM6 ITM2C CEACAM5 ITM2C CEACAM6 SLC26A2 CEACAM6 CHP2 CEACAM6 SLC26A3

[0232] In some embodiments, the immunoreactive cell comprises a bicistronic chimeric antigen receptor system construct. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and CEACAM1 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and CEACAM5 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and CEACAM6 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and CEACAM5 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and CEACAM6 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and CEACAM6 CAR.

[0233] In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and VSIG2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and CPM CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and ITM2C CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and SLC26A2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and SLC4A4 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and GPA33 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and PLA2G2A CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and ABCA8 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and ATP1A2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and CHP2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEA CAR and SLC26A3 CAR.

[0234] In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and VSIG2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and CPM CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and ITM2C CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and SLC26A2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and SLC4A4 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and GPA33 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and PLA2G2A CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and ABCA8 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and ATP1A2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and CHP2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM1 CAR and SLC26A3 CAR.

[0235] In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and VSIG2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and CPM CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and ITM2C CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and SLC26A2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and SLC4A4 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and GPA33 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and PLA2G2A CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and ABCA8 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and ATP1A2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and CHP2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM5 CAR and SLC26A3 CAR.

[0236] In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and VSIG2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and CPM CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and ITM2C CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and SLC26A2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and SLC4A4 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and GPA33 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and PLA2G2A CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and ABCA8 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and ATP1A2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and CHP2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and CHP2 CAR. In some embodiments, the bicistronic chimeric antigen receptor system construct comprises CEACAM6 CAR and SLC26A3 CAR.

[0237] In some embodiments, the bicistronic chimeric antigen receptor system construct comprises [surface] Any pair of antigens provided in

[10] . cells expressing chimeric inhibitory receptors

[0238] In some embodiments, a cell (eg, an immunoreactive cell) of the invention comprises one or more chimeric inhibitory receptors of the invention. In some embodiments, each of the one or more chimeric inhibitory receptors comprises an antigen binding domain that binds an antigen expressed on normal cells but not on tumor cells such as solid tumor cells. In some embodiments, the one or more chimeric inhibitory receptors bind an antigen expressed on a non-tumor cell derived from a tissue selected from the group consisting of: brain, neurons Tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, bladder, male reproductive organs, female reproductive organs, fat, soft tissue, and skin.

[0239] In some embodiments, chimeric inhibitory receptors and one or more chimeric receptors (such as chimeric TCR or CAR) expressed on cells of the invention (such as immune response cells) can be used as NOT logic gates to control, Modulating or otherwise inhibiting one or more activities of the one or more chimeric receptors. In some embodiments, chimeric receptors of the invention inhibit one or more activities of cells of the invention (eg, immunoreactive cells). In some embodiments, a chimeric inhibitory receptor is combined with one or more chimeric receptors of the invention to combine an OR logic gate with a NOT logic gate and / or an AND logic gate with a NOT logic gate.

[0240] In some embodiments, the chimeric inhibitory receptor binds one or more antigens selected from the group consisting of VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, and SLC26A3.

[0241] In some embodiments, the chimeric receptor binds a CEA antigen and the chimeric inhibitory receptor binds a VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, or SLC26A3 antigen.

[0242] In some embodiments, the chimeric receptor binds the CEACAM1 antigen and the chimeric inhibitory receptor binds the VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, or SLC26A3 antigen.

[0243] In some embodiments, the chimeric receptor binds the CEACAM5 antigen and the chimeric inhibitory receptor binds the VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, or SLC26A3 antigen.

[0244] In some embodiments, the chimeric receptor binds the CEACAM6 antigen and the chimeric inhibitory receptor binds the VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, or SLC26A3 antigen.

[0245] In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0246] In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM6 antigen.

[0247] In some embodiments, the chimeric inhibitory receptor binds the ITM2C antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds ITM2C antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the ITM2C antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds ITM2C antigen and the chimeric receptor binds CEACAM6 antigen.

[0248] In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0249] In some embodiments, the chimeric inhibitory receptor binds SLC4A4 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC4A4 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC4A4 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC4A4 antigen and the chimeric receptor binds CEACAM6 antigen.

[0250] In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0251] In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM6 antigen.

[0252] In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0253] In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0254] In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0255] In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM6 antigen.

[0256] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-VSIG2 antibody. The VSIG2 antibody can be any suitable VSIG2 antibody known in the art.

[0257] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-CPM antibody. The CPM antibody can be any suitable CPM antibody known in the art.

[0258] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-ITM2C antibody. The ITM2C antibody can be any suitable ITM2C antibody known in the art.

[0259] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-SLC26A2 antibody. The SLC26A2 antibody can be any suitable SLC26A2 antibody known in the art.

[0260] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-SLC4A4 antibody. The SLC4A4 antibody can be any suitable SLC4A4 antibody known in the art.

[0261] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-GPA33 antibody. The GPA33 antibody can be any suitable GPA33 antibody known in the art.

[0262] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-PLA2G2A antibody. The PLA2G2A antibody can be any suitable PLA2G2A antibody known in the art.

[0263] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-ABCA8 antibody. The ABCA8 antibody can be any suitable ABCA8 antibody known in the art.

[0264] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-ATP1A2 antibody. The ATP1A2 antibody can be any suitable ATP1A2 antibody known in the art.

[0265] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-CHP2 antibody. The CHP2 antibody can be any suitable CHP2 antibody known in the art.

[0266] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain derived from an anti-SLC26A3 antibody. The SLC26A3 antibody can be any suitable SLC26A3 antibody known in the art. costimulatory ligand

[0267] In some embodiments, cells of the invention (eg, immune response cells, eg, NK cells) may further include one or more recombinant or exogenous costimulatory ligands. For example, the cell can be further transduced with one or more costimulatory ligands such that the cell co-expresses or is induced to co-express one or more chimeric receptors of the invention and one or more costimulatory ligands . Without wishing to be bound by theory, it is believed that the interaction between one or more chimeric receptors and one or more co-stimulatory ligands can provide non-antigen-specific signals important for full cell activation. Examples of suitable costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is an interleukin involved in systemic inflammation and stimulates the acute phase response. Its main function is to regulate immune cells. Members of the TNF superfamily share many common characteristics. Most TNF superfamily members are synthesized as type II transmembrane proteins containing a short cytoplasmic segment and a relatively long extracellular domain (extracellular C-terminus). Examples of suitable TNF superfamily members include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL ), CD30L / CD153, tumor necrosis factor β (TNFP) / lymphotoxin α (LTa), lymphotoxin β (LTP), CD257 / B cell activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced Type TNF receptor ligand (GITRL) and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF 14). The immunoglobulin (Ig) superfamily is a large group of cell surface soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share many structural features with immunoglobulins and have immunoglobulin domains (folds). Examples of suitable immunoglobulin superfamily ligands include, but are not limited to, the CD28 ligands CD80 and CD86, and the PD-1 ligand PD-L1 / (B7-H1). In certain embodiments, the one or more costimulatory ligands are selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. effector molecule

[0268] In some embodiments, cells of the invention (eg, immune response cells, eg, NK cells) comprise one or more chimeric receptors and may further comprise one or more immunomodulatory effector molecules. Non-limiting examples of effector molecules encompassed by the invention include interleukins, antibodies, chemokines, nucleotides, peptides, enzymes and oncolytic viruses. For example, cells can be engineered to express and secrete in a regulated manner at least one, two, three or more of the following immunomodulatory effector molecules: IL-12, IL-16, IFN-β, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, IL-21, OX40 ligand, CD40L, anti-PD-1 antibody, anti-PD-L1 antibody, Anti-CTLA-4 antibodies, anti-TGFβ antibodies, anti-TNFR2, MIP1α (CCL3), MIP1β (CCL5), CCL21, CpG oligodeoxynucleotides, and antitumor peptides (e.g., antimicrobial peptides with antitumor activity, see e.g. Gaspar, D. et al., Front Microbiol. 2013; 4: 294; Chu, H. et al., PLoS One. 2015; 10(5): e0126390, and website: aps.unmc.edu / AP / main.php) .

[0269] In some embodiments, the effector molecule is an immunomodulatory effector molecule that: stimulates T cell signaling, activity and / or recruitment, stimulates antigen presentation and / or processing, stimulates natural killer cell-mediated cytotoxic signaling, activity and / or recruit, stimulate dendritic cell differentiation and / or maturation, stimulate immune cell recruitment, stimulate macrophage signaling, stimulate matrix degradation, stimulate production of immunostimulatory metabolites, or stimulate type I interferon signaling; and at least one Proteins, including effector molecules that: inhibit negative co-stimulatory signaling, inhibit pro-apoptotic signaling of anti-tumor immune cells, inhibit T regulatory (Treg) cell signaling, activity and / or recruitment, inhibit tumor checkpoints Molecules, activate Stimulator of Interferon Genes (STING) signaling, inhibit myeloid-derived suppressor cell signaling, activity and / or recruitment, degrade immunosuppressive factors / metabolites, inhibit vascular endothelial growth factor signaling, or directly kill tumor cells .

