Compositions and methods for treating mesothelin-positive cancers

Abstract

The present disclosure provides immune cells comprising a first activator receptor specific for mesothelin and a second inhibitory receptor specific for lost ligands in mesothelin-positive cancer cells, as well as methods of making the immune cells and using the immune cells to treat cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 304,409, filed on January 28, 2022. The contents of the above application are expressly incorporated herein by reference in their entirety, including any drawings. Technical Field

[0003] The present disclosure relates to the field of adoptive cell therapy and cancer treatment. Background Art

[0004] Cell therapy is a powerful tool for treating various diseases, especially cancer. In traditional adoptive cell therapy, immune cells are engineered to express specific receptors, such as chimeric antigen receptors (CAR) or T cell receptors (TCR), which direct the activity of immune cells to cell targets through the interaction of receptors with ligands expressed by target cells. Since many targets are expressed in normal tissues, it remains challenging to identify suitable target molecules. When transplanted cells target normal tissues expressing target molecules, this expression can lead to toxicity. Therefore, there is a need in the art for compositions and methods for treating diseases, especially cancer, by adoptive cell therapy.

[0005] Mesothelin (MSLN) was proposed as a cancer target in 1992 (Chang et al. Cancer Res 52: 181-86), but there is still no feasible therapy using MSLN. It is expressed not only in most mesotheliomas, but also in most subgroups of ovarian, cervical, uterine, gastric, pancreatic and lung adenocarcinomas (Hassan et al. J Clin Oncol 34: 4171-79). In normal adults, MSLN is only present in the mesothelium, while the mesothelial tissue itself is optional. Several investigational therapies for MSLN have been tested; for example, immunotoxin conjugates, antibody-drug conjugates, bispecific antibodies, CAR-T and hybrid TCR-scFv.

[0006] All active systemic therapies are toxic. Therefore, there is a need in the art for compositions and methods related to the treatment of MSLN(+) cancer. Summary of the invention

[0007] Provided herein are compositions and methods related to the treatment of MSLN(+) cancer. Advantageously, the compositions and methods disclosed herein can utilize loss of heterozygosity (LOH) to treat MSLN(+) cancer. In some cases, the compositions and methods disclosed herein can avoid systemic toxicity to normal tissues by pairing MSLN-targeted activator receptors with blocker receptors. Without being bound by theory, differential expression of blocker antigens in tumor tissues and normal tissues caused by LOH at the locus encoding the blocker antigen can confer high selectivity for tumor killing.

[0008] The present disclosure provides an immune cell comprising: (a) a first receptor comprising an extracellular ligand binding domain specific for mesothelin (MSLN); and (b) a second receptor comprising an extracellular ligand binding domain specific for HLA-A*03, wherein the first receptor is an activator receptor responsive to MSLN; and wherein the second receptor is an inhibitory receptor responsive to HLA-A*03.

[0009] In some embodiments of the immune cells of the present disclosure, HLA-A*03 is lost in MSLN+ cancer cells through loss of heterozygosity.

[0010] In some embodiments, the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6; or CDR sequences having up to 1, 2 or 3 substitutions, deletions or insertions relative to the CDRs of Table 6 or Table 7. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of the following sequences: (i) SEQ ID NOs: 638, 645, 650, 657, 676, and 693; (ii) SEQ ID NOs: 638, 645, 650, 658, 677, and 694; (iii) SEQ ID NOs: 638, 645, 650, 659, 678, and 695; (iv) SEQ ID NOs: 638, 645, 650, 660, 678, and 696; (v) SEQ ID NOs: 638, 645, 650, 661, 679, and 697; (vi) SEQ ID NOs: NO:639, 646, 651, 657, 676 and 698; (vii) SEQ ID NO:638, 645, 650, 657, 676 and 699; (viii) SEQ ID NO:639, 646, 651, 657, 676 and 700; (ix) SEQ ID NO:638, 645, 650, 662, 680 and 701; (x) SEQ ID NO:639, 646, 651, 657, 676 and 702; (xi) SEQ ID NO:638, 645, 650, 661, 679 and 703; (xii) SEQ ID NO:640, 647, 652, 657, 676 and 704; (xiii) SEQ ID NO:639, 646, 651, 657, 676 and 704 NO:641, 648, 653, 663, 681 and 705; (xiv) SEQ ID NO:638, 645, 650, 664, 682 and 706; or (xv) SEQ ID NO:1260-1265; or CDR sequences having up to 1, 2 or 3 substitutions, deletions or insertions relative to the CDRs of the following sequences: (i) SEQ ID NO:638, 645, 650, 657, 676 and 693; (ii) SEQ ID NO:638, 645, 650, 658, 677 and 694; (iii) SEQ ID NO:638, 645, 650, 659, 678 and 695; (iv) SEQ ID NO:638, 645, 650, 660, 678 and 696; (v) SEQ ID NO: NO: 638, 645, 650, 661, 679 and 697;(vi) SEQ ID NOs: 639, 646, 651, 657, 676 and 698; (vii) SEQ ID NOs: 638, 645, 650, 657, 676 and 699; (viii) SEQ ID NOs: 639, 646, 651, 657, 676 and 700; (ix) SEQ ID NOs: 638, 645, 650, 662, 680 and 701; (x) SEQ ID NOs: 639, 646, 651, 657, 676 and 702; (xi) SEQ ID NOs: 638, 645, 650, 661, 679 and 703; (xii) SEQ ID NOs: 640, 647, 652, 657, 676 and 704; (xiii) SEQ ID NOs: NO:641, 648, 653, 663, 681 and 705; or (xiv) SEQ ID NO:638, 645, 650, 664, 682 and 706; or (xv) SEQ ID NO:1260-1265. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of the following sequences: (i) SEQ ID NOs: 638, 645, 650, 657, 676, and 693; (ii) SEQ ID NOs: 638, 645, 650, 658, 677, and 694; (iii) SEQ ID NOs: 638, 645, 650, 659, 678, and 695; (iv) SEQ ID NOs: 638, 645, 650, 660, 678, and 696; (v) SEQ ID NOs: 638, 645, 650, 661, 679, and 697; (vi) SEQ ID NOs: NO:639, 646, 651, 657, 676 and 698; (vii) SEQ ID NO:638, 645, 650, 657, 676 and 699; (viii) SEQ ID NO:639, 646, 651, 657, 676 and 700; (ix) SEQ ID NO:638, 645, 650, 662, 680 and 701; (x) SEQ ID NO:639, 646, 651, 657, 676 and 702; (xi) SEQ ID NO:638, 645, 650, 661, 679 and 703; (xii) SEQ ID NO:640, 647, 652, 657, 676 and 704; (xiii) SEQ ID NO:639, 646, 651, 657, 676 and 704 NO: 641, 648, 653, 663, 681 and 705;or (xiv) SEQ ID NOs: 638, 645, 650, 664, 682, and 706. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 1260-1265. In some embodiments, the extracellular ligand binding domain of the second receptor comprises a polypeptide sequence selected from the polypeptide sequences disclosed in Table 5; or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain of the second receptor comprises any one of SEQ ID NOs: 615-628 or SEQ ID NO: 1259, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain of the second receptor comprises any one of SEQ ID NOs: 615-628. In some embodiments, the extracellular ligand binding domain of the second receptor comprises SEQ ID NO: 1259. ;

[0011] In some embodiments of the immune cells of the present disclosure, the first receptor is a chimeric antigen receptor (CAR). In some embodiments, the extracellular ligand binding domain of the first receptor comprises complementary determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 disclosed in Table 2; or CDR sequences with a maximum of 1, 2 or 3 substitutions, deletions or insertions relative to the CDRs of Table 2. In some embodiments, the extracellular ligand binding domain of the first receptor comprises: a variable heavy chain (VH) portion comprising a sequence shown in Table 3; and a variable light chain (VL) portion comprising a sequence shown in Table 4; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy chain (VH) portion and a variable light chain (VL) portion, wherein the variable heavy chain (VH) portion comprises SEQ ID NO: 233 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereof, and the variable light chain (VL) portion comprises SEQ ID NO: 279 or a sequence having 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereof. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NO: 3-6, 80 and 154-215, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereof. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the scFv sequence of SEQ ID NO: 171; or a sequence at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto.

[0012] In some embodiments of the immune cells of the present disclosure, the first receptor comprises a hinge domain, a transmembrane domain and an intracellular domain. In some embodiments, the hinge domain comprises a CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises a sequence of SEQ ID NO:7, or a sequence with at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises a sequence of SEQ ID NO:11, or a sequence with at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain and a CD3ζ activation domain. In some embodiments, the intracellular domain comprises a sequence of SEQ ID NO: 285, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first receptor comprises a sequence of SEQ ID NO: 303, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0013] In some embodiments of the immune cells of the present disclosure, the second receptor comprises a LILRB1 intracellular domain or a functional variant thereof. In some embodiments, the LILRB1 intracellular domain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 70, or a sequence that is identical to SEQ ID NO: 70. In some embodiments, the second receptor comprises a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 74, or a sequence that is identical to SEQ ID NO: 74. In some embodiments, the second receptor comprises a LILRB1 hinge domain or a functional variant thereof. In some embodiments, the LILRB1 hinge domain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 73, or a sequence that is identical to SEQ ID NO: 73. In some embodiments, the second receptor comprises a LILRB1 intracellular domain, a LILRB1 transmembrane domain, a LILRB1 hinge domain, a functional variant of any of these domains, or a combination thereof. In some embodiments, the LILRB1 hinge domain, the LILRB1 intracellular domain, and the LILRB1 transmembrane domain comprise SEQ ID NO: 71, or a sequence at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 71, or a sequence identical to SEQ ID NO: 71. In some embodiments, the second receptor comprises the sequence of SEQ ID NO: 1268, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0014] In some embodiments of the immune cells of the present disclosure, the MSLN+ cancer cells are mesothelioma cancer cells, ovarian cancer cells, cervical cancer cells, colorectal cancer cells, esophageal cancer cells, head and neck cancer cells, renal cancer cells, uterine cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, colorectal cancer cells or bile duct cancer cells, or any cancer cells expressing MSLN. In some embodiments, the MSLN+ cancer cells are mesothelioma cancer cells, ovarian cancer cells, cervical cells, uterine cancer cells, gastric cancer cells, pancreatic cancer cells or lung adenocarcinoma cells.