[0270] In some embodiments, the one or more effector molecules include an interleukin selected from the group consisting of IL-15, IL-12 (e.g., IL12p70 fusion protein), IL-18, and IL-21. chemokine receptor

[0271] In some embodiments, cells of the invention (eg, immune response cells, such as NK cells) comprise one or more chimeric receptors and may further comprise one or more chemokine receptors. For example, transgenic expression of the chemokine receptors CCR2b or CXCR2 in cells such as T cells enhances communication with CCL2-secreting or CXCL1-secreting solid tumors (Craddock et al., J Immunother. 2010 Oct; 33 (8):780-8, and Kershaw et al., Hum Gene Ther. 2002 Nov 1; 13(16): 1971-80). Without wishing to be bound by theory, it is believed that the chemokine receptors expressed on the chimeric receptor-expressing cells of the invention recognize chemokines secreted by the tumor and improve the targeting of the cell to the tumor, thereby facilitating The cells infiltrate the tumor and enhance the anti-tumor potency of the cells. Chemokine receptors of the present invention may include naturally occurring chemokine receptors, recombinant chemokine receptors, or chemokine-binding fragments thereof. Examples of suitable chemokine receptors that may be expressed on cells of the invention include, but are not limited to, CXC chemokine receptors, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7; CC chemokine receptors, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11; CX3C chemokine receptors, such as CX3CR1; XC chemokine receptors, such as XCR1; and chemokine binding fragments thereof . In some embodiments, the chemokine receptors to be expressed on the cell are selected based on the chemokines secreted by the tumor. Chimeric receptor regulation

[0272] Some embodiments of the invention relate to modulating the activity of one or more chimeric receptors in cells expressing the chimeric receptors of the invention. There are several methods by which the activity of chimeric receptors can be modulated. In some embodiments, tunable chimeric receptors may be desired, in which the activity of one or more chimeric receptors can be controlled to optimize the safety and / or efficacy of chimeric receptor therapies. For example, apoptosis is induced using caspases fused to dimerization domains (see e.g. Di et al., N Engl. J. Med. 2011 Nov 3; 365(18): 1673-1683 ) can be used as a safety switch for chimeric receptor therapy. In some embodiments, cells expressing chimeric receptors of the invention can also be expressed upon administration of, for example, rimiducid (IUPAC name: (2S)-1-[(2S)-2-(3,4 ,5-Trimethoxyphenyl)butyl]piperidine-2-carboxylic acid [(1R)-3-(3,4-dimethoxyphenyl)-1-[3-[2-[2-[ [ 2-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxy phenyl)butyl]piperidine-2-carbonyl]oxypropyl]phenoxy]acetyl]amino]ethylamino]-2-side oxyethoxy]phenyl]propyl] Inducible caspase-9 (iCaspase-9) is a dimerized drug that induces caspase-9 activation and leads to apoptosis. In some embodiments, iCaspase-9 contains a binding domain containing a chemical inducer of dimerization (CID) that mediates dimerization in the presence of CID, thereby resulting in inducible selective depletion of cells expressing the chimeric receptor.

[0273] Alternatively, in some embodiments, the chimeric receptors of the invention can be modulated by using small molecules or antibodies that inactivate or otherwise inhibit the activity of the chimeric receptors. For example, antibodies can deplete cells expressing chimeric receptors by inducing antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, cells expressing chimeric receptors of the invention may further express antigens recognized by molecules capable of inducing cell death by ADCC or complement-induced cell death. For example, cells expressing chimeric receptors of the invention may further express receptors that can be targeted by antibodies or antibody fragments. Examples of suitable receptors that can be targeted by antibodies or antibody fragments include, but are not limited to, EpCAM, VEGFR, integrins (eg ανβ3, α4, αΙ¾β3, α4β7, α5β1, ανβ3, αν), TNF receptor superfamily members (eg TRAIL-R1 and TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a / LFA-1, CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5, CD319 / SLAMF7 and EGFR, as well as their truncated forms.

[0274] In some embodiments, cells expressing chimeric receptors of the invention may also express truncated epidermal growth factor receptors (EGFR) that lack signaling capabilities but retain epitopes recognized by molecules capable of inducing ADCC (e.g., WO2011 / 056894).

[0275] In some embodiments, the chimeric receptor-expressing cells of the invention further comprise a highly expressed tight marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in the chimeric receptor-expressing cells , thereby binding to an anti-CD20 antibody (such as rituximab), resulting in ADCC selective depletion of cells expressing the chimeric receptor. Other methods of depleting cells expressing chimeric receptors of the invention may include, but are not limited to, administration of a monoclonal anti-CD52 antibody that selectively binds to and targets cells expressing chimeric receptors by inducing ADCC performs destruction. In some embodiments, chimeric receptor ligands, such as anti-atopic antibodies, can be used to selectively target cells expressing the chimeric receptor. In some embodiments, the anti-atopic antibody can elicit effector cell activity, such as ADCC or ADC activity. In some embodiments, the chimeric receptor ligand can be further coupled to an agent that induces cell killing, such as a toxin. In some embodiments, cells expressing chimeric receptors of the invention may further express target proteins recognized by cell depleting agents of the invention. In some embodiments, the target protein is CD20 and the cell depleting agent is an anti-CD20 antibody. In such embodiments, the cell depleting agent is administered once it is desired to reduce or eliminate cells expressing the chimeric receptor. In some embodiments, the cell-depleting agent is an anti-CD52 antibody.

[0276] In some embodiments, a modulated chimeric receptor comprises a set of polypeptides, wherein components of the chimeric receptors of the invention are partitioned on separate polypeptides or members. For example, the set of polypeptides can include a dimerization switch that, in the presence of a dimeric molecule, allows the polypeptides to couple to each other to form a functional chimeric receptor. [Nucleic Acid Constructs Encoding Chimeric Receptors] []

[0277] Certain aspects of the invention pertain to nucleic acids (eg, isolated nucleic acids) encoding one or more chimeric receptors of the invention. In some embodiments, the nucleic acid is an RNA construct, such as a messenger RNA (mRNA) transcript or a modified RNA. In some embodiments, the nucleic acid is a DNA construct.

[0278] In some embodiments, nucleic acids of the invention encode chimeric receptors comprising one or more antigen binding domains (wherein each domain binds to a target antigen (e.g., a solid tumor antigen)), a transmembrane domain and one or more intracellular signaling domains. In some embodiments, the nucleic acid encodes a chimeric receptor comprising an antigen binding domain, a transmembrane domain, a primary signaling domain (e.g., CD3-ζ domain), and one or more co-stimulatory signals conduction domain. In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a spacer. In some embodiments, the antigen binding domain is linked to the transmembrane domain by the spacer. In some embodiments, the spacer comprises a group selected from [surface] A nucleic acid sequence of any one of the nucleic acid sequences listed in [9]. In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[0279] Nucleic acids of the invention may be obtained using any suitable recombinant method known in the art, including but not limited to, by screening libraries from cells expressing the gene of interest, by obtaining the gene of interest from a vector known to include the gene, or by The relevant gene is isolated directly from cells and tissues containing the gene using standard techniques. Alternatively, the relevant gene can be produced synthetically.

[0280] In some embodiments, nucleic acids of the invention are contained within vectors. In some embodiments, nucleic acids of the invention are expressed in cells via transposons, CRISPR / Cas9 systems, TALENs, or zinc finger nucleases.

[0281] In some embodiments, expression of a nucleic acid encoding a chimeric receptor of the invention can be achieved by operably linking the nucleic acid to a promoter and incorporating the construct into an expression vector. Suitable vectors can replicate and integrate in eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences and promoters that can be used to regulate expression of the desired nucleic acid.

[0282] In some embodiments, the expression constructs of the invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols (eg, US5399346, US5580859, and US5589466). In some embodiments, the vectors of the invention are gene therapy vectors.

[0283] Nucleic acids of the invention can be cloned into many types of vectors. For example, the nucleic acid can be colonized into a vector including, but not limited to, a plastid, a phage plastid, a phage derivative, an animal virus, or a myxosome. In some embodiments, the vector can be an expression vector, a replication vector, a probe generation vector, or a sequencing vector.

[0284] In some embodiments, the plastid vector comprises a transposon / translocase system for incorporation of the nucleic acid of the invention into the host cell genome. Methods for expressing proteins in immune cells using the transposon and translocase plastid systems are generally described in Chicaybam L, Hum Gene Ther. 2019 Apr;30(4):511-522. doi: 10.1089 / hum.2018.218; and Ptáčková P, Cytotherapy. 2018 Apr;20(4):507-520. doi: 10.1016 / j.jcyt.2017.10.001, each of which is incorporated herein by reference in its entirety. In some embodiments, the transposon system is Sleeping Beauty transposon / translocase or piggyBac transposon / translocase.

[0285] In some embodiments, the expression vectors of the invention can be provided to cells in the form of viral vectors. Suitable viral vector systems are well known in the art. For example, viral vectors can be derived from retroviruses (such as gamma retroviruses and lentiviruses), adenoviruses, adeno-associated viruses, and herpesviruses. In some embodiments, the vectors of the invention are retroviral vectors. Types of retroviral vectors include lentiviral vectors and gamma retroviral vectors. In some embodiments, the vectors of the invention are lentiviral vectors. Lentiviral vectors are derived from lentiviruses, such as human immunodeficiency virus (HIV), and are suitable for long-term gene transfer because such vectors generally allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors may be superior to other retroviral vectors (e.g., murine leukemia virus) for transducing certain cell types because lentiviral vectors often transduce non-proliferating cells. In some embodiments, the vectors of the invention are gamma retroviral vectors. Gamma retrovirus vectors are derived from viruses of the genus Gamma retrovirus, including murine leukemia virus (MLV) and feline leukemia virus.

[0286] Viral particles produced using retroviral vectors, such as lentiviral and γ-retroviral vectors, are often pseudotyped to include the retroviral exogenous envelope protein. A commonly used envelope protein for pseudotyping is the vesicular stomatitis virus (VSV) G glycoprotein. In some embodiments, a retroviral vector (eg, a lentiviral vector or a gamma retroviral vector) is pseudotyped with the baboon endogenous retrovirus (BaEV) envelope protein. The BaEV envelope protein may be the wild-type BaEV envelope, or it may be a mutant BaEV envelope, such as a chimeric BaEV in which the cytoplasmic tail is replaced by an MLV-A viral cytoplasmic tail or truncated at the C-terminus to remove the R-peptide (eg Anais Girard-Gagnepain et al., Blood 2014; 124 (8): 1221–1231. doi: https: / / doi.org / 10.1182 / blood-2014-02-558163).

[0287] In some embodiments, the vector of the present invention is an adenovirus vector (A5 / 35). In some embodiments, vectors of the invention contain an origin of replication functional in at least one organism, a promoter sequence, suitable restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and US6326193). A number of virus-based systems have been developed for gene transfer into mammalian cells. The selected gene can be inserted into a vector and encapsulated in retroviral particles using techniques known in the art. Recombinant virus can then be isolated and delivered to mammalian cells in vivo or ex vivo. Many retroviral systems are known in the art.

[0288] In some embodiments, the vectors of the invention include additional promoter elements, such as enhancers that regulate the frequency of transcription initiation. Enhancers are typically located in a region 30-110 bp upstream of the initiation site, but many promoters have been shown to also contain functional elements downstream of the initiation site. The spacing between promoter elements can be flexible so that promoter function is preserved when the elements are reversed or moved relative to each other. For example, in the thymidine kinase (tk) promoter, the spacing between promoter elements can increase to 50 bp, after which activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription. Exemplary promoters may include, but are not limited to, the SFFV gene promoter, the EFS gene promoter, the CMV IE gene promoter, the EF1a promoter, the ubiquitin C promoter, and the phosphoglycerol kinase (PGK) promoter.

[0289] In some embodiments, the promoter capable of expressing the nucleic acid of the invention in mammalian cells, such as the immunoreactive cells of the invention, is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex responsible for the enzymatic delivery of amide-based tRNAs to the ribosome. The EF1a promoter has been used extensively in mammalian expression plastids and has been shown to be effective in driving expression of chimeric receptors from nucleic acids cloned into lentiviral vectors.

[0290] In some embodiments, a promoter capable of expressing a nucleic acid of the invention in a mammalian cell, such as an immunoreactive cell of the invention, is a constitutive promoter. For example, a suitable constitutive promoter is the immediate early cytomegalovirus (CMV) promoter. The CMV promoter is a strong constitutive promoter capable of driving high levels of expression of any polynucleotide sequence operably linked to the promoter. Other suitable constitutive promoters include, but are not limited to, ubiquitin C (UbiC) promoter, Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long term Repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, elongation factor la promoter, hemoglobin promoter and creatine kinase promoter.

[0291] In some embodiments, a promoter capable of expressing a nucleic acid of the invention in a mammalian cell, such as an immunoreactive cell of the invention, is an inducible promoter. The use of an inducible promoter can provide a molecular switch capable of inducing or repressing the expression of a nucleic acid of the invention when the promoter is operably linked to the nucleic acid. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters.

[0292] In some embodiments, the vectors of the invention may further comprise a signal sequence to promote secretion, a polyadenylation signal and a transcription terminator, elements allowing episomal replication and / or elements allowing selection. Exemplary signal sequences are provided at [surface]

[11] . [surface]

[11] [Amino acid sequence] [SEQ ID NO:] [describe] MALPVTALLLPLALLLLHAARP 76 CD8 signal sequence

[0293] In some embodiments, the vectors of the present invention may further comprise selectable marker genes and / or reporter genes to facilitate identification and selection of cells expressing chimeric receptors from populations of cells transduced with the vectors. In some embodiments, a selectable marker can be encoded by a nucleic acid isolated from the vector and used in the co-transfection procedure. A selectable marker or reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell. Examples of selectable markers include, but are not limited to, antibiotic resistance genes such as neo and the like.

[0294] In some embodiments, reporter genes can be used to identify transduced cells and assess the functionality of regulatory sequences. As disclosed herein, a reporter gene is a gene that is absent or not expressed in a recipient organism or tissue and that encodes a polypeptide whose expression results in a readily detectable property, such as enzymatic activity. The expression of the reporter gene can be analyzed at a suitable time after the nucleic acid has been introduced into the recipient cell. Examples of reporter genes include, but are not limited to, the gene encoding luciferase, the gene encoding beta-galactosidase, the gene encoding chloramphenicol acetyltransferase, the gene encoding secreted alkaline phosphatase, and the gene encoding green fluorescent Photoprotein gene. Suitable performance systems are well known in the art and can be prepared using known techniques or are commercially available. In some embodiments, the construct with the smallest 5' flanking region that shows the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be ligated to a reporter gene and used to assess the ability of the agent to regulate promoter-driven transcription.

[0295] In some embodiments, a vector comprising a nucleic acid sequence encoding a chimeric receptor of the invention further comprises a second nucleic acid encoding a polypeptide that increases the activity of the chimeric receptor.

[0296] In embodiments where a cell expressing a chimeric receptor comprises two or more chimeric receptors, a single nucleic acid may contain each encoding chimeric receptor under a single regulatory control element (e.g., a promoter) or within the nucleic acid. Two or more chimeric receptors are encoded under separate regulatory control elements in the receptor nucleotide sequence. In some embodiments where the cell expressing the chimeric receptor contains two or more chimeric receptors, each chimeric receptor can be encoded by a separate nucleic acid. In some embodiments, each individual nucleic acid contains its own control elements (eg, promoter). In some embodiments, a single nucleic acid encodes two or more chimeric receptors, and the nucleotide sequences encoding the chimeric receptors are in the same reading frame and represent a single polypeptide chain. In such embodiments, the two or more chimeric receptors may be separated by one or more peptide cleavage sites such as autocleavage sites or intracellular protease substrates. Suitable peptide cleavage sites may include, but are not limited to, T2A peptide cleavage sites, P2A peptide cleavage sites, E2A peptide cleavage sites, and F2A peptide cleavage sites. In some embodiments, the two or more chimeric receptors comprise a T2A peptide cleavage site. In some embodiments, the two or more chimeric receptors comprise an E2A peptide cleavage site. In some embodiments, the two or more chimeric receptors comprise T2A and E2A peptide cleavage sites.

[0297] Methods of introducing genes into cells and expressing them are well known in the art. For example, in some embodiments, the expression vector can be transferred into the host cell by physical, chemical or biological means. Examples of physical means for introducing nucleic acid into host cells include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Examples of chemical methods for introducing nucleic acids into host cells include, but are not limited to, colloidal dispersion systems, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, gels, Beams, mixed micelles and liposomes. Examples of biological means for introducing nucleic acids into host cells include, but are not limited to, the use of DNA and RNA vectors.

[0298] In some embodiments, liposomes can be used as a non-viral delivery system to introduce nucleic acids or vectors of the invention into host cells in vitro, ex vivo, or in vivo. In some embodiments, the nucleic acid can be associated with the lipid, for example, by encapsulation in an aqueous solution inside the liposome, interspersed within the lipid bilayer of the liposome, via association with both the liposome and the nucleic acid. The associated linker molecule is attached to the liposome, encapsulated in the liposome, complexed with the liposome, dispersed in a solution containing the lipid, mixed with the lipid, combined with the lipid, contained in the lipid as a suspension In the substance, containing micelles or complexed with micelles, or otherwise associated with lipids. As disclosed herein, lipid-associated nucleic acid or carrier compositions are not limited to any specific structure in solution. In some embodiments, such compositions may exist in a bilayer structure, be in the form of micelles, or have a "collapsed" structure. Such compositions can also be simply dispersed in solution to form aggregates that are not uniform in size or shape. As disclosed herein, lipids are fatty substances that may occur naturally or be synthesized. In some embodiments, lipids may include lipid droplets naturally occurring in the cytoplasm or a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Suitable lipids are available from commercial sources and include, but are not limited to, dimyristyl phosphatidyl choline ("DMPC"), dicetyl phosphate ("DCP"), cholesterol, and dimyristyl phosphatidyl glycerol ("DMPC"). "DMPG"). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Use chloroform as the solvent because it evaporates more easily than methanol. As used herein, "liposome" can encompass a variety of unilamellar and multilamellar lipid vehicles formed by creating encapsulated lipid bilayers or aggregates. In some embodiments, liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. In some embodiments, multilamellar liposomes can have multiple lipid layers separated by an aqueous medium. Multilamellar liposomes form spontaneously when phospholipids are suspended in excess aqueous solution. In some embodiments, the lipid components can rearrange themselves before forming a closed structure and can trap water and dissolved solutes between the lipid bilayers. In some embodiments, the lipid may exhibit a microcellular structure or exist only as heterogeneous aggregates of lipid molecules.

[0299] In some embodiments, a nucleic acid or vector of the invention is introduced into a mammalian host cell, such as an immune-responsive cell of the invention. In some embodiments, the presence of the nucleic acid or vector of the invention in the host cell can be confirmed by any suitable assay known in the art, including but not limited to Southern blot analysis, Northern blot analysis, RT- PCR, PCR, ELISA analysis and western blot analysis.

[0300] In some embodiments, a nucleic acid or vector of the invention is stably transduced into an immune response cell of the invention. In some embodiments, the cells exhibiting stable expression of the nucleic acid or vector express the encoded chimeric receptor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks after transduction. , at least 7 weeks, at least 8 weeks, at least 3 months, at least 6 months, at least 9 months, or at least 12 months.

[0301] In embodiments where the chimeric receptor of the present invention is transiently expressed in cells, the nucleic acid or vector encoding the chimeric receptor of the present invention is transfected into the immune response cells of the present invention. In some embodiments, the immunoreactive cells express the chimeric receptor about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days after transfection. , about 12 days, about 13 days, about 14 days, or about 15 days.

[0302] In some embodiments, the nucleic acid construct encodes a bicistronic chimeric antigen receptor comprising a chimeric receptor and a chimeric inhibitory receptor. In some embodiments, the nucleic acid construct comprises a multicistronic chimeric antigen receptor comprising two or more chimeric receptors and a chimeric inhibitory receptor. In some embodiments, multiple nucleic acid constructs are used, with one construct encoding a chimeric receptor and one construct encoding a chimeric inhibitory receptor.

[0303] In some embodiments, the chimeric receptor binds the CEACAM6 antigen and the chimeric inhibitory receptor binds the VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, or SLC26A3 antigen.

[0304] In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0305] In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM6 antigen.

[0306] In some embodiments, the chimeric inhibitory receptor binds the ITM2C antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds ITM2C antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the ITM2C antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds ITM2C antigen and the chimeric receptor binds CEACAM6 antigen.