[0015] In some embodiments, the MSLN+ cancer cell is an epithelial cancer cell. An epithelial cancer is a cancer that originates from an epithelial cell. In some embodiments, the MSLN+ epithelial cancer is a carcinoma.

[0016] In some embodiments of the immune cells of the present disclosure, the MSLN+ cancer cell is an MSLN+ / HLA-A*03- cancer cell that does not express HLA-A*03. In some embodiments, the MSLN+ / HLA-A*03- cancer cell is derived from an MSLN+ / HLA-A*03+ cell that has lost HLA-A*03 by loss of heterozygosity at HLA-A. In some embodiments, in the presence of an MSLN+ / HLA-A*03- cancer cell with loss of heterozygosity, the first receptor and the second receptor together specifically activate the immune cell. In some embodiments, in the presence of an MSLN+ cell that has not lost HLA-A*03 by loss of heterozygosity, the first receptor and the second receptor together do not specifically activate the immune cell.

[0017] In some embodiments of the immune cells of the present disclosure, the immune cells are T cells. In some embodiments, the T cells are CD8+CD4- T cells or CD8-CD4+ T cells.

[0018] In some embodiments of the immune cells of the present disclosure, the expression and / or function of MHC class I genes have been reduced or eliminated. In some embodiments, MHC class I genes are beta-2-microglobulin (B2M). In some embodiments, immune cells also include interfering RNA, and the interfering RNA includes a sequence complementary to the B2M mRNA sequence. In some embodiments, the interfering RNA includes a sequence selected from the sequence shown in Table 13, or a sequence with a maximum of 1, 2, 3 or 4 substitutions, insertions or deletions relative to it. In some embodiments, interfering RNA can induce RNAi-mediated degradation of B2M mRNA. In some embodiments, interfering RNA is a short hairpin RNA (shRNA). In some embodiments, shRNA includes: (a) a first sequence, which has a sequence complementary to the B2M mRNA sequence from 5' to 3'; and (b) a second sequence, which has a sequence complementary to the first sequence from 5' to 3', wherein the first sequence and the second sequence form shRNA. In some embodiments, the shRNA is encoded by a sequence comprising GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 349) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 350), or a sequence at least 80%, at least 90%, or at least 95% identical thereto.

[0019] In some embodiments of the immune cells of the present disclosure, the expression and / or function of MHC class I genes have been reduced or eliminated. In some embodiments, the MHC class I gene is beta-2-microglobulin (B2M). In some embodiments, the immune cells include one or more modifications to the sequence encoding B2M, wherein one or more modifications reduce the expression of B2M and / or eliminate the function of B2M. In some embodiments, one or more modifications include one or more inactivating mutations of the endogenous gene encoding B2M. In some embodiments, one or more inactivating mutations include deletions, insertions, substitutions or frameshift mutations. In some embodiments, one or more inactivating mutations are introduced by a nucleic acid-guided endonuclease compounded with at least one guide. In some embodiments, at least one guide is a guide nucleic acid (gNA) of a sequence of an endogenous gene specifically targeting encoding B2M. In some embodiments, gNA includes a sequence selected from the sequences shown in Table 12, or a sequence with up to 1, 2, 3 or 4 substitutions, insertions or deletions relative thereto.

[0020] In some embodiments of the immune cells of the present disclosure, the expression and / or function of MHC class I genes have been reduced or eliminated. In some embodiments, the MHC class I gene is HLA-A*03. In some embodiments, the immune cells comprise a polynucleotide containing an interfering RNA, the interfering RNA comprising a sequence complementary to the HLA-A*03mRNA sequence. In some embodiments, the interfering RNA is capable of inducing RNA interference (RNAi)-mediated degradation of HLA-A*03mRNA. In some embodiments, the interfering RNA is a short hairpin RNA (shRNA), comprising: (a) a first sequence having a sequence complementary to the HLA-A*03mRNA sequence from the 5' end to the 3' end; and (b) a second sequence having a sequence complementary to the first sequence from the 5' end to the 3' end, wherein the first sequence and the second sequence form shRNA. In some embodiments, the shRNA comprises the sequence shown in Table 14. In some embodiments, the immune cell comprises one or more modifications to a sequence of an endogenous gene encoding HLA-A*03, wherein the one or more modifications reduce the expression of HLA-A*03 and / or eliminate the function of HLA-A*03. In some embodiments, the one or more modifications comprise one or more inactivating mutations of an endogenous gene encoding HLA-A*03. In some embodiments, the one or more inactivating mutations are introduced by a nucleic acid-guided endonuclease complexed with at least one guide nucleic acid (gNA) that specifically targets a sequence of an endogenous gene encoding HLA-A*03. In some embodiments, the gNA comprises a sequence shown in Table 11.

[0021] In some embodiments of the immune cells of the present disclosure, the first receptor comprises a sequence of SEQ ID NO: 164, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and the second receptor comprises a sequence of SEQ ID NO: 1259, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the immune cell comprises a shRNA encoded by a sequence comprising the following sequence: GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 349) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 350), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the first receptor and the second receptor are encoded by a single polynucleotide, and wherein the sequences encoding the first and second receptors are separated by a sequence encoding a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351).

[0022] In some embodiments of the immune cells of the present disclosure, the immune cells are autologous.

[0023] In some embodiments of the immune cells of the present disclosure, the immune cells are allogeneic.

[0024] The present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the immune cells of the present disclosure. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0025] The present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the immune cells of the present disclosure, for use as a medicament for treating MSLN+ cancer.

[0026] The present disclosure provides a polynucleotide or a polynucleotide system comprising one or more polynucleotides, wherein the polynucleotide comprises a polynucleotide sequence encoding the following receptors: (a) a first receptor comprising an extracellular ligand binding domain specific for mesothelin (MSLN); and (b) a second receptor comprising an extracellular ligand binding domain specific for HLA-A*03, wherein the first receptor is an activator receptor responsive to MSLN on MSLN+ cancer cells; and wherein the second receptor is an inhibitory receptor responsive to HLA-A*03.

[0027] In some embodiments of the polynucleotides or polynucleotide systems of the present disclosure, the polynucleotides or polynucleotide systems comprise one or more polynucleotides for generating immune cells of the present disclosure, the polynucleotides comprising polynucleotide sequences encoding the first receptor and the second receptor. In some embodiments, the polynucleotides or polynucleotide systems comprise sequences encoding shRNAs specific to B2M. In some embodiments, the sequences encoding the first receptor, the second receptor, and the shRNAs specific to B2M are encoded by the same polynucleotides. In some embodiments, (a) the sequence encoding the shRNA specific for B2M comprises GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGT GC (SEQ ID NO:349) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO:350), or a sequence at least 80%, at least 90%, or at least 95% identical thereto; (b) the sequence encoding the first receptor comprises a sequence encoding a polypeptide of SEQ ID NO:303, or a sequence at least 80%, at least 90%, or at least 95% identical thereto; and (c) the sequence encoding the second receptor comprises a sequence encoding a polypeptide of SEQ ID NO:1268, or a sequence at least 80%, at least 90%, or at least 95% identical thereto.

[0028] The present disclosure provides a vector comprising one or more polynucleotides of the present disclosure.

[0029] The present disclosure provides a method for killing MSLN+ cancer cells having loss of heterozygosity at the HLA-A*03 locus, comprising administering an effective amount of the immune cell or pharmaceutical composition of the present disclosure to a subject.

[0030] The present disclosure provides a method of treating MSLN+ cancer in a subject having an MSLN+ tumor with loss of heterozygosity at the HLA-A*03 locus, comprising administering to the subject an effective amount of an immune cell or pharmaceutical composition of the present disclosure.

[0031] The present disclosure provides a method for treating cancer in a subject, comprising: (a) determining the HLA-A genotype or expression of normal cells and multiple cancer cells of the subject; (b) optionally, determining MSLN expression in multiple cancer cells of the subject; and (c) if the normal cells express HLA-A*03 and the multiple cancer cells do not express HLA-A*03, and the multiple cancer cells are MSLN positive, administering to the subject an effective amount of the immune cells or pharmaceutical composition of the present disclosure.

[0032] In some embodiments of the disclosed methods, the subject is a heterozygous HLA-A*03 patient with a malignant tumor that expresses MSLN (MSLN+) and has lost HLA-A*03 expression. In some embodiments, the subject is a heterozygous HLA-A*03 patient with a recurrent unresectable or metastatic solid tumor that expresses MSLN and has lost HLA-A*03 expression. In some embodiments, the cancer comprises mesothelioma, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or bile duct cancer. In some embodiments, the cancer comprises mesothelioma, ovarian cancer, cervical cancer, uterine cancer, gastric cancer, pancreatic cancer, or lung adenocarcinoma. In some embodiments, the subject's cancer has recurred. In some embodiments, the cancer is refractory to one or more previously administered anticancer therapies. In some embodiments, the cancer is metastatic.

[0033] In some embodiments of the disclosed methods, the cancer cells comprise MSLN+ / HLA-A*03- cancer cells that do not express HLA-A*03. In some embodiments, the MSLN+ / HLA-A*03- cancer cells are derived from MSLN+ / HLA-A*03+ cells that have lost HLA-A*03 by loss of heterozygosity at HLA-A. In some embodiments, in the presence of MSLN+ / HLA-A*03- cancer cells, the first receptor and the second receptor together specifically activate immune cells. In some embodiments, in the presence of MSLN+ cells that have not lost HLA-A*03, the first receptor and the second receptor together do not specifically activate immune cells.

[0034] In some embodiments of the disclosed methods, administering immune cells or pharmaceutical compositions reduces the size of a subject's tumor. In some embodiments, the tumor is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%. In some embodiments, the tumor is eliminated.

[0035] In some embodiments of the disclosed methods, administering the immune cells or pharmaceutical compositions prevents tumor growth in the subject.

[0036] In some embodiments of the disclosed methods, administration of the immune cells or pharmaceutical composition reduces the number of tumors in the subject.