[0307] In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0308] In some embodiments, the chimeric inhibitory receptor binds SLC4A4 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC4A4 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the SLC4A4 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the SLC4A4 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0309] In some embodiments, the chimeric inhibitory receptor binds a GPA33 antigen and the chimeric receptor binds a CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0310] In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM6 antigen.

[0311] In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0312] In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0313] In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0314] In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM6 antigen.

[0315] In some embodiments, the encoded bicistronic chimeric antigen receptor system comprises a [surface] CAR of the antigen-binding domain of any antigen provided in [1]. In some embodiments, the encoded bicistronic chimeric antigen receptor system comprises a gene derived from, e.g. [surface] CAR of the antigen-binding domain of the antibody provided in [2]. In some embodiments, the encoded bicistronic chimeric antigen receptor system comprises a dicistronic chimeric antigen receptor system comprising: [surface] The CAR of the antigen-binding domain of scFv provided in [3]. In some embodiments, the encoded bicistronic chimeric antigen receptor system comprises two or more targeted [surface] CAR of the antigen-binding domain of any antigen pairing provided in

[10] .

[0316] In some embodiments, the nucleic acid construct encodes a bivalent chimeric antigen receptor comprising a chimeric receptor and a chimeric inhibitory receptor. In some embodiments, the chimeric receptor binds the CEACAM6 antigen and the chimeric inhibitory receptor binds the VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2, or SLC26A3 antigen.

[0317] In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the VSIG2 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0318] In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds CPM antigen and the chimeric receptor binds CEACAM6 antigen.

[0319] In some embodiments, the chimeric inhibitory receptor binds the ITM2C antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds ITM2C antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the ITM2C antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds ITM2C antigen and the chimeric receptor binds CEACAM6 antigen.

[0320] In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0321] In some embodiments, the chimeric inhibitory receptor binds SLC4A4 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC4A4 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the SLC4A4 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the SLC4A4 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0322] In some embodiments, the chimeric inhibitory receptor binds a GPA33 antigen and the chimeric receptor binds a CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the GPA33 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0323] In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the PLA2G2A antigen and the chimeric receptor binds the CEACAM6 antigen.

[0324] In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEA antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds the ABCA8 antigen and the chimeric receptor binds the CEACAM6 antigen.

[0325] In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds ATP1A2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0326] In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds CHP2 antigen and the chimeric receptor binds CEACAM6 antigen.

[0327] In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEA antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM1 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM5 antigen. In some embodiments, the chimeric inhibitory receptor binds SLC26A3 antigen and the chimeric receptor binds CEACAM6 antigen.

[0328] In some embodiments, the encoded bivalent chimeric antigen receptor comprises a protein that targets [surface] CAR of the antigen-binding domain of any antigen provided in [1]. In some embodiments, the encoded bivalent chimeric antigen receptors comprise genes with a target derived from, e.g. [surface] CAR of the antigen-binding domain of the antibody provided in [2]. In some embodiments, the encoded bivalent chimeric antigen receptors comprise [surface] The CAR of the antigen-binding domain of scFv provided in [3]. In some embodiments, the encoded bivalent chimeric antigen receptor comprises a protein that targets [surface] A CAR of two or more antigen-binding domains for any antigen pairing provided in

[10] . [Pharmaceutical compositions and administration] []

[0329] Certain aspects of the invention relate to compositions (eg, pharmaceutical compositions) comprising one or more chimeric receptors of the invention or immune responsive cells of the invention expressing the one or more chimeric receptors. In some embodiments, compositions comprising chimeric receptors or genetically modified immune-responsive cells expressing such chimeric receptors can be provided systemically or directly to an individual for the treatment of proliferative disorders, such as solid tumors.

[0330] Compositions comprising the genetically modified cells of the present invention may be administered in any physiologically acceptable vehicle, such as intravascularly, but they may also be introduced into bone or the genetically modified cells may be found to be suitable for regeneration and Other suitable sites of differentiation (e.g. thymus). Compositions comprising genetically modified cells of the invention may comprise purified cell populations. Methods for determining the percentage of genetically modified cells in a cell population are well known in the art and include, but are not limited to, fluorescence-activated cell selection (FACS). In some embodiments, the purity of the genetically modified cells in the cell population can be about 50%, about 55%, about 60%, or about 65%, about 70%, about 75%, about 80%, about 80% in the cell population. 85%, about 90%, about 95%, about 97%, about 98%, about 99% or more cells. Dosage can be readily adjusted by those skilled in the art (e.g., reduced purity may require an increase in dose).

[0331] In certain embodiments, the compositions are pharmaceutical compositions comprising genetically modified cells, such as immune response cells or progenitor cells thereof, and a pharmaceutically acceptable carrier. Administration can be autologous or allogeneic. [treatment method] []

[0332] Certain aspects of the invention pertain to methods of using the chimeric receptors of the invention and genetically modified cells expressing such chimeric receptors (eg, immune reactive cells, such as NK cells) to treat individuals in need thereof. In some embodiments, the methods of the invention can be used to treat cancer in an individual, wherein the cancer expresses CEA (is CEA+). In some embodiments, methods of the invention can be used to treat cancer in an individual, wherein the cancer expresses CEACAM1 (is CEACAM1+). In some embodiments, methods of the invention can be used to treat cancer in an individual, wherein the cancer expresses CEACAM5 (is CEACAM5+). In some embodiments, methods of the invention can be used to treat cancer in an individual, wherein the cancer expresses CEACAM6 (is CEACAM6+). In some embodiments, the methods of the invention can be used to treat cancer, such as solid tumors, in an individual.

[0333] In some embodiments, the solid tumors are lung cancer, pancreatic cancer, gastrointestinal cancer, and colon cancer. In some embodiments, the solid tumor is lung adenocarcinoma. In some embodiments, the solid tumor is pancreatic cancer. In some embodiments, the solid tumor is gastrointestinal cancer (including but not limited to esophageal cancer, gastric cancer, small bowel cancer, colon cancer, or rectal cancer). In some embodiments, the solid tumor is colon cancer. In some embodiments, the solid tumor is colorectal cancer.

[0334] In some embodiments, the methods of the present invention may include administering Genetically modified cells of the present invention. In some embodiments, an effective amount may be provided in one or a series of administrations of the genetically modified cells (eg, immune responsive cells, eg, NK cells) of the invention. In some embodiments, an effective amount may be provided in bolus form or by continuous infusion.

[0335] As disclosed herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to achieve a beneficial or desired clinical result following treatment. An effective amount can be administered to an individual in one or more doses. For treatment, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse or slow the progression of the disease or otherwise lessen the pathological consequences of the disease. Effective amounts are generally determined by the physician on a case-by-case basis and are within the capabilities of those skilled in the art. Several factors are typically considered in determining the appropriate dosage to achieve an effective amount. Such factors include the age, sex, and weight of the individual, the condition being treated, the severity of the condition, and the form and effective concentration of the immunoreactive cells administered.

[0336] In some embodiments, the method reduces the number of tumor cells. In some embodiments, the method reduces tumor size. In some embodiments, the method reduces tumor volume. In some embodiments, the method increases the progression-free survival time of the individual. In some embodiments, the method increases the survival time of the individual. therapeutic treatment

[0337] In some embodiments, the methods of the invention increase the immune response in an individual in need thereof. In some embodiments, the methods of the invention include methods for treating and / or preventing solid tumors in an individual. In some embodiments, the individual is a human. In some embodiments, human subjects amenable to therapy may include two treatment groups distinguishable by clinical criteria. Individuals with "advanced disease" or "high tumor burden" are those with clinically measurable tumors. A clinically measurable tumor is one that can be detected based on tumor mass (e.g., based on percentage of tumor cells, by palpation, CAT scan, sonogram, mammography, or X-ray; autopositive biochemical or histopathological markers are insufficient to identify this group). In some embodiments, the pharmaceutical compositions of the invention are administered to such individuals to elicit an anti-tumor response with the goal of alleviating their condition. In some embodiments, tumor mass reduction occurs as a result of administration of the pharmaceutical composition, but any clinical improvement will constitute a benefit. In some embodiments, clinical improvement includes reducing tumor risk or rate of progression or mitigating its pathological consequences. In some embodiments, the second group of suitable human subjects is an "adjuvanted group" of subjects. Such individuals are those with a history of solid tumors who have responded to another treatment modality. Prior therapy may include, but is not limited to, surgical resection, radiation therapy, and / or traditional chemotherapy. Therefore, these individuals do not have clinically measurable tumors. However, it is suspected to be at risk of disease progression near the original tumor site or due to metastasis. In some embodiments, this group can be further subdivided into high-risk individuals and low-risk individuals. Segmentation can be based on characteristics observed before and after initial treatment. These characteristics are known in the clinical field and are appropriately defined for each different solid tumor. The high-risk subgroup is typically characterized by tumor invasion of adjacent tissues or subgroups showing lymph node involvement.

[0338] In any and all aspects of increasing an immune response as described herein, any increase or decrease or alteration of a characteristic or aspect of function is compared to cells that have not been contacted with an immune responsive cell as described herein.

[0339] Increasing an immune response can be enhancing an immune response or inducing an immune response. For example, increasing an immune response encompasses initiating or initiating an immune response, or increasing or amplifying an ongoing or existing immune response. In some embodiments, the treatment induces an immune response. In some embodiments, the induced immune response is an adaptive immune response. In some embodiments, the induced immune response is an innate immune response. In some embodiments, the treatment enhances the immune response. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response. In some embodiments, the treatment increases the immune response. In some embodiments, the increased immune response is an adaptive immune response. In some embodiments, the increased immune response is an innate immune response.

[0340] In some embodiments, another group of individuals are those individuals who have a genetic predisposition to a solid tumor disorder but do not yet have evidence of clinical signs of a solid tumor. For example, women who test positive for gene mutations associated with cancers of the female reproductive organs (e.g., breast cancer, ovarian cancer) but are still of reproductive age may benefit from receiving one or more cells of the invention prophylactically at the time of treatment ( Such as immune response cells, such as NK cells) to prevent the occurrence of cancer of the female reproductive organs until it is suitable for preventive surgery. In some embodiments, an individual may have an advanced form of the disease, in which case treatment goals may include reducing or reversing disease progression and / or ameliorating side effects. In some embodiments, the individual may have a history of the condition and has been treated, in which case the goals of treatment typically may include reducing or delaying the risk of relapse.