[0037] In some embodiments of the disclosed methods, administration of immune cells or pharmaceutical compositions causes selective killing of cancer cells in subjects but does not kill normal cells. In some embodiments, at least about 60% of the killed cells are cancer cells, about 65% of the killed cells are cancer cells, about 70% of the killed cells are cancer cells, about 75% of the killed cells are cancer cells, about 80% of the killed cells are cancer cells, about 85% of the killed cells are cancer cells, about 90% of the killed cells are cancer cells, about 95% of the killed cells are cancer cells or about 100% of the killed cells are cancer cells. In some embodiments, administration of immune cells or pharmaceutical compositions causes at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or all of the cancer cells in subjects to be killed.

[0038] In some embodiments of the disclosed methods, administration of the immune cell or pharmaceutical composition results in fewer side effects to the subject than administration of an otherwise equivalent immune cell comprising the first activator receptor but not the second inhibitory receptor.

[0039] The present disclosure provides a method for preparing a plurality of immune cells, comprising: (a) providing a plurality of immune cells, and (b) transforming the plurality of immune cells using a polynucleotide, polynucleotide system or vector of the present disclosure.

[0040] The present disclosure provides a kit comprising the immune cells or pharmaceutical compositions of the present disclosure. In some embodiments, the kit further comprises instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a table showing the expression of candidate blocker genes from the TCGA database. (*) indicates genes previously identified as expressed in the mesothelium.

[0042] Figure 2 This is a graph showing the expression of mesothelin (MSLN) in normal tissues.

[0043] Figure 3Figure 1 shows the expression of MSLN in TCGA cancers (including tumor and normal samples). Abbreviations: BLCA (bladder cancer), BRCA (breast cancer), CESC (cervical squamous cell carcinoma and cervical adenocarcinoma), CHOL (choledochal carcinoma), COAD (colon adenocarcinoma), ESCA (esophageal cancer), GBM (glioblastoma multiforme), HNSC (head and neck squamous cell carcinoma), KICH (chromophobe renal cell carcinoma), KIRP (renal papillary cell carcinoma), LIHC (hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PAAD (pancreatic cancer), PRAD (prostate cancer), PCPG (pheochromocytoma and paraganglioma), READ (rectal adenocarcinoma), SARC (sarcoma), SKCM (cutaneous melanoma), THCA (thyroid cancer), THYM (thymoma), STAD (gastric adenocarcinoma), UCEC (endometrial carcinoma).

[0044] Figure 4 is a graph showing the expression of MSLN in CCLE cell line.

[0045] Figure 5 is a graph showing LRRN4 expression in normal tissues.

[0046] Figure 6 is a graph showing the expression of LRRN4 in TCGA cancers (including tumor and normal samples).

[0047] Figure 7 is a graph showing the expression of LRRN4 in TCGA tumors.

[0048] Figure 8 is a graph showing the expression of LRRN4 in CCLE cell line.

[0049] Fig. 9 is a graph showing the distribution of the 10 leucine-rich repeats (LRRs) and type III fibronectin domains relative to the transmembrane domain of LRRN4. Most of these domains are likely to be located on the cell surface.

[0050] Fig. 10A We show that the pMHC HLA-A*02scFv LIR-1 based inhibitory receptor can cis-inhibit activation of Jurka T cells in a cell-free bead-based assay.

[0051] Fig. 10B It was shown that using the leukemia cell line K562 as target cells, the inhibitory receptor based on pMHC HLA-A*02scFv LIR-1 could inhibit the activation of Jurkat cells by MSLN scFv CAR.

[0052] Fig.11Figure (left) and statistical graph (right) showing that the pMHC HLA-A*02scFv LIR-1 based inhibitory receptor can inhibit the activation of JurkaT cells by MSLN scFv CAR using the pMHC HLA-A*02scFv LIR-1 based inhibitory receptor and HLA-A*02+HeLa and SiHa cells as target cells, as measured by the fold induction of IFNγ.

[0053] Fig.12 It was shown that the pMHC HLA-A*02scFv LIR-1 based inhibitory receptor inhibited killing caused by the MSLN CAR activator using HLA-A*02+SiHa cells but not HLA-A*02-SiHa cells.

[0054] Fig.13 is a bioinformatics flow chart for identifying potential inhibitory receptor targets lost in cancer cells due to loss of heterozygosity.

[0055] Fig.14 is a bioinformatics flowchart for identifying potential inhibitory receptor targets that are not expressed in cancer cells.

[0056] Fig.15A A pair of graphs showing that HLA-A*02 blockers inhibit MSLN CAR activation against high-density antigen MSLN. Jurkat-cells transfected with MSLN LBD1-CAR or MSLN LBD1-CAR&A2-LIR-1 co-cultured with K562 cells expressing MSLN or MSLN&HLA-A*02 show that activation caused by high-density antigen is blocked using A2-LIR-1 blockers only in the presence of HLA-A*02.

[0057] Fig. 15B is a pair of graphs showing that HLA-A*02 blockade inhibits MSLN CAR activator targeting the high-density antigen MSLN. In the presence of HLA-A*02, A2-LIR-1 blockade was used to block the killing of endogenous MSLN+HeLa cells by MSLN LBD1-CAR T cells.

[0058] Fig. 15C is a pair of graphs showing that HLA-A*02 blockade inhibits MSLN CAR activators targeting the high-density antigen MSLN. Killing of endogenous MSLN+HeLa cells by MSLN LBD2-CAR T cells is shown. The effect of A2-LIR-1 blockade on T cell killing is partially controlled by the activator LBD, suggesting that further optimization of the blocker module or activator / blocker pair is needed.

[0059] Fig.16 is a series of graphs showing that the HLA-A*02LIR1 inhibitory receptor (PA2.1, mouse and humanized) effectively blocks killing of T cells expressing the MSLN 3rd generation CAR in the presence of HeLa cells expressing MSLN and HLA-A*02. Top row: MSLN+HLA-A*02+HeLa target cells; bottom row: MSLN+HLA-A*02-HeLa cells (control). The mouse SS1 3rd generation CAR (upper right, boxed) provides a better window than the humanized M5 and humanized SS1 CARs.

[0060] Fig.17 is a series of graphs showing that the HLA-A*02LIR1 inhibitory receptor (PA2.1, mouse and humanized) effectively blocks killing by T cells expressing the MSLN 3rd generation CAR in the presence of MSLN+HLA-A*02+Capan-2 cells.

[0061] Fig.18 is a pair of figures showing that the killing of MSLN+HLA-A*02+HeLa cells (left) or HCT116 wild-type (WT) cells that are naturally MSLN+HLA-A*02+ by T cells expressing the second-generation CAR with mouse SS1 scFv can be effectively blocked by the HLA-A*02 scFv LIR1 inhibitory receptor.

[0062] Fig.19 is a series of fluorescence activated cell sorting (FACS) images showing the expression of the murine SS1 second generation CAR by T cells with and without co-transduction of the HLA-A*02scFv LIR-1 blocker.

[0063] Fig. 20A is a graph showing the effect of the LIR-1 hinge on the ability of the HLA-A*02 inhibitory receptor to block KRAS TCR activation of Jurkat cells. H: hinge, T: transmembrane domain, ICD: intracellular domain, s: short. Fig. 20B The LIR-1 constructs are described in more detail in Humanized PA2.1 and humanized BB7.2 have a shorter LIR-1 hinge block that resembles the original longer hinge.

[0064] Fig. 20B Is a graph and table showing the change in EC50 (+ / - HLA-A*02 target cells) for Jurkat cells expressing KRAS TCR activators and the HLA-A*02 scFv LIR-1 inhibitory receptor (SEQ ID NOs: 352-356) shown in the bottom table.

[0065] Fig.21Ais a graph showing the effect of the LIR-1 hinge on the ability of the HLA-A*02 inhibitory receptor to block KRAS TCR activation of Jurkat cells. H: hinge, TM: transmembrane domain, ICD: intracellular domain, s: short; tr: truncated. Fig. 21B The LIR-1 constructs are described in more detail. Mouse PA2.1 with a slightly longer hinge functions similarly to the original LIR-1 hinge in the T2-Jurkat assay.

[0066] Fig. 21B Graph and a pair of tables showing the EC50 changes (+ / - HLA-A*02 target cells) of Jurka T cells expressing KRAS TCR activators and the HLA-A*02 scFv LIR-1 inhibitory receptors (lengths as shown in the left table) (SEQ ID NOs: 357-361) shown in the bottom table.

[0067] Fig.22A and Fig. 22B Shown is a Tmod approach to achieve selective cytotoxicity through two targets (Tmod refers to immune cells that express a combination of activator and inhibitor receptors). Fig.22A The lungs (and other important organs) are shown to be surrounded by an MSLN(+) mesothelial lining, which makes MSLN-targeted drugs have a high risk of off-target, non-tumor toxicity. By selecting patients who are HLA-A*02 heterozygous (whose tumors have lost this allele due to LOH), there is an opportunity to target MSLN-activated CAR-T cells to specifically kill tumor cells and preserve normal mesothelium. Fig. 22B The molecular composition of the MSLN-targeted Tmod construct is shown (Tmod refers to paired activator and inhibitory receptors). The two receptors are co-expressed in a single construct, and the encoded fusion protein is cleaved in the cell to produce the activator and the blocker.

[0068] Fig.23A , Fig. 23B , Fig.23C and Fig.23D Isolation and characterization of selective MSLN binders are shown. The on-target probe was labeled with soluble MSLN (Acro Bio); the off-target probe used for counter-selection was a mixture of soluble CEA and EGFR proteins. Fig.23A Enrichment of the IgG library is shown. Fig. 23B Enrichment of the scFv library is shown. Fig.23CSurface expression of MSLN CAR (Gen3) in Jurkat cells is shown. Cells were transfected with CAR constructs and stained with Protein L or monomeric soluble MSLN (see Methods). Benchmark and CAR1-6 expression histograms are labeled. PE, phycoerythrin; NA, neutravidin; SA, streptavidin. "On-target NGS" corresponds to a cell population that is collected and DNA sequenced to determine the genotype enrichment of a single individual. Fig.23D Characterization of MSLN binders in a solid-state Jurkat cell assay in which MSLN protein is attached to the surface (see Hamburger et al., 2020). 62 CAR constructs (Gen3) with different scFvs were transiently transfected in Jurkat cells to express the CAR and functional responses to surface-bound recombinant human sMSLN (Acro Bio) were assessed 6 hours later. Most induced some degree of response.