[0341] In some embodiments, the activity of a CEA CAR (eg, CEACAM1 CAR, CEACAM5 CAR, or CEACAM6 CAR) is inhibited. In some embodiments, the activity of an immune cell expressing a CEA CAR (eg, CEACAM1 CAR, CEACAM5 CAR, or CEACAM6 CAR) is inhibited. In certain embodiments, the activity of a CEA CAR is inhibited using an inhibitory chimeric receptor. In certain embodiments, the activity of an immune cell expressing a CEA CAR is inhibited using an inhibitory chimeric receptor. In some embodiments, the inhibitory chimeric receptor binds a VSIG2 antigen. In some embodiments, the inhibitory chimeric receptor binds a CPM antigen. In some embodiments, the inhibitory chimeric receptor binds an ITM2C antigen. In some embodiments, the inhibitory chimeric receptor binds the SLC26A2 antigen. In some embodiments, the inhibitory chimeric receptor binds the SLC4A4 antigen. In some embodiments, the inhibitory chimeric receptor binds the GPA33 antigen. In some embodiments, the inhibitory chimeric receptor binds the PLA2G2A antigen. In some embodiments, the inhibitory chimeric receptor binds the ABCA8 antigen. In some embodiments, the inhibitory chimeric receptor binds the ATP1A2 antigen. In some embodiments, the chimeric receptor inhibits binding to CHP2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the SLC26A3 antigen. In some embodiments, the chimeric receptor is a multispecific receptor comprising two or more antigen-binding domains, such that the chimeric receptor can bind two or more antigens. combination therapy

[0342] In some embodiments, genetically modified cells of the invention (eg, immune response cells, such as NK cells) expressing one or more chimeric receptors of the invention can be used in combination with other known agents and therapies. In some embodiments, combination therapies of the invention comprise genetically modified cells of the invention that can be administered in combination with one or more other therapeutic agents. In some embodiments, genetically modified cells and one or more other therapeutic agents can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the genetically modified cells can be administered first, followed by one or more other agents, or the order of administration can be reversed. In some embodiments, genetically modified cells are further modified to express one or more additional therapeutic agents. [Set] []

[0343] Certain aspects of the invention relate to kits for the treatment and / or prevention of solid tumors. In certain embodiments, the kit includes a therapeutic or prophylactic composition comprising an effective amount of one or more chimeric receptors of the invention, an isolated nucleic acid of the invention, a vector of the invention, and / or Cells of the invention (e.g. immune response cells). In some embodiments, the kit includes a sterile container. In some embodiments, such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing the drug.