[0069] Fig.24A The sensitivity of MSLN CAR and benchmark CAR M5, SS1 and m912 is shown. Except SS1 (Gen2), all constructs are Gen3. Jurkat cell dose-response (RLU) was measured to evaluate the sensitivity in the 6-hour co-culture assay: (1) titrated MSLN encoding mRNA was used to transfect HEK293 cells; (2) QIFIKIT (quantitative analysis kit, Agilent) was used to convert surface expression based on flow cytometry to MSLN molecules / cells; and (3) by fitting the dose response-curve, the molecular / cell sensitivity (EC50) of 6 new CARs and 3 benchmark CARs was calculated. For those CARs whose sensitivity was below the detection limit in the assay, EC50 was reported as <3000MSLN molecules / cell. The maximum signal (Emax) of each construct was also recorded. The experiment was repeated 1-4 times.

[0070] Fig. 24B CAR3 selectivity is shown. Example of MSLN CAR3 selectivity using M5CAR as a benchmark. CAR activation was measured in a Jurkat cell functional assay by MSLN(+) or MSLN(-) cell lines. For the MSLN(+) cell line, a variant MSLN(-) version was generated by MSLN knockout for comparison. For more detailed off-target characterization, see Fig.32B .

[0071] Fig.25A Expression of MSLN in human cell lines assessed by staining with MSLN mAb and flow cytometry is shown. Although no functional responses were observed to the CAR3 or M5 benchmark CARs, K562 exhibited some cross-reactivity with the anti-MSLN antibody.

[0072] Fig.25B The plotted levels of MSLN and A*02 mRNA (CCLE) and protein (QIFIKIT) are shown to be correlated. The conversion between protein and mRNA levels was calculated using a standard curve (see method in Example 8 below).

[0073] Fig.26A A summary of the cell lines used in this study is shown. Quantification of surface density of MSLN and A*02 in various cancer cell lines and the corresponding reported mRNA levels in normal lung tissue (GTEx). Surface MSLN and A*02 of engineered and wild-type tumor cell lines were quantified using QIFIKIT (Quantitative Analysis Kit, Agilent). TPM values ​​were divided by 2 when the cell line HLA-A haplotype was heterozygous for A*02. Note that in some cases, the HLA-A allele copy number was unknown. The PM value of MS751+ transduced A*02 (438 TPM) was estimated by using a standard curve to measure its surface A*02 protein level. Cell lines transduced with HLA-A*02 better mimic the A*02:MSLN ratio of normal lung tissue compared to cell lines expressing endogenous protein levels (bold black box). TPM, transcripts per million; na, not applicable; A*02:HLA-A*02.

[0074] Fig.26B Quantification of MSLN molecules / cell using QIFIKIT is shown. Anti-human MSLN mouse antibody clone 618923 (R&D Systems) was used to stain approximately 100,000 cells. After washing the cells, anti-mouse IgG F(ab')2 secondary antibody (Invitrogen A21237) was used to stain the cells and QIFI beads. The number of MSLN molecules on the surface was quantified using the QIFI antigen standard curve.

[0075] Fig.27A Characterization of MSLN CAR Tmod constructs in Jurkat cell functional assays is shown. Six HuTARG-derived MSLN activators (CAR1-6) and benchmark CAR M5 and SS1 activators were paired with A*02 blockers (filled circles) or empty vector controls (open circles). Jurkat NFAT luciferase cells expressing CAR+ / - blockers were co-cultured with wild-type endogenous MSLN(+) HeLa cells transfected with A*02:01 mRNA titration. Functional response (RLU) was assessed after 6 hours of co-culture. Quantification of titrated antigen molecules on the surface was performed using QIFIKIT. IC50 (molecules / cell) values ​​are indicated in the figure. CAR1-6 is Gen3; CAR M5 and SS1 are Gen2.

[0076] Fig.27B Two-dimensional titration of MSLN and A*02 mRNA in MSLN(-)HeLa target cells is shown to determine the EC50 of the MSLN CAR3Tmod construct in Jurkat cells. MSLN(-)HeLa target cells were transfected with serially diluted MSLN mRNA and constant A*02 mRNA, and Jurkat cells were transiently transfected to express MSLN CAR3 and A*02 blockers. Functional response (RLU) was assessed after 6 hours of co-culture.

[0077] Fig.27C Shown are transfections of MSLN(-)HeLa target cells with serially diluted A*02 mRNA and constant MSLN mRNA to determine the IC50 of the MSLN CAR3Tmod construct in Jurkat cells.

[0078] Fig.28A The plotted levels of MSLN and A*02 mRNA and protein are shown. The EC50 and IC50 of the constructs are correlated with the expression levels of MSLN and HLA-A in normal (GTEx database) and tumor tissues and cell lines (TCGA, CCLE databases). Fig.25B The conversion between protein and mRNA levels was calculated using the standard curve shown in (Methods). The HeLa and MS751A*02 transgenic cell line variants shown better mimic the activator and blocker target ratios in normal tissues.

[0079] Fig.28B Cytotoxicity of MSLN CAR and CAR3Tmod in primary T cells is shown. As shown, primary T cells transduced with CAR or Tmod were co-cultured with tumor or normal target cells at an effector-target ratio (E:T) = 1:1 for 48 hours. The A*02:MSLN (B:A) target antigen ratio ranged from 2-27:1. M5 is a Gen2CAR; the others are Gen3. Tumor = MSLN (+) A*02 (-) target cells; Normal = MSLN (+) A*02 (+) target cells.

[0080] Fig.29AComparison of the lead CAR3 receptor paired with A*02 blocker to the benchmark CAR in the cytotoxicity assay is shown. SS1CAR is a Gen2 construct; the others are Gen3. Primary T cells transduced with various CARs + / - A*02 blockers using 2 different lentiviral vectors were cultured with endogenous MSLN(+) A*02(-) tumors or MSLN(+) A*02(+) normal HeLa cells to assess cytotoxicity. Transduced primary T cells were normalized to a constant activator or activator-blocker double-positive population percentage [15% A(+) or A(+) B(+)] cells by diluting the transduced primary T cells with untransduced T cells to a final effective effector-target ratio (E:T) of 0.6:1 or 0.3:1. When the T cells also expressed the A*02 blocker, both E:T ratios resulted in selective killing in the presence of the A*02 antigen.

[0081] Fig.29B IFN-g secretion after 48 hours of co-culture with tumor or normal target cells is shown. Significance was calculated using two-way ANOVA with multiple comparison test (n=3 technical replicates).

[0082] Fig.29C Shown are T cell activation assessed by measuring forward scatter shift after 48 h of co-culture of transduced T cells with tumor or "normal" target cells.

[0083] Fig. 30A MSLN CAR3Tmod cells are shown to selectively kill RFP(+) tumor cells and retain GFP(+) normal cells in mixed tumor and normal cell co-cultures. Due to the adhesion properties of the HeLa cell line, the killed targets tend to remain on the cell surface in clusters. White arrows point to examples of some killed RFP(+) tumor cells. Examples of E:T of 0.6:1 and normal: tumor = 1:1.

[0084] Fig. 30B Demonstrated cytotoxicity in mixed normal and tumor co-cultures with E:T = 1:1 and normal:tumor = 9:1 (for other ratios see Fig.31B ). Primary T cells transduced with CAR3 or CAR3Tmod constructs were co-cultured with HeLa target cells for 48 h and imaged using GFP and RFP expressed in MSLN(+)A*02(+) normal cells and MSLN(+)A*02(-) tumor cell lines, respectively.

[0085] Fig.31AMSLN CAR3Tmod cells were shown to selectively kill RFP(+) tumor cells in mixed tumor and normal cell co-cultures. Primary T cells transduced with the CAR3Tmod construct were co-cultured with HeLa cells for 48 hours and imaged using GFP and RFP expressed in MSLN(+)A*02(+) normal cells and MSLN(+)A*02(-) tumor cell lines, respectively.

[0086] Fig.31B The cytotoxicity of CAR3 and CAR3Tmod was shown in mixed normal and tumor co-cultures with normal:tumor ranging from 9:1 to 1:9; E:T = 0.6:1.

[0087] Fig.32A MSLN CAR3Tmod constructs were shown to mediate selective, sustained, and reversible cytotoxicity. Primary T cells transduced with CAR3 or CAR3Tmod were co-cultured with tumor cells or normal target cells at an E:T = 1.2:1 for 48 h. T cells were then collected, depleted of dead or non-adherent target cells, and re-inoculated onto fresh tumor cells or normal target cells for an additional 48 h. RACA, repeated antigen challenge assay. R1, round 1; R2, round 2.

[0088] Fig.32B Shown in Jurkat cell functional assay, MSLN CAR3Tmod construct selectivity for MSLN(+) and MSLN(-) control target cell subsets showed no off-target activity (see method in Example 8 below). Bar height corresponds to the mean of technical replicates.

[0089] Fig.33A Schematic diagram showing RACA (repeated antigen attack assay) and reversible assay. Primary T cells transduced with CAR3 or CAR3Tmod were co-cultured with tumor or normal targets at E:T=1.2:1 for 48 hours. T cells were then collected, cleared of all dead or shed target cells, and re-inoculated onto fresh tumor or normal target cells for an additional 48 hours.

[0090] Fig.33B Soluble circulating MSLN (sMSLN) was shown to have no effect on CAR-T activity. The presence of 500 ng / mL sMSLN (Acro Bio) did not affect the acute cytotoxicity of M5 benchmark CAR or CAR3 against tumor cells or normal target cells.

[0091] Fig.33C Staining of transiently transfected CAR(+) Jurkat cells with labeled sMSLN monomer or tetramer by flow cytometry analysis is shown, demonstrating that sMSLN is structurally intact and able to bind the receptor.

[0092] Fig.34A , Fig.34B and Fig.34C Tmod construct-mediated selective killing of tumor cells in a xenograft model was demonstrated. Fig.34A Schematic diagram showing the bilateral flank tumor and normal MS751 xenograft model. Fig.34B Bioluminescence values ​​are shown to the right of the color scale and are expressed in units of photons / sec / cm2 / sr. Day 0 = before T cell injection; Day 8 and Day 15 = after T cell injection. Fig.34C Implant dimensions assessed by caliper measurement are shown (see results of Example 8 below).

[0093] Fig.35A , Fig.35B and Fig.35C The MSLN CAR Tmod was shown to selectively kill tumors in a xenograft model. Fig.35A Primary T cells transduced with MSLN CAR or CAR3Tmod were shown to be co-cultured in vitro for 48 hours with HLA-A KO tumor or A*02 transgenic normal MS751 target cells at E:T=1.4:1. M5 is Gen2CAR; others are Gen3. Tumor = MSLN(+)A*02(-) target cells; Normal = MSLN(+)A*02(+) target cells. Fig.35B show Fig.34C Data shown in Figure 2 are individual mouse xenograft growth curves. Fig.35C Shown are BLI quantification of normal and tumor cells after T cell injection.

[0094] Fig.36 Cis-binding of A*02 blockers in A*02(+) or (-) T cells was shown to abrogate function. Binding of A*02 tetramers to blockers in A*02(+) Jurkat cells and primary T cells was significantly reduced due to cis-binding of autologous A*02. Reduced binding (due to reduced availability of blockers) correlated with reduced blocker activity. Cytotoxicity assays are shown at E:T=0.5:1.

[0095] Fig.37A , Fig.37B and Fig.37C showed that the MSLN Tmod system can be expanded to autologous T cells. Fig.37A We show that cis-binding of autologous A*02 in A*02(+) donors abolishes binding to A*02 tetramers. B2M knockout (KO) by CRISPR restores blocker availability, as evidenced by binding to A*02 tetramers, similar to levels observed in A*02(-) donors. Fig.37BCytotoxicity assays are shown showing activator-only and MSLN SS1CAR Tmod primary T cells cultured with tumor (solid) or normal (open) target cells. The MSLN SS1CAR Tmod construct kills MSLN(+)A*02(-) tumor HeLa target cells, but no longer blocks in the presence of autologous A*02 due to cis-binding. For A*02(+) donors, blocking was achieved only by B2M CRISPR KO. E:T=1.2:1. Fig.37C Representative images at 48 h are shown. Fig.37D We showed that, similar to SS1Tmod, CAR3Tmod had reduced binding to A*02 tetramers in A*02(+) T cells. Knockdown (KD) of B2M using shRNA restored the availability of the blocker. Fig.37E It was shown that B2M shRNA also restored the blockade of cytotoxicity against MSLN(+)A*02(+) "normal" HeLa cells. Fig.37F We showed that CAR3 paired with humanized A*02 blockade retained the ability to block “normal” cell killing in A*02(+) donor T cells even in the absence of B2M KO or KD.

[0096] Fig.38A Enrichment of anti-HLA-A*11 binders by multiple rounds of cell sorting from a scFv library is shown. The on-target probe is labeled with HLA-A*11 tetramers; the off-target proteins are a mixture of unrelated MHC tetramers.

[0097] Fig.38B Shown is Jurkat cell activation in mRNA titration assay: HeLa target cells were transfected with serially diluted HLA-A*11 mRNA and Jurkat T cells were transiently transfected to express HLA-A*11 CAR4. Functional responses (RLU) were assessed after 6 h of co-culture. PE, phycoerythrin.

[0098] Fig.39A It is shown that Jurkat cells expressing MSLN CAR3 and A*03, A*11 or B*07 blocking constructs are blocked on endogenous MSLN(+) HeLa target cells in the presence of increasing blocker antigen.

[0099] Fig.39B and Fig.39CPrimary T cell cytotoxicity assays of MSLN CAR3+A*11 blocker are shown. Primary T cells transduced with CAR3 and A*11:01 directed blocker effectively blocked HeLa target cells containing A*11:01 and killed wild-type HeLa cells as effectively as CAR-only cells. Transduced primary T cells were co-cultured with HeLa cells with or without HLA-A*11:01 at E:T=0.8:1. Note that the tumor and normal target cells used here express GFP. Fig.39C Display for Fig.39B Representative co-culture images at 48 hr.

[0100] Fig.40 MSLN CAR3Tmod killing of tumor target cells and blockade of "normal" target cells were shown to be unaffected by the presence of PD-L1 induced by overnight treatment of HeLa target cells with 50 ng / mL IFN-γ. Blockade with anti-PD-1 antibody also had no effect on CAR3Tmod.

[0101] Fig.41 Characterization of MSLN CAR A*03Tmod construct in Jurkat cell functional assay is shown. mBA GAP-A3 = construct with mouse blocker and activator (no shRNA).

[0102] Fig.42 Functional characterization of the MSLN CAR A*03Tmod construct on MS751 target cells in primary T cells from 5 different donors is shown. mBAsh(GAP-A3) = construct with mouse blocker and activator (plus β2M shRNA).

[0103] Fig.43 Functional characterization of MSLN CAR A*03Tmod constructs on HeLa target cells in primary T cells from 5 different donors is shown. mBAsh(GAP-A3) = construct with mouse blocker and activator (plus β2M shRNA).

[0104] Fig.44A and Fig.44B The Tmod construct was shown to mediate selective killing of tumor cells in a xenograft model. Fig.44A Schematic diagram showing the bilateral flank tumor and normal MS751 xenograft model. Fig.44B Xenograft size assessed by caliper measurement is shown. Day 0 = xenograft injection. Horizontal dashed line indicates time of T cell injection. mBAsh(A3) = construct with mouse blocker and activator (plus β2M shRNA). DETAILED DESCRIPTION

[0105] Provided herein are compositions and methods for treating cancer using immune cells, wherein the immune cells include a dual receptor system in response to the gene expression differences of ligands between cancer and normal (i.e., healthy or wild-type) cells. These differences in expression may be due to the loss of heterozygosity in cancer cells. Alternatively, the difference in expression may be because the gene is not expressed in cancer cells, or the expression level in cancer cells is lower than that in normal cells. The dual receptor system is expressed in immune cells (e.g., immune cells for adoptive cell therapy), and the activity of these immune cells is targeted to cancer cells showing loss of heterozygosity or expression differences. In the two receptor systems, the first receptor (activator receptor, sometimes referred to as A module herein) activates or promotes the activation of immune cells, while the second receptor (inhibitory receptor, sometimes referred to as blocker, inhibitor receptor or B module herein) acts to inhibit the activation of immune cells by the first receptor. Each receptor contains a ligand binding domain (LBD) that binds to a specific ligand. When the ligand binds, the signals emitted by the two receptors are integrated by immune cells. Differential expression of ligands for the first and second receptors in cancer and normal cells, such as by loss of heterozygosity or differences in transcriptional levels of a locus encoding an inhibitory ligand in cancer cells, mediates activation of immune cells by target cancer cells that express the first activator ligand but not the second inhibitory ligand.

[0106] Loss of heterozygosity (LOH) due to large-scale chromosomal deletions is a source of genetic differences in tumors. LOH is a common event during tumorigenesis, affecting almost every locus in the genome, with an average of about 20% of genes showing LOH in tumors. Because all malignant cells found in tumors lack certain germline alleles, LOH provides a way to clearly distinguish tumors from normal tissues. One locus where LOH occurs is the human leukocyte antigen (HLA) locus, which encodes polymorphic, abundant, ubiquitous surface antigens. The dual receptor system described herein uses one receptor to activate T cells exposed to tumor antigen-positive tumor cells (sometimes referred to as an "activator module"), and uses a second receptor to prevent the activation of immune cells in the presence of surface blocker antigens (e.g., HLA-A*02 or HLA-A*03 proteins). The dual receptor system described herein (sometimes referred to herein as "Tmod") has other advantageous properties as a cell therapy, including but not limited to reversible activation / blocking of immune cells, and selectivity in a mixture of tumors and "normal" cells.

[0107] In specific embodiments of the compositions and methods provided herein, immune cells comprising two receptor systems described herein are used to treat mesothelin (MSLN) positive cancers. This includes mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or bile duct cancer. In some embodiments, the subject's cancer has recurred. In some embodiments, the cancer is refractory to one or more previously administered anticancer therapies. In some embodiments, the cancer is metastatic. For MSLN positive cancers, the target antigen of the activator receptor is MSLN or a peptide antigen thereof complexed with major histocompatibility complex class I (MHC-I). MSLN is expressed in normal fat, fallopian tubes, lungs, and salivary gland tissues, etc. ( Figure 2 ). Because MSLN is expressed in certain tumors, it is an attractive tumor-specific antigen that could mediate selective killing of MSLN+ tumors if these cancer cells could be specifically targeted by appropriate therapeutic approaches. However, normal MSLN expression in non-cancer (non-target) cells prevents the effective use of MSLN for targeted therapies such as adoptive cell therapy. By pairing MSLN activator receptors with inhibitory receptors, the methods provided herein increase the specificity of adoptive cell therapies and reduce the deleterious effects associated with these therapies, such as dose-limiting toxicities.

[0108] In some embodiments, the ligand of the activator is an MSLN peptide complexed with MHC class I. In the methods described herein, this MSLN-targeted activator receptor is paired with an inhibitory receptor to increase the safety window of the activator by blocking its cytolytic effect on normal MSLN-positive tissues. However, since tumor cells do not express the ligand of the inhibitor or blocker receptor, the activator receptor still directs the targeted killing of tumor cells by immune cells containing a dual receptor system. The target of the second inhibitory receptor is expressed by MSLN-positive tissues such as lung, mesothelial and adipose tissue, but not in cancer cells, and the inhibitory receptor recognizes this "non-target antigen" as an inhibitory stimulus. The exemplary target of the second inhibitory receptor is expressed by lung tissue and is lost from MSLN-positive cancer cells due to loss of heterozygosity (LOH) or other mechanisms, leaving a single allele form in the cancer cell, which can be distinguished from other alleles by the allele-specific ligand binding domain on the inhibitory receptor. Exemplary targets of inhibitory receptors include, but are not limited to, major histocompatibility complex (MHC) proteins such as human leukocyte antigen A (HLA-A). HLA-B, HLA-C, and other HLA. HLA is encoded by variant genes such as HLA-A*01, HLA-A*02, HLA-A*03, HLA-C*07, etc., which can be lost from MSLN-positive cancer cells by loss of heterozygosity. Alternatively, further exemplary targets of inhibitory receptors include, but are not limited to, intercellular adhesion molecule 1 (ICAM1), catechol-O-methyltransferase (COMT), and CXC motif chemokine ligand 16 (CXCL16). Each of these has common non-synonymous variant forms, and the amino acid changes in its extracellular domain can be contacted by antibodies and can be used as inhibitory receptors or blocking receptor targets for cell integrators designed to safely treat MSLN-positive cancer patients using engineered T cells activated by activator receptors such as MSLN or MSLN pMHC-responsive activator receptors.