[0344] In some embodiments, therapeutic or prophylactic compositions are provided for use in administering therapeutic or prophylactic compositions to individuals with or at risk of developing solid tumors, e.g., for the treatment and / or prevention of colorectal cancer, pancreatic cancer, cancer, lung adenocarcinoma and / or gastric cancer. In some embodiments, instructions may include information regarding use of the composition to treat and / or prevent a condition. In some embodiments, the instructions include, but are not limited to, a description of the therapeutic or prophylactic composition, dosage schedule, dosing schedule for treating or preventing the condition or symptoms thereof, precautions, warnings, indications, counter-indications, Overdose information, adverse reactions, animal pharmacology, clinical studies and / or references. In some embodiments, instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, brochure, card, or folder provided in or with the container. [Example] [] 1. An inhibitory chimeric receptor comprising an extracellular antigen-binding domain that binds to an antigen selected from the group consisting of: VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2 and SLC26A3. 2. Inhibiting chimeric receptors as in Example 1, wherein the antigen is VSIG2. 3. Inhibiting chimeric receptors as in embodiment 1, wherein the antigen is CPM. 4. Inhibiting chimeric receptors as in embodiment 1, wherein the antigen is ITM2C. 5. Inhibiting chimeric receptors as in Example 1, wherein the antigen is SLC26A2. 6. Inhibiting chimeric receptors as in embodiment 1, wherein the antigen is SLC4A4. 7. Inhibiting the chimeric receptor as in embodiment 1, wherein the antigen is GPA33. 8. Inhibiting chimeric receptors as in embodiment 1, wherein the antigen is PLA2G2A. 9. Inhibiting chimeric receptors as in embodiment 1, wherein the antigen is ABCA8. 10. Inhibiting chimeric receptors as in embodiment 1, wherein the antigen is ATP1A2. 11. Inhibiting chimeric receptors as in embodiment 1, wherein the antigen is CHP2. 12. Inhibiting chimeric receptors as in embodiment 1, wherein the antigen is SLC26A3. 13. The inhibitory chimeric receptor of any one of embodiments 1 to 12, wherein when expressed on a cell, the inhibitory chimeric receptor inhibits one or more activities of the cell. 14. The inhibitory chimeric receptor of any one of embodiments 1 to 13, wherein the antigen is not expressed on tumor cells, or the antigen is expressed on tumor cells at a lower level than the expression on non-tumor cells. 15. The inhibitory chimeric receptor of any one of embodiments 1 to 14, wherein the antigen is expressed on non-tumor cells, or the antigen is expressed on non-tumor cells at a level higher than that on the corresponding tumor cells superior. 16. The inhibitory chimeric receptor of embodiment 15, wherein the antigen is expressed on non-tumor cells, and the non-tumor cell line is derived from a tissue selected from the group consisting of: lung, pancreas, gastrointestinal tract, colon , brain, neuronal tissue, endocrine, bone, bone marrow, immune system, muscle, liver, gallbladder, kidneys, bladder, male reproductive organs, female reproductive organs, fat, soft tissue and skin. 17. The inhibitory chimeric receptor of any one of embodiments 1 to 16, wherein the inhibitory chimeric receptor comprises one or more intracellular inhibition domains, and the one or more intracellular inhibition domains are selected from the following composition Group: PD-1, CTLA4, TIGIT, LAIR1, GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, BTLA, LIR1, NKG2A, KIR3DL1, GITR, PD-L1, CSK, SHP-1 , PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, RasGAP, CD94 and CD161. 18. The inhibitory chimeric receptor of any one of embodiments 1 to 17, wherein the antigen-binding domain comprises one or more antibodies, antigen-binding fragments of antibodies, F(ab) fragments, F(ab') fragments, Single chain variable fragment (scFv) or single domain antibody (sdAb). 19. The inhibitory chimeric receptor of any one of embodiments 1 to 18, wherein the antigen binding domain comprises one or more single chain variable fragments (scFv). 20. The inhibitory chimeric receptor of embodiment 19, wherein the one or more scFvs each comprise a heavy chain variable domain (VH) and a light chain variable domain (VL). 21. The inhibitory chimeric receptor of embodiment 20, wherein the VH and the VL are separated by a peptide linker. 22. The inhibitory chimeric receptor of embodiment 21, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 77. 23. The inhibitory chimeric receptor of any one of embodiments 20 to 22, wherein the one or more scFvs each comprise the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain , L is the peptide linker, and VL is the light chain variable domain. 24. An isolated cell comprising an inhibitory chimeric receptor as in any one of embodiments 1 to 23. 25. The isolated cell of embodiment 24, wherein the inhibitory chimeric receptor system recombinantly expresses. 26. The isolated cell of embodiment 24 or embodiment 25, wherein the inhibitory chimeric receptor system is expressed by a vector or a selected locus from the genome of the cell. 27. The isolated cell of any one of embodiments 24 to 26, wherein the cell further comprises a chimeric receptor comprising one or more extracellular antigen binding domains, wherein the one or more extracellular antigen binding domains Binds to one or more antigens selected from the group consisting of: CEA, CEACAM1, CEACAM5 and CEACAM6. 28. An isolated cell comprising: (a) an inhibitory chimeric receptor comprising an extracellular antigen binding domain that binds to an antigen selected from the group consisting of: VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, ATP1A2, CHP2 and SLC26A3; and (b) A chimeric receptor comprising one or more extracellular antigen binding domains, wherein the one or more extracellular antigen binding domains bind one or more additional antigens selected from the group consisting of The group consisting of: CEA, CEACAM1, CEACAM5 and CEACAM6. 29. The isolated cell of embodiment 27 or embodiment 28, wherein the chimeric receptor is a chimeric T cell receptor or a chimeric antigen receptor (CAR). 30. The isolated cell of embodiment 29, wherein the chimeric receptor is a CAR. 31. The isolated cell of embodiment 30, wherein the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from the group consisting of: CD3ζ chain intracellular signaling Transduction domain, CD3ε chain intracellular signaling domain, CD97 intracellular signaling domain, CD11a-CD18 intracellular signaling domain, CD2 intracellular signaling domain, ICOS intracellular signaling domain, CD27 intracellular signaling domain, CD154 cells Intracellular signaling domain, CD8 intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, ZAP40 intracellular signaling domain, CD30 intracellular signaling domain, GITR Intracellular signaling domain, HVEM intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, MyD88 intracellular signaling domain, 2B4 intracellular signaling domain, NKp46 intracellular signaling domain, NKp30 cellular Intracellular signaling domain, NKp44 intracellular signaling domain, NKG2D intracellular signaling domain, CD226 intracellular signaling domain and CD160 intracellular signaling domain. 32. The isolated cell of embodiment 30 or embodiment 31, wherein the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain , CD25 transmembrane domain, CD7 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain Membrane domain, LAX transmembrane domain, LAT transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, TIM3 transmembrane domain, KIR3DS1 transmembrane domain, KIR3DL1 transmembrane domain, NKG2D transmembrane domain, NKG2A transmembrane domain, TIGIT transmembrane domain, 2B4 transmembrane domain and BTLA transmembrane domain. 33. The isolated cell of any one of embodiments 30 to 32, wherein the CAR comprises a spacer, the spacer is between the antigen binding domain and the transmembrane domain, and the spacer is selected from The amino acid sequence of the group consisting of SEQ ID NO: 49-58. 34. The isolated cell of any one of embodiments 27 to 33, wherein the inhibitory chimeric receptor and / or the antigen binding domain of the chimeric receptor comprises one or more antibodies, antigen binding fragments of antibodies , F(ab) fragment, F(ab') fragment, single chain variable fragment (scFv) or single domain antibody (sdAb). 35. The isolated cell of any one of embodiments 27 to 33, wherein the inhibitory chimeric receptor and / or the antigen binding domain of the chimeric receptor comprises one or more single chain variable fragments (scFv ). 36. The isolated cell of embodiment 35, wherein the one or more scFvs each comprise a heavy chain variable domain (VH) and a light chain variable domain (VL). 37. The isolated cell of embodiment 36, wherein the VH and the VL are separated by a peptide linker. 38. The isolated cell of embodiment 37, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 77. 39. The isolated cell of any one of embodiments 36 to 38, wherein each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. 40. The isolated cell of any one of embodiments 28 to 39, wherein the one or more additional antigens are CECAM1. 41. The isolated cell of embodiment 40, wherein the antigen binding domain that binds to CEACAM1 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 Amino acid sequence of the light chain variable domain (VL). 42. The isolated cell of any one of embodiments 28 to 39, wherein the one or more additional antigens are CECAM5. 43. The isolated cell of embodiment 42, wherein the antigen-binding domain that binds to CEACAM5 comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) VH comprising the amino acid sequence of SEQ ID NO: 3 and VL comprising the amino acid sequence of SEQ. ID NO: 4; (b) VH comprising the amino acid sequence of SEQ ID NO: 9 and VL comprising the amino acid sequence of SEQ. ID NO: 10; (c) VH comprising the amino acid sequence of SEQ ID NO: 11 and VL comprising the amino acid sequence of SEQ. ID NO: 12; (d) VH comprising the amino acid sequence of SEQ ID NO: 13 and VL comprising the amino acid sequence of SEQ. ID NO: 14; (e) VH comprising the amino acid sequence of SEQ ID NO: 78 and VL comprising the amino acid sequence of SEQ. ID NO: 79; and (f) VH comprising the amino acid sequence of SEQ ID NO: 15 and VL comprising the amino acid sequence of SEQ. ID NO: 16. 44. The isolated cell of embodiment 42, wherein the antigen-binding domain that binds to CEACAM5 comprises a heavy chain (HC) and a light chain (LC) selected from the group consisting of: (a) HC comprising the amino acid sequence of SEQ ID NO: 5 and LC comprising the amino acid sequence of SEQ. ID NO: 6; and (b) HC comprising the amino acid sequence of SEQ ID NO: 7 and LC comprising the amino acid sequence of SEQ. ID NO: 8. 45. The isolated cell of any one of embodiments 28 to 39, wherein the one or more additional antigens are CECAM6. 46. ​​The isolated cell of embodiment 45, wherein the antigen binding domain bound to CEACAM6 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 18 Amino acid sequence of the light chain variable domain (VL). 47. The isolated cell of any one of embodiments 28 to 39, wherein the cell is an immunoreactive cell. 48. The isolated cell of any one of embodiments 28-47, wherein the inhibition of chimeric receptor binding to the antigen is capable of inhibiting the immune response cell. 49. The isolated cell of any one of embodiments 28-48, wherein binding of the chimeric receptor to the one or more additional antigens is capable of activating the immune reactive cell. 50. The isolated cell of any one of embodiments 28-49, wherein the chimeric receptor binds to the one or more additional antigens with low binding affinity. 51. The isolated cell of any one of embodiments 28 to 50, wherein the chimeric receptor binds to the one or more antigens with a lower binding affinity than the binding affinity that inhibits binding of the chimeric receptor to the antigen Additional antigens. 52. The isolated cell of any one of embodiments 28-51, wherein the inhibitory chimeric receptor binds to the antigen with low binding affinity. 53. The isolated cell of any one of embodiments 27 to 52, wherein the chimeric receptor is expressed recombinantly. 54. The isolated cell of any one of embodiments 27 to 53, wherein the chimeric receptor is expressed by a vector or a selected locus from a gene body of the cell. 55. The isolated cell of any one of embodiments 24 to 54, wherein the cell line is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells cells, natural killer T (NKT) cells, bone marrow cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells and induced pluripotent stem cells (iPSCs), and iPSC-derived cells. 56. The isolated cell of any one of embodiments 24 to 55, wherein the cell is autologous. 57. The isolated cell of any one of embodiments 24 to 55, wherein the cell is allogeneic. 58. An isolated nucleic acid encoding the inhibitory chimeric receptor of any one of embodiments 1-23. 59. A carrier comprising the nucleic acid as in embodiment 58. 60. A genetically modified cell comprising a nucleic acid such as embodiment 58 or a carrier such as embodiment 59. 61. A method of treating an individual in need thereof, the method comprising administering the isolated cells of any one of embodiments 24-57. 62. A method of stimulating a cell-mediated immune response against tumor cells in an individual, the method comprising administering to an individual having a tumor the isolated cells of any one of embodiments 24-57. 63. A method of providing anti-tumor immunity in an individual, the method comprising administering to an individual in need thereof the isolated cells of any one of embodiments 24-57. 64. A method of reducing tumor burden in an individual, the method comprising administering to the individual the isolated cells of any one of embodiments 24-57. 65. The method of embodiment 64, wherein the method reduces the number of tumor cells. 66. The method of embodiment 64, wherein the method reduces tumor size. 67. The method of embodiment 64, wherein the method reduces tumor volume. 68. The method of embodiment 64, wherein the method eliminates the tumor in the individual. 69. A method of treating an individual suffering from a tumor, the method comprising administering the isolated cells of any one of embodiments 24 to 57. 70. A method of treating or preventing colorectal cancer in an individual, the method comprising administering to the individual the isolated cells of any one of embodiments 24-57. 71. A method of treating or preventing pancreatic cancer in an individual, the method comprising administering to the individual the isolated cells of any one of embodiments 24-57. 72. A method of treating or preventing lung adenocarcinoma in an individual, the method comprising administering to the individual the isolated cells of any one of embodiments 24-57. 73. A method of treating or preventing gastric cancer in an individual, the method comprising administering to the individual the isolated cells of any one of embodiments 24 to 57. 74. The method of any one of embodiments 61 to 73, wherein the isolated cell lines are administered in an effective amount. 75. The method of any one of embodiments 69 to 74, wherein the method increases progression-free survival time of the individual. 76. The method of any one of embodiments 69 to 75, wherein the method increases survival time of the individual. 77. A pharmaceutical composition comprising an effective amount of the isolated cells of any one of embodiments 24 to 57 and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof . 78. The pharmaceutical composition according to embodiment 77, wherein the pharmaceutical composition is used for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma and / or gastric cancer. 79. A set for the treatment and / or prevention of colorectal cancer, pancreatic cancer, lung adenocarcinoma and / or gastric cancer, comprising the isolated cells as in any one of embodiments 24 to 57 or as implemented The pharmaceutical composition of Example 77. 80. The kit of embodiment 79, wherein the kit further comprises written instructions for using the isolated cells to treat and / or prevent colorectal cancer, pancreatic cancer, lung adenocarcinoma and / or gastric cancer in an individual. 81. A kit for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma and / or gastric cancer, comprising the isolated nucleic acid of embodiment 58 or the vector of embodiment 59. 82. The kit of embodiment 81, wherein the kit further comprises a method for using the nucleic acid to produce one or more methods for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma and / or gastric cancer in an individual. Written instructions for antigen-specific cells. Example

[0345] The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be practiced given the general description provided herein.

[0346] The following are examples of specific embodiments for carrying out the claimed subject matter. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but a certain degree of experimental error and deviation should of course be allowed. [example] [1] [:] [Bioinformatics screening for solid tumor antigens] []

[0347] CEA was selected for further study due to its established role as a tumor antigen coupled with its limited therapeutic efficacy due to on-target and off-tissue toxicity. Analyze the performance levels of CEACAM1, CEACAM5 and CEACAM6 in microarray data, RNA-seq data and proteomic data from selected solid tumor and normal tissue samples, as well as data from off-target tissues. [picture] A comparative analysis of CEA family members is depicted in [1]. Analysis of data from The Cancer Genome Atlas (TCGA) showed that CEACAM5 and CEACAM6 expression was higher in both colorectal cancer (CRC) and normal tissue ( [picture] [2]). This analysis allows comparisons between different groups. The y-axis of the log2 scale allows determination of relative differences in performance between different tumors and normal tissue. These findings were obtained through bioinformatics analysis of patients using the RNA dataset provided by single cell RNAseq of colon cancer and healthy colon as disclosed in Lee et al. (2020) Nat Genetics, 52(1):56-73. confirmed that CEACAM5 expression was shown in [picture] [3A] and CEACAM6 expression is shown in [picture] In [3B], it is revealed that the performance between cancer cells and normal cells is comparable. Protein analysis in normal and cancer tissues was evaluated by immunohistochemical staining of tissue microarrays, and the expression of CEA family proteins was confirmed at the protein level ( [picture] [4]). Although expression is also found to be higher in normal tissues, the high level of CEACAM family members in solid tissue types highlights CEACAM1, CEACAM5 and CEACAM6 as suitable targets for solid tumor therapy. For further systematic validation, a set of human colon cancer cell lines were purchased from ATCC, and flow cytometry was used to determine the performance of different CEACAM isoforms (CEA family members: CEACAM1, CEACAM5 and CEACAM6) ( [picture] [5]). [example] [2] [:for inhibitory antigen target] [(] [NOT]" [Target] [)] [Biological information screening] []

[0348] Then to [example] The CEACAM1, CEACAM5 and CEACAM6 antigens identified in [1] were matched for NOT gate selection. Using scRNA sequencing data, NOT targets were identified using the following criteria: NOT targets have lower representation in solid tumor tissue and higher representation in the tissue of interest. Potential NOT antigens were identified by differential gene expression (DEG) analysis of scRNAseq data. Targets were initially filtered by log fold change (LFC) >1 healthy:tumor tissue. Targets were then filtered and only putative membrane or cell surface proteins were considered. These were then manually prioritized and investigated based on literature searches etc. Similarly, suitable NOT targets were confirmed by examining the expression relative to the solid tumor antigens CEACAM1, CEACAM5 or CEACAM6. Selected NOT targets have low expression in tumor cells and high expression in healthy epithelial cells when antigen expression in solid tumors is high.

[0349] "NOT" gating targets identified by this strategy include VSIG2, CPM, ITM2C, SLC26A2, SLC4A4, GPA33, PLA2G2A, ABCA8, and ATP1A2. "NOT" targets are also described in [surface] [9] in.

[0350] For ABCA8 ( [picture] [6A]) and ATP1A2 ( [picture] [6B]), showing RNA analysis from the TCGA dataset. Single cell RNA sequencing (scRNAseq) results for ABCA8 are shown in [picture] [7A] and [picture] [7B].

[0351] Using those provided in Li et al. (2017) Nat. Genetics, 11(23):6861-6873 (GSE81861), Lee et al. (2020) Nat. Genetics, 52(1):56-73 (GSE132465) and GSE144735 The following RNA data sets were analyzed for VSIG2 expression in tumor cells and various normal cells, wherein the analysis was performed as described in Example 1 above. like [picture] As shown in [8], VSIG2 expression was not detected in tumor cells. In contrast, normal tissues showed detectable expression of VSIG2 in the four subsets of normal cells examined, demonstrating that VSIG2 meets the criteria for being a NOT target. In an independent scRNAseq dataset, we further analyzed the specific expression of VSIG2 expression in the tissue epithelial compartment. Specific cell subpopulations of scRNAseq VSIG2 expression were identified by overlay using principal component analysis (PCA). like [picture] [9A] to [picture] As shown in [9B], VSIG2 expression was higher in a subset of normal epithelial compartments ( [picture] [9A] outline area, left panel), while in tumor cell epithelial compartment subpopulation ( [picture] [9A] is lower in the outlined region, right panel) and is lower throughout the tumor tissue, again demonstrating that VSIG2 meets the criteria for being a NOT target.

[0352] Comparative gene expression studies of VSIG2 and CEACAM5 were also examined in normal and tumor tissues. like [picture] [10A] to [picture] As shown in [10E], this analysis demonstrated an inverse correlation of performance across a number of tissue types. A similar pattern of expression was observed for VSIG2 when compared to CEACAM6. The scRNAseq analysis was also performed on many cell types and tissues for VSIG2, and the analysis was consistent with the results described above ( [picture]

[11] ). and [picture] [10A] to [picture] The comparative performance analysis in [10E] is consistent, and VSIG2 is related to the expression of CEACAM5 and CEACAM6 in healthy lung tissue, as shown in [picture] shown in [12A].

[0353] Determination of putative VSIG2 protein expression using tissue microarrays with multiple gastrointestinal tumor and healthy tissue samples ( [picture] [13A]). VSIG2 protein expression was determined by immunohistochemistry (IHC) using two different commercially available antibodies (monoclonal antibody OTI2D8 and polyclonal antibody ab252969). Representative images of healthy intestinal mucosa showing positive VSIG2 immunoreactivity in epithelial cells ( [picture] [13B] to [picture] [13C]). Co-localization of aCAR target CEACAM5 and iCAR target VSIG2 in healthy gastrointestinal epithelial cells was observed using multiplex IHC ( [picture] [14A] to [picture] [14B]). Importantly, co-localization of the aCAR target CEACAM5 and the iCAR target VSIG2 was not observed in grade 3 stage IIB colon cancer samples, where most VSIG2 signals were observed to be low or nucleated, and thus the iCAR NOT gate technique would not confers protection on cancerous tissue, but will specifically protect healthy tissue ( [picture]

[15] ). Co-expression of aCAR target (CEACAM5) and iCAR target (VSIG2) in the stromal compartment and immune cells in colon samples was determined by analyzing scRNAseq data from two datasets ( [picture] [16A] to [picture] [16C]).

[0354] Gene expression of another potential NOT target CPM was analyzed similarly. In colon cancer and normal tissues ( [picture] [17A]) and normal lung tissue ( [picture] [17B]) for performance analysis. The comparative gene expression study of CPM and CEACAM5 and CEACAM6 in normal and tumor tissues showed an inverse correlation, such as [picture] shown in [17C].

[0355] Putative gene expression analysis was also performed on NOT candidate antigens to determine expression in normal and tumor tissues: GPA33 ( [picture] [18A-] [picture] [18B]), PLA2G2A ( [picture] [19A] [to picture] [19B]), ITM2C ( [picture] [20A] [to picture] [20B]), CHP2 ( [picture] [21A] [to picture] [21B]), SLC26A2 ( [picture] [22A] [to picture] [22B]), SLC4A4 ( [picture] [23A] [to picture] [23B]) and SLC26A3 ( [picture] [24A] [to picture] [24B]). [example] [3] [:Performance] [CEA aCAR] [Of] [T] [Cells exhibit increased cytokines] [ / ] [Chemokine induction and target cell killing] []

[0356] CAR expression, cytokine expression, and target cell killing of T cells transduced with a construct encoding a CEA-activated CAR ("aCAR") lentivirus were assessed. Naive donor T cells from two different donors transduced with CEA CAR constructs including CD28 co-stimulatory domain, CD3ζ intracellular domain, and various anti-CEA scFvs (derived from hMN14, hMFE23, MG7, and MRG1) to test. Each CAR construct encodes in the N-terminal to C-terminal direction a CD8 signal sequence (SEQ ID NO: 76), a scFv domain (i.e., "binder"), a Myc epitope tag (SEQ ID NO: 75), CD8 hinge (SEQ ID NO: 50), transmembrane domain derived from CD28 (SEQ ID NO: 46) or OX40 (SEQ ID NO: 47), derived from CD28 (SEQ ID NO: 71) or OX40 (SEQ ID NO: 72), and the CD3ζ activation domain (SEQ ID NO: 73). A description of each binder, transmembrane domain, and co-stimulatory domain used in each construct is provided in [surface]

[12] . T cells were transduced with an MOI of lentivirus / T cells of 0.6 or 0.3 pg (as measured by p24 assay). Naive T cells were obtained from two different donors (designated "Donor 1" and "Donor 2") and used to assess construct transduction efficiency. CAR expression was assessed by staining for the presence of an epitope tag (Myc) via flow cytometry. Cells were stained at 4 and 7 days post-transduction to determine the stability of construct expression. A gate for determining % Myc expression was drawn using a non-transduced control (no virus) such that <1% Myc+ cells were in the positive gate. The median fluorescence intensity (MFI) was determined to quantify and assess CAR performance levels. The expression levels of each CAR in donor 1 and donor 2 T cells on day 4 are shown in [surface]

[13] . The expression levels of each CAR in donor 1 and donor 2 T cells on day 8 are shown in [surface]

[14] . [surface]

[12] [construct] [ID] [Binding agent] [(scFv)] [,] [Orientation and] [SEQ ID NO] [TM] [and co-stimulatory domain] [SB02463] hMN14 VL-VH (SEQ ID NO: 19) CD28 [SB02465] hMFE23 VH-VL (SEQ ID NO: 20) [SB02467] MG7 VL-VH (SEQ ID NO: 21) [SB02470] MRG1 VH-VL (SEQ ID NO: 22) [SB02779] hMN14 L / H (SEQ ID NO: 19) OX40 [SB02782] hMFE23 VH-VL (SEQ ID NO: 20) [SB02785] MG7 L / H (SEQ ID NO: 21) [SB02788] MRG1 H / L (SEQ ID NO: 22) [surface]

[13] [No.] [4] [sky] [Construction] [ID] [MOI (pg)] [Myc+%] [Myc+] [of cells] [MFI] SB02463 0.6 67.1 39083 [Donor] [1] SB02465 0.6 93.2 56281 SB02467 0.6 71.1 14712 SB02470 0.6 77.8 28766 SB02476 0.6 71.2 5370 virus free 0 0.47 2680 SB02463 0.3 60.1 36351 SB02465 0.3 91 47832 SB02467 0.3 67.8 12812 SB02470 0.3 73.2 26101 SB02476 0.3 64 4950 SB02463 0.6 58.9 40008 [Donor] [2] SB02465 0.6 89.9 48857 SB02467 0.6 62 13511 SB02470 0.6 77.9 26231 SB02476 0.6 70.1 5392 virus free 0 0.89 2329 SB02463 0.3 53.4 37398 SB02465 0.3 89.9 49248 SB02467 0.3 72.1 17351 SB02470 0.3 74.2 25653 SB02476 0.3 63.1 4910 [surface]

[14] [No.] [8] [sky] [Construction] [ID] [MOI] [Myc+%] [Myc+] [of cells] [MFI] SB02463 0.6 65 40931 [Donor] [1] SB02465 0.6 93.3 37665 SB02467 0.6 81.8 23874 SB02470 0.6 74.5 31633 SB02476 0.6 87.2 6797 virus free 0 0.21 2557 SB02463 0.3 58.3 40180 SB02465 0.3 91.1 33318 SB02467 0.3 78.9 22621 SB02470 0.3 71 29855 SB02476 0.3 83.1 6449 SB02463 0.6 61.9 71269 [Donor] [2] SB02465 0.6 94.1 68204 SB02467 0.6 72.1 41184 SB02470 0.6 81.7 65274 SB02476 0.6 93.7 12608 virus free 0 0.34 2389 SB02463 0.3 54.7 57595 SB02465 0.3 93.6 56880 SB02467 0.3 80.4 38961 SB02470 0.3 79.1 60171 SB02476 0.3 92.3 11386

[0357] The killing activity of T cells transduced with CEA aCAR was assessed. T cells transduced with CEA aCAR were incubated overnight (16 to 18 hours) with target cells of a colon cancer cell line (LS174t) at a 2:1 E:T ratio. Ls174t target cell lines include "parental" LS174t cells (left panel) and "mKate" cell lines that have been transduced to express the fluorescent reporter gene mKate (right panel). Potential CAR activity was assessed using two T cell donors, Donor 1 and Donor 2. Cell culture supernatants were collected and tested for killing. To assess cell death due to killing activity, LDH levels in cell culture supernatants were measured. Increased LDH release indicates target cell killing. LDH activity was measured following the manufacturer's instructions (CyQUANT LDH Cytotoxicity Assay, Thermo Fisher). Maximal killing activity of chemically lysed target cells is determined, while spontaneous LDH release from individual target cells is determined. Calculation of parental target cells co-cultured with construct-transduced Donor 1 T cells ( [picture] [25A]), mKate target cells co-cultured with construct-transduced Donor 1 T cells ( [picture] [25B]), parental target cells co-cultured with construct-transduced donor 2 T cells ( [picture] [26A]), and mKate target cells co-cultured with construct-transduced donor 2 T cells ( [picture] [26B]) (as a percentage of maximum LDH release). All CEA aCARs exhibited significant killing activity relative to untransduced controls (no virus), indicating CAR-mediated killing of homologous target cells. Killing activity was comparable in the two target cell lines tested.