[0109] The compositions and methods disclosed herein can reduce or eliminate dose-limiting toxicity (DLT) caused by the expression of MSLN on normal tissues. The present disclosure provides a method for treating MSLN-positive cancers using adoptive cell therapy by adding a second inhibitory receptor to target MSLN in cancer cells, wherein the second inhibitory receptor blocks the activation of adoptive immune cells in the presence of a second ligand (a ligand other than MSLN, referred to as a non-target antigen or a blocker antigen). Using the compositions and methods described herein, tumor cells expressing MSLN are attacked by adoptive cells (such as immune cells) expressing both receptors because these tumor cells only express the activator ligand MSLN. In contrast, normal cells expressing MSLN and non-target antigens are protected from attack by adoptive immune cells. Inhibitory receptor responses to non-target antigens on normal cells can prevent MSLN-targeted activator receptors from activating immune cells. This dual-targeting approach creates a therapeutic window that enables MSLN-directed cell therapy to be safely and effectively performed in patients with MSLN-positive cancers.

[0110] The present disclosure provides methods and compositions that allow the use of potent MSLN CARs and TCRs that induce on-target toxicity and render these MSLN-targeted receptors useful as therapeutic agents by mitigating their toxicity.

[0111] In variations, the compositions and methods described herein can be used to kill target cells and / or treat subjects who have partially or completely reduced expression of non-target antigens due to reasons other than loss of heterozygosity, including but not limited to partial gene deletions, epigenetic silencing, and point or truncating mutations in the sequence encoding the non-target antigen.

[0112] The methods and compositions described in U.S. Patent Application Publication No. 2022 / 0370497 are incorporated herein by reference in their entirety.

[0113] definition

[0114] Before describing the present disclosure in greater detail, it may be helpful to understand the present disclosure by providing definitions of certain terms used herein.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For purposes of the present disclosure, the following terms are defined below. Other definitions are set forth in the present disclosure.

[0116] As used herein, the term "about" or "approximately" refers to an amount, level, value, amount, frequency, percentage, dimension, size, quantity, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, amount, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of an amount, level, value, amount, frequency, percentage, dimension, size, quantity, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to a reference amount, level, value, amount, frequency, percentage, dimension, size, quantity, weight, or length.

[0117] As used herein, the term "isolated" means material that is substantially or essentially free from components that normally accompany it in its native state. In specific embodiments, the terms "obtained" or "derived" are used synonymously with isolated.

[0118] The terms "subject", "patient" and "individual" are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro. "Subject", "patient" or "individual" as used herein include any animal that exhibits pain that can be treated with the vectors, compositions and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits or guinea pigs), farm animals and livestock or pets (such as cats or dogs). Non-human primates are included, preferably human patients are included.

[0119] As used herein, "treatment" or "treating" includes any beneficial or desired effect and may include even minimal amelioration of symptoms. "Treatment" does not necessarily mean complete eradication or cure of a disease or condition or its associated symptoms.

[0120] As used herein, "prevent" and similar terms such as "prevented," "preventing," and the like refer to methods used to prevent, inhibit, or reduce the likelihood of disease symptoms. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of disease symptoms.

[0121] As used herein, "preventing" and similar terms also include reducing the intensity, effects, symptoms and / or burden of a disease prior to the onset or recurrence of the disease.

[0122] As used herein, the term "amount" refers to "an amount effective" or "an effective amount" of a virus to achieve beneficial or desired prophylactic or therapeutic results, including clinical results.

[0123] A "therapeutically effective amount" of a virus or cell can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the virus or cell. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient).

[0124] An "increase" or "enhancement" of a physiological response (e.g., electrophysiological activity or cellular activity) is generally a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the level of activity in untreated cells.

[0125] The amount of a "reduction" or "reduction" in a physiological response (e.g., electrophysiological activity or cellular activity) is generally a "statistically significant" amount and can include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimal points between and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) relative to the level of activity in untreated cells.

[0126] "Maintain" or "preserve" or "maintenance" or "no change" or "no significant change" or "no significant decrease" generally refers to a physiological response that is comparable to the response elicited by a vehicle or control molecule / composition. A comparable response is one that is not significantly different or measurably different from a reference response.

[0127] Generally, "sequence identity" or "sequence homology" refers to the precise nucleotide-nucleotide or amino acid-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Generally, the technology used to determine sequence identity includes determining the nucleotide sequence of the polynucleotide and / or determining the amino acid sequence encoded therein, and comparing these sequences with a second nucleotide sequence or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by measuring their "percent identity". The percent identity of two sequences (whether nucleic acid sequences or amino acid sequences) is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Percent identity can also be determined, for example, by using advanced BLAST computer programs (including version 2.2.9, available from the National Institutes of Health) to compare sequence information. The BLAST program is based on the alignment method of Karlin and Altschul, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (usually nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine the percent identity over the entire length of the compared proteins. Default parameters are provided to optimize searches using short query sequences, for example, in the blastp program. The program also allows the use of SEG filters to mask segments of query sequences determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The desired degree of sequence identity ranges from about 80% to 100% and integer values ​​therebetween. Typically, the percent identity between the disclosed sequence and the claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0128] As used herein, a "polynucleotide system" refers to one or more polynucleotides. One or more polynucleotides can be designed to work in conjunction with a specific application, or to produce a desired transformed cell.

[0129] As used herein, the term "exogenous" refers to any molecule originating from outside an organism, including nucleic acids, proteins or peptides, small molecule compounds, etc. In contrast, the term "endogenous" refers to any molecule originating from within an organism (ie, naturally produced by the organism).

[0130] The term "MOI" is used herein to refer to the multiplicity of infection, which is the ratio of agent (eg, viral particles) to infected target (eg, cells).

[0131] In this specification, unless otherwise specified, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range, and where appropriate, include its fractions (such as one tenth and one hundredth of an integer). When the term "about" is immediately before a number or value, it means a range of up to ±10% of the number or value.

[0132] As used herein, "target cell" refers to a cell targeted by adoptive cell therapy. For example, a target cell can be a cancer cell, which can be killed by a transplanted T cell of adoptive cell therapy. The target cells of the present disclosure express a target antigen as described herein, but do not express a non-target antigen.

[0133] As used herein, "non-target cells" refer to cells that are not targeted by adoptive cell therapy. For example, in adoptive cells targeting cancer cells, normal, healthy, non-cancerous cells are non-target cells. Some or all non-target cells in a subject may express both target antigens and non-target antigens. Non-target cells in a subject may express non-target antigens regardless of whether these cells also express the target antigen.

[0134] As used herein, "non-target allelic variant" refers to an allele of a gene whose product is expressed by non-target cells but not by target cells. For example, a non-target allelic variant is an allele of a gene expressed by a normal non-cancer cell of a subject but not by a cancer cell of a subject. Expression of a non-target allelic variant can be lost in cancer cells by any mechanism, including but not limited to loss of heterozygosity, mutation, or epigenetic modification of the gene encoding the non-target allelic variant.

[0135] As used herein, "specific for" or "specifically binds to" when used with respect to a ligand binding domain, such as an antigen binding domain, refers to a ligand binding domain that has high specificity for a given target. Antibody specificity can be viewed as the goodness of fit between a ligand binding domain and the corresponding ligand, or a measure of the ability of the ligand binding domain to discriminate between similar or even dissimilar ligands. In contrast to specificity, affinity is a measure of the strength of binding between a ligand binding domain and a ligand, such that low affinity ligand binding domains bind weakly, while high affinity ligand binding domains bind strongly. A ligand binding domain that is specific for a target allele is one that is able to discriminate between different alleles of a gene. For example, a ligand binding domain that is specific for HLA-A*02 does not bind, or only weakly binds, to other HLA-A alleles, such as HLA-A*01 or HLA-A*03. Those skilled in the art will appreciate that a ligand binding domain can be said to be specific for a particular target and still have low levels of binding to one or more additional targets that do not affect its function in the receptor systems described herein.

[0136] As used herein, "target antigen", whether referring to the term antigen or the name of a specific antigen, refers to an antigen expressed by a target cell, such as a cancer cell. The expression of the target antigen is not limited to the target cell. The target antigen can be expressed by cancer cells and normal non-cancerous cells in a subject.

[0137] As used herein, "non-target antigen" (or "blocker antigen"), whether referring to the term antigen or the name of the specific antigen, refers to an antigen expressed by normal non-cancerous cells and not expressed in cancer cells. This difference in expression allows inhibitory receptors to inhibit immune cell activation in the presence of non-target cells but not in the presence of target cells.

[0138] Polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population. Polymorphism may comprise one or more base changes, insertions, repetitions or deletions. Polymorphisms include, for example, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), which are variations that occur when a single nucleotide of adenine (A), thymine (T), cytosine (C) or guanine (G) changes.

[0139] As used herein, "affinity" refers to the strength of binding of a ligand to a single ligand binding site on a receptor, e.g., an antigen for an antigen binding domain of any receptor described herein. A ligand binding domain can have a weaker interaction (low affinity) or a stronger interaction (high affinity) with its ligand.

[0140] Kd or dissociation constant is an equilibrium constant that measures the tendency of a larger object to reversibly separate into smaller components, such as when a macromolecular complex containing a receptor and its cognate ligand separates into ligand and receptor. When Kd is high, it means that a high concentration of ligand is required to occupy the receptor and the receptor has a low affinity for the ligand. Conversely, a low Kd means that the ligand has a high affinity for the receptor.

[0141] As used herein, a "responsive" or "responsive to" receptor refers to a receptor comprising an intracellular domain that, when bound to a ligand (i.e., an antigen), generates a signal corresponding to a known function of the intracellular domain. An activator receptor bound to a target antigen can generate a signal that causes activation of an immune cell expressing the activator receptor. An inhibitory receptor bound to a non-target antigen can generate an inhibitory signal that prevents or reduces activation of an immune cell expressing the activator receptor. The responsiveness of a receptor and its ability to activate or inhibit an immune cell expressing the receptor can be determined by any method known in the art and described herein, including but not limited to reporter gene assays and cytotoxicity assays.