[0358] The interleukin activity of CEA aCAR-transduced T cells was assessed. Together with as measured by LDH release and shown in [picture] [25A], [picture] [25B], [picture] [26A] and [picture] Along with the killing assay in [26B], T cell interleukin activity of the same cell culture supernatants was also assessed. Cell culture supernatants collected from the killing assay using Donor 2 were assessed using Luminex. Since the difference in the parental target cell comparison performance of mkate's target cell lines in the LDH analysis was minimal, the two target cell lines in the same group were evaluated as technically repeated experiments. A multiplex immunoassay (ProcartaPlex, Thermo Fisher) was used to measure IL-2 ( [picture] [27A]), IFNγ ( [picture] [27B]), TNFα ( [picture] [27C]), caspase 3 ( [picture] [27D]), perforin ( [picture] [27E]) and granzyme B ( [picture] [27F]) to further evaluate T cell activity. like [picture] [27A] [to picture] As shown in [27F], T cells expressing CEA aCAR exhibited significantly higher interleukin and chemokine activities than untransduced control T cells (without virus), indicating that CAR-mediated T cells recognize homologous target cells. Cell activation. [Example] [4] [: Lentiviral Transduction] [CEA aCAR NK] [Cells display increased target...

Claims

1. An isolated cell comprising: (a) an inhibitory chimeric receptor comprising an extracellular antigen-binding domain that binds to an antigen, wherein the antigen is VSIG2; and (b) a chimeric receptor comprising one or more extracellular antigen-binding domains, wherein the one or more extracellular antigen-binding domains bind CEACAM5.

2. The separated cells as described in claim 1, wherein: (a) The antigen-binding domain of the inhibitory chimeric receptor comprises one or more single-stranded variable fragments (scFv); (b) The inhibitory chimeric receptor comprises a transmembrane domain selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain, CD25 transmembrane domain, CD7 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, LAX transmembrane domain, LAT transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, TIM3 transmembrane domain, KIR3DS1 transmembrane domain, KIR3DL1 transmembrane domain, NKG2D transmembrane domain, NKG2A transmembrane domain, TIGIT transmembrane domain, 2B4 transmembrane domain, and BTLA transmembrane domain; (c) The inhibitory chimeric receptor includes a spacer region between the antigen-binding domain and the transmembrane domain; and / or (d) the inhibitory chimeric receptor includes one or more intracellular inhibitory domains selected from the group consisting of: PD-1, CTLA4, TIGIT, LAIR1, GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, BTLA, LIR1, NKG2A, KIR3DL1, GITR, PD-L1, CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, RasGAP, CD94, and CD161.

3. The isolated cells of claim 1, wherein the chimeric receptor is a chimeric antigen receptor (CAR), comprising: (a) one or more intracellular signaling domains, wherein the one or more intracellular signaling domains are selected from the group consisting of: CD3ζ chain intracellular signaling domain, CD3ε chain intracellular signaling domain, CD97 intracellular signaling domain, CD11a-CD18 intracellular signaling domain, CD2 intracellular signaling domain, ICOS intracellular signaling domain, CD27 intracellular signaling domain, CD154 intracellular signaling domain, CD8 intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, CD40 ... Intracellular signal transduction domains: ZAP40 intracellular signal transduction domain, CD30 intracellular signal transduction domain, GITR intracellular signal transduction domain, HVEM intracellular signal transduction domain, DAP10 intracellular signal transduction domain, DAP12 intracellular signal transduction domain, MyD88 intracellular signal transduction domain, 2B4 intracellular signal transduction domain, NKp46 intracellular signal transduction domain, NKp30 intracellular signal transduction domain, NKp44 intracellular signal transduction domain, NKG2D intracellular signal transduction domain, CD226 intracellular signal transduction domain, and CD160 intracellular signal transduction domain; (b) A transmembrane domain selected from the group consisting of: CD8 transmembrane domain, CD28 transmembrane domain, CD25 transmembrane domain, CD7 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, LAX transmembrane domain, LAT transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, TIM3 transmembrane domain, KIR3DS1 transmembrane domain, KIR3DL1 transmembrane domain, NKG2D transmembrane domain, NKG2A transmembrane domain, TIGIT transmembrane domain, 2B4 transmembrane domain, and BTLA transmembrane domain; (c) A spacer region located between the antigen-binding domain and the transmembrane domain, and the spacer region having a region selected from SEQ ID NO: The amino acid sequence of the group consisting of 49-58.

4. The isolated cells of claim 1, wherein the inhibitory chimeric receptor and / or the antigen-binding domain of the chimeric receptor comprises one or more single-chain variable fragments (scFv), each of the one or more scFv comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) separated by peptide linkers.

5. The isolated cells of claim 4, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO:

77.

6. The isolated cells of claim 2, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NO: 49-58.

7. The separated cells as described in claim 1, wherein: (a) The antigen-binding domain to CEACAM5 includes a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (i) a VH containing the amino acid sequence of SEQ ID NO: 3 and a VL containing the amino acid sequence of SEQ ID NO: 4; (ii) a VH containing the amino acid sequence of SEQ ID NO: 9 and a VL containing the amino acid sequence of SEQ ID NO: 10; (iii) a VH containing the amino acid sequence of SEQ ID NO: 11 and a VL containing the amino acid sequence of SEQ ID NO: 12; (iv) a VH containing the amino acid sequence of SEQ ID NO: 13 and a VL containing the amino acid sequence of SEQ ID NO: 14; (v) a VH containing the amino acid sequence of SEQ ID NO: 78 and a VL containing the amino acid sequence of SEQ ID NO: 79; and (vi) a VH containing the amino acid sequence of SEQ ID NO:

79. The VH of the amino acid sequence SEQ ID NO: 15 and the VL of the amino acid sequence SEQ ID NO: 16; or (b) the antigen-binding domain of CEACAM5 includes a heavy chain (HC) and a light chain (LC) selected from the group consisting of: (i) an HC of the amino acid sequence SEQ ID NO: 5 and an LC of the amino acid sequence SEQ ID NO: 6; and (ii) an HC of the amino acid sequence SEQ ID NO: 7 and an LC of the amino acid sequence SEQ ID NO:

8.

8. The isolated cells as requested in item 1, wherein the cells are immune response cells.

9. The separated cells as requested in item 1, wherein: The chimeric receptor binds to one or more additional antigens with a binding affinity at least 5 times lower than that of the inhibitory chimeric receptor binding to the antigen.

10. The isolated cells of claim 1, wherein the chimeric recipient system exhibits recombination.

11. The isolated cells as claimed in claim 1, wherein the cell line is selected from the following groups: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, natural killer T (NKT) cells, bone marrow cells, macrophages, human embryonic stem cells (ESC), ESC-derived cells, pluripotent stem cells and induced pluripotent stem cells (iPSC) and iPSC-derived cells.

12. An isolated nucleic acid encoding an inhibitory chimeric receptor in an isolated cell as described in any one of claims 1 to 11.

13. A vector comprising the nucleic acid as claimed in claim 12.

14. A genetically modified cell comprising nucleic acid as claimed in claim 12 or a vector as claimed in claim 13.

15. A pharmaceutical composition comprising an effective amount of any one of claims 1 to 11 cells, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

16. Use of a cell as claimed in any one of claims 1 to 11 and 14, for the preparation of a drug that stimulates a cell-mediated immune response against tumor cells in an individual.

17. Use of a cell as claimed in any one of claims 1 to 11 and 14 for the preparation of a drug that provides antitumor immunity in an individual.

18. Use of a cell as claimed in any one of claims 1 to 11 and 14 for the preparation of a drug that reduces tumor burden in an individual.

19. Use of a cell as claimed in any one of claims 1 to 11 and 14 for the preparation of a medicament for treating an individual suffering from a tumor.

20. Use of a cell as claimed in any one of claims 1 to 11 and 14, for the preparation of a medicament for the treatment or prevention of cancer in an individual.

21. As requested in paragraph 20, wherein the cancer is selected from the group consisting of: colorectal cancer, pancreatic cancer, lung adenocarcinoma and stomach cancer.

22. As used in request item 19, wherein: (a) The drug increases the individual’s progression-free survival; and / or (b) The drug increases the individual’s survival time.

23. A kit for treating and / or preventing colorectal cancer, pancreatic cancer, lung adenocarcinoma and / or gastric cancer, comprising cells as claimed in any one of claims 1 to 11 and 14, and a written instruction for using the cells to treat and / or prevent colorectal cancer, pancreatic cancer, lung adenocarcinoma and / or gastric cancer in an individual.

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