[0142] As used herein, the "activation" of immune cells or immune cells are "activated" refers to immune cells that can perform one or more characteristic functions of immune response. These functions include proliferation, cytokine release and cytotoxicity, i.e., killing target cells. Activated immune cells express markers that are obvious to those skilled in the art. For example, activated T cells can be one or more of CD69, CD71, CD25 and HLA-DR. When immune cells expressing activator receptors (e.g., MSLN CAR) respond to the binding of receptors to target antigens (e.g., MSLN) expressed by target cells, they can be activated by activator receptors. "Target antigen" may also be referred to as "activation antigen" and may be separated or expressed by target cells. When inhibitory receptors respond to non-target antigens (e.g., HLA-A*02), even when activator receptors bind to target activator ligands, the activation of immune cells expressing inhibitory receptors may be prevented. "Non-target antigens" may also be referred to as "inhibitory ligands" or "blockers" and may be separated or expressed by target cells.

[0143] Receptor expression on immune cells can be verified by reporting the presence of activator receptors and inhibitory receptors as described herein. For example, immune cell populations can be stained with labeled molecules (e.g., fluorophore-labeled receptor-specific antibodies or fluorophore-labeled receptor-specific ligands) and quantified using fluorescence activated cell sorting (FACS) flow cytometry. This method allows the percentage of immune cells in immune cell populations to be characterized as expressing activator receptors, inhibitory receptors, or two receptors. The ratio of activator receptors and inhibitory receptors expressed by immune cells described herein can be measured by, for example, digital droplet PCR. These methods can be used to characterize cell populations for producing and manufacturing immune cells, pharmaceutical compositions, and kits as described herein. For immune cells, pharmaceutical compositions, and kits as described herein, it should be understood that the appropriate percentage of immune cells expressing both activator receptors and inhibitory receptors is determined specifically for the methods described herein. For example, the appropriate percentage of immune cells expressing activator receptors and inhibitory receptors can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. As another example, 50% to 99%, 60% to 95%, 65% to 95%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 80% to 99%, 85% to 99%, 90% to 99%, or 95% to 99% of the immune cells may express both activator receptors and inhibitory receptors. For example, a suitable ratio of activator receptors to inhibitory receptors in immune cells may be about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5. It should be understood that purification, enrichment, and / or depletion steps may be used for immune cell populations to meet suitable values ​​for the immune cells, pharmaceutical compositions, and kits described herein.

[0144] The responsive receptors expressed by immune cells described herein can be verified by measuring the generation of the signal generated by the intracellular domain of the receptor. Reporter cell lines such as Jurkat-luciferase NFAT cells (Jurkat cells) can be used to characterize responsive receptors. Jurkat cells are derived from T cells and contain a stably integrated nuclear factor of activated T cells (NFAT)-inducible luciferase reporter system. NFAT is a family of transcription factors required for immune cell activation, and its activation can be used as a signal transduction marker for T cell activation. Jurkat cells can be transduced or transfected with activator receptors and / or inhibitory receptors as described herein. If Jurkat cells express a luciferase reporter gene, the activator receptor responds to the binding of the ligand, and the level of response can be determined by the level of reporter gene expression. The presence of luciferase can be measured using any known luciferase detection reagent (such as luciferin). If when co-expressed with the activator receptor in Jurkat cells, the inhibitory receptor responds to the binding of the ligand, it prevents the immune cells of normal response from expressing luciferase that responds to the activator receptor. For example, the responsiveness of inhibitory receptors can be determined and quantified in Jurkat cells expressing both activators and inhibitors by observing the following: 1) Jurkat cells express luciferase in the presence of activator receptor ligands and in the absence of inhibitory receptor ligands; and 2) in the presence of activator receptor ligands and inhibitory receptor ligands, luciferase expression in Jurkat cells is reduced or eliminated. This method can be used to measure the sensitivity, efficacy and selectivity of specific pairs of activator receptors and activator receptors and inhibitory receptors. Dose-response experiments can be used to quantify sensitivity, efficacy and selectivity by EC50 or IC50 values, wherein activator receptor ligands and / or inhibitory receptor ligands are titrated into Jurkat cell cultures expressing specific pairs of activator receptors or activators and inhibitory receptors. Alternatively, EC50 and IC50 values ​​can be measured in co-cultures of immune cells (e.g., Jurkat cells or primary immune cells) expressing specific pairs of activator receptors or activators and inhibitory receptors and target cells expressing increasing amounts of activator ligands or inhibitory ligands. Increasing amounts of activator ligand or inhibitor ligand can be achieved in target cells by, for example, titrating activator ligand or inhibitor ligand encoding mRNA into target cells, or using target cells that naturally express different levels of target ligand. Exemplary suitable EC50 and IC50 values ​​for activators and inhibitory receptors determined using target cells expressing different amounts of target ligand and non-target ligand include EC50s of 10 transcripts per million (TPM) or less for activator receptors, such as EC50s of 2 to 10 TPM, and IC50s of 25 TPM or less for inhibitory receptors, such as IC50s of 5-21 TPM.

[0145] The activation of the immune cells described herein that express a specific pair of activator receptors or activators and inhibitory receptors can be further determined by measuring the assay of target cell viability after co-incubation with the immune cells. Immune cells, sometimes referred to as effector cells, are co-incubated with target cells expressing activator receptor ligands, inhibitory receptor ligands, or both activators and inhibitory receptor ligands. After co-incubation, the viability of the target cells is measured using any method that measures cell culture viability. For example, a mitochondrial function assay that measures active mitochondrial enzymes using tetrazolium salt substrates can be used to determine viability. Vitality can also be determined using imaging-based methods. Target cells can express fluorescent proteins, such as green fluorescent protein or red fluorescent protein. A reduction in total cell fluorescence indicates a reduction in target cell viability. After incubation with immune cells that express a specific pair of activator receptors or activators and inhibitory receptors, the reduction in target cell viability is interpreted as the activation of target cell-mediated immune cells. The measurement of immune cell selectivity can also be determined using this method. If the following is observed, then the immune cells expressing the activator receptor and inhibitory receptor pair are selective: 1) the viability is reduced in target cells that express the activator receptor ligand but do not express the inhibitory receptor ligand; 2) the viability is not reduced in target cells that express both the activator receptor ligand and the inhibitory receptor ligand. From these measurements, a "specific killing" value can be derived, which quantifies the percentage of immune cell activation based on the reduction in target cell viability as a percentage of the negative control (immune cells that do not express the activator receptor). In addition, a "selectivity ratio" value can be derived from these measurements, which represents the ratio of specific killing observed in target cells expressing activator receptor ligands in the absence of inhibitory receptor ligands to specific killing observed in target cells expressing both activator receptor ligands and inhibitory receptor ligands. This method can be used to characterize cell populations used to produce and prepare immune cells, pharmaceutical compositions, and kits described herein. Suitable specific killing values ​​for immune cells, pharmaceutical compositions and kits can be, for example, the following standards: 1) at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% specific killing after 48 hours of co-incubation of immune cells expressing activator receptor ligands and target cells in the absence of inhibitory receptor ligands; and 2) specific killing of target cells expressing activator receptor ligands and inhibitory receptor ligands is less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, less than or equal to 5%, less than or equal to 3% or less than or equal to 1%.

[0146] As another example, suitable specific killing values ​​for immune cells, pharmaceutical compositions and kits can be the following standards: 1) 30% to 99%, 40% to 99%, 50% to 99%, 55% to 95%, 60% to 95%, 60% to 90%, 50% to 80%, 50% to 70% or 50% to 60% of target cells expressing activator ligands but not inhibitor ligands are killed; and 2) 1% to 40%, 3% to 40%, 5% to 40%, 5% to 30%, 10% to 30%, 15% to 30% or 5% to 20% of target cells expressing activator ligands and inhibitor ligands are killed. As another example, suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits can be, for example, the following criteria: 1) at least 50% specific killing after 48 hours of co-incubation of immune cells expressing activator receptor ligands and target cells in the absence of inhibitory receptor ligands; and 2) less than or equal to 20% specific killing of target cells expressing activator receptor ligands and inhibitory receptor ligands. As another example, the immune cell is capable of killing at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% of target cells expressing the activator ligand but not the inhibitor ligand within a time period of 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours or 60 hours, while killing less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3% or less than 1% of target cells expressing both the activator ligand and the inhibitor ligand within the same time period.

[0147] For immune cells, pharmaceutical compositions and kits, suitable specific killing values ​​for target cells expressing activator ligands in the absence of inhibitory ligand values ​​can be, for example, at least about 50% to at least about 95%. For immune cells, pharmaceutical compositions and kits, suitable specific killing values ​​for target cells expressing activator ligands in the absence of inhibitory ligand values ​​can be, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%. For immune cells, pharmaceutical compositions and kits, suitable specific killing values ​​for target cells expressing activator ligands in the absence of inhibitory ligand values ​​can be, for example, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90% or at most about 95%. For immune cells, pharmaceutical compositions and kits, suitable specific killing values ​​for target cells expressing both activator receptor ligands and inhibitory receptor ligands can be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. Suitable specific killing values ​​for immune cells, pharmaceutical compositions and kits can be determined after the immune cells and target cells are co-incubated for about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, or about 72 hours.

[0148] For immune cells, pharmaceutical compositions and kits, suitable specific killing values ​​for target cells expressing activator ligands in the absence of inhibitory ligand values ​​can be, for example, at least about 50% to at least about 95%. For immune cells, pharmaceutical compositions and kits, suitable specific killing values ​​for target cells expressing activator ligands in the absence of inhibitory ligand values ​​can be, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%. For immune cells, pharmaceutical compositions and kits, suitable specific killing values ​​for target cells expressing activator ligands in the absence of inhibitory ligand values ​​can be, for example, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90% or at most about 95%. For immune cells, pharmaceutical compositions and kits, suitable specific killing values ​​for target cells expressing both activator receptor ligands and inhibitory receptor ligands can be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. Suitable specific killing values ​​for immune cells, pharmaceutical compositions and kits can be determined after the immune cells and target cells are co-incubated for about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, or about 72 hours.

[0149] As used herein, the term "functional variant" refers to a protein having one or more amino acid substitutions, insertions or deletions compared to a parent protein and retaining one or more desired activities of the parent protein. A functional variant can be a protein fragment (i.e., a variant having an N-terminal and / or C-terminal deletion) that retains one or more desired activities of the parent protein.

[0150] All publications and patents mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application (including any definitions herein) shall prevail. However, the mention of any reference, article, publication, patent, patent disclosure and patent application cited herein is not and should not be regarded as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0151] Activator receptor

[0152] The present disclosure provides a first receptor, which includes a first extracellular ligand binding domain specific to a target antigen, the target antigen including a cancer cell-specific antigen or a peptide antigen complexed with a major histocompatibility complex class I (MHC-I). The first receptor is an activator receptor, and mediates the activation of immune cells expressing the first receptor when the extracellular ligand binding domain of the first receptor binds to the target antigen. The first receptor responds to the target antigen (i.e., the activator ligand). For example, when the target antigen binds or contacts the first receptor, the first receptor responds and activates immune cells expressing the first receptor when the extracellular ligand binding domain of the first receptor binds to the target antigen. In some embodiments, the first receptor is a chimeric antigen receptor (CAR). In some embodiments, the first receptor is a T cell receptor (TCR).

[0153] In some embodiments, the first receptor is humanized. As used herein, "humanization" refers to replacing a sequence or subsequence in a transgenic isolated from or derived from a non-human species with a homologous or functionally equivalent human sequence. For example, a humanized antibody can be produced by transplanting mouse CDRs into human framework sequences, followed by reverse replacement of the corresponding mouse residues from the source antibody with certain human framework residues.

[0154] Activator Target

[0155] In some embodiments, the target antigen of the first receptor is a cancer cell specific antigen. Any cell surface molecule expressed by the target cancer cell can be a suitable target antigen for the first receptor ligand binding domain. For example, a cell adhesion molecule, a cell-cell signaling molecule, an extracellular domain, a molecule related to chemotaxis, a glycoprotein, a G protein coupled receptor, a transmembrane protein, a neurotransmitter receptor, or a voltage-gated ion channel can be used as a target antigen.

[0156] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen complexed with major histocompatibility complex class I (MHC-I). Any molecule expressed by the target cancer cell and presented as a peptide antigen (pMHC) by major histocompatibility complex class I (MHC-I) on the surface of the cancer cell can be a suitable target antigen for the first receptor extracellular ligand binding domain.

[0157] In some embodiments, the cancer cell-specific antigen is mesothelin (MSLN) or a peptide antigen thereof complexed with major histocompatibility complex class I (MHC-I).

[0158] Major histocompatibility complex class I (MHC-I) is a protein complex that displays antigens to immune system cells, triggering an immune response. The human leukocyte antigens (HLA) corresponding to MHC-I are HLA-A, HLA-B, and HLA-C.

[0159] Cancer cell specific pMHC antigens comprising any one of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G are considered to be within the scope of the present disclosure. In some embodiments, the cancer cell specific antigen comprises HLA-A. The HLA-A receptor is a heterodimer comprising a heavy α chain and a smaller β chain. The α chain is encoded by a variant of HLA-A, while the β chain (β2-microglobulin) is constant. There are thousands of HLA-A gene variants, all of which fall within the scope of the present disclosure. In some embodiments, MHC-I comprises a human leukocyte antigen A*02 allele (HLA-A*02).

[0160] In some embodiments, the cancer cell specific antigen comprises HLA- B. Hundreds of forms (alleles) of the HLA-B gene are known, each of which is assigned a specific number (eg, HLA-B*27).

[0161] In some embodiments, the cancer cell specific antigen comprises HLA-C. HLA-C belongs to the HLA class I heavy chain paralog. The class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). More than 100 HLA-C alleles are known in the art.

[0162] In some embodiments, the cancer cell specific antigen is an ovarian cancer antigen, a pancreatic cancer antigen, a lung cancer antigen, a colorectal cancer antigen, or a mesothelioma antigen. In some embodiments, the cancer cell specific antigen is a colorectal cancer antigen. In some embodiments, the cancer cell specific antigen is MSLN or a peptide antigen thereof.

[0163] In some embodiments, the cancer cell specific antigen is MSLN or a peptide antigen thereof complexed with major histocompatibility complex class I (MHC-I). MSLN is a 40KDa protein that is normally expressed in mesothelial cells as well as in the lungs, fallopian tubes, salivary glands, and adipose tissue ( Figure 2 MSLN is expressed in a variety of human tumor types, including mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or bile duct cancer. In some embodiments, the subject's cancer has recurred. In some embodiments, the cancer is refractory to one or more previously administered anticancer therapies. In some embodiments, the cancer is metastatic.

[0164] All MSLN isoforms are considered cancer cell specific antigens of the present disclosure. The MSLN isoform 1 proprotein is described in NCBI Record No. NP_005814.2, the contents of which are incorporated herein by reference. In some embodiments, MSLN comprises the following amino acid sequence:

[0165]

[0166] In some embodiments, MSLN comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1.

[0167] MSLN isoform 2 proprotein is described in NCBI Record No. NP_037536.2, the contents of which are incorporated herein by reference. In some embodiments, MSLN comprises the following amino acid sequence:

[0168]

[0169]

[0170] In some embodiments, MSLN comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2.

[0171] In some embodiments, the cancer cell specific antigen is a peptide antigen derived from MSLN. In some embodiments, the peptide antigen comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a subsequence of SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the peptide antigen comprises a sequence that is identical to a subsequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0172] Extracellular ligand binding domain

[0173] The present disclosure provides a first receptor comprising a first extracellular ligand binding domain specific for a target antigen. In some embodiments, the target antigen comprises a cancer cell specific antigen.

[0174] In some embodiments, the cancer cell-specific antigen is MSLN or a MSLN-derived peptide antigen complexed with MHC-I, and the ligand binding domain of the first receptor recognizes and binds to the MSLN antigen.

[0175] Any type of ligand binding domain that can modulate receptor activity in a ligand-dependent manner is considered to be within the scope of the present disclosure. In some embodiments, the ligand binding domain is an antigen binding domain. Exemplary antigen binding domains particularly include scFv, SdAb, only Vβ domains, and TCR antigen binding domains derived from TCRα and β chain variable domains.

[0176] Any type of antigen binding domain is considered to be within the scope of the present disclosure.

[0177] For example, the first extracellular ligand binding domain can be part of a continuous polypeptide chain, the continuous polypeptide chain including, for example, only a Vβ domain, a single domain antibody fragment (sdAb) or a heavy chain antibody HCAb, a single chain antibody (scFv) derived from a mouse, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor; NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426). In some aspects, the first extracellular ligand binding domain comprises an antigen binding domain, which comprises an antibody fragment. In a further aspect, the first extracellular ligand binding domain comprises an antibody fragment comprising a scFv or a sdAb.

[0178] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins or fragments thereof of polyclonal or monoclonal origin, and can be derived from natural or recombinant sources.

[0179] The term "antibody fragment" or "antibody binding domain" refers to at least a portion of an antibody or a recombinant variant thereof that contains an antigen binding domain, i.e., the antigen-determining variable region of an intact antibody, which is sufficient to confer recognition and specific binding to a target such as an antigen and its defined epitope on the antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, single-chain (sc) FV ("scFv") antibody fragments, linear antibodies, single domain antibodies (abbreviated as "sdAb") (VL or VH), Camelidae VHH domains, and multispecific antibodies formed from antibody fragments.

[0180] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected by a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0181] A "heavy chain variable region" or "VH" with respect to antibodies (or "VHH" in the case of single domain antibodies, such as Nanobodies) refers to the fragment of the heavy chain that contains the three CDRs inserted between flanking stretches called framework regions, which are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0182] Unless otherwise specified, a scFv used herein may have the VL and VH variable regions in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0183] In some embodiments, the antigen binding domain of the activator receptor and / or inhibitory receptor comprises an scFv. In some embodiments, the scFv comprises a VL region and a VH region connected by a joint. In some embodiments, the joint comprises a glycine serine joint, such as GGGGSGGGGSGGGGSGG (SEQ ID NO: 152). In some embodiments, the scFv further comprises a signal sequence at the N-terminus of the scFv. Exemplary signal sequences include MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 362), which is encoded by ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGT (SEQ ID NO: 153).

[0184] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa ("K") and lambda ("λ") light chains refer to the two major antibody light chain isotypes.

[0185] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. The term should also be interpreted as referring to an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein, or an amino acid sequence specific to the antibody, wherein the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0186] The term "Vβ domain", "Vβ domain only", "β chain variable domain" or "single variable domain TCR (svd-TCR)" refers to an antigen binding domain consisting essentially of a single T cell receptor (TCR) β variable domain that specifically binds to an antigen in the absence of a second TCR variable domain. Only the Vβ domain engages the antigen using the complementary determining region (CDR). Each Vβ domain alone contains three complement determining regions (CDR1, CDR2 and CDR3). Additional elements can be combined provided that the Vβ domain is configured to bind to an epitope in the absence of a second TCR variable domain.

[0187] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an antibody fragment, a single-chain Fv antibody fragment (scFv), or a beta chain variable domain (Vβ).

[0188] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a TCR alpha chain variable domain and a TCR beta chain variable domain.

[0189] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a scFv antigen binding domain. Exemplary MSLN scFvs are shown in Table 1 below.

[0190] Table 1: Exemplary MSLNscFv domains

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221] In some embodiments, the extracellular ligand binding domain of the first receptor is a scFv. In some embodiments, the scFv domain binds to MSLN. In some embodiments, the scFv is the ligand binding domain of CAR. Exemplary scFv domains specific for MSLN are shown in Table 1 above. In Table 1, underlines represent CDR sequences.

[0222] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an antigen binding domain that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to a sequence of SEQ ID NO: 3-6, 80, or 154-215, or a sequence as shown in Table 1. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an antigen binding domain that comprises a sequence of SEQ ID NO: 3-6, 80, or 154-215 as shown in Table 1.

[0223] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a binding domain that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to the sequence of SEQ ID NO: 171. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a binding domain comprising the sequence of SEQ ID NO: 171.

[0224] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a scFv antigen binding domain having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, or at least 99% identity to any one of SEQ ID NOs: 3-6. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a scFv antigen binding domain comprising a sequence of any one of SEQ ID NOs: 3-6 or 80. In some embodiments, the extracellular ligand binding domain of the first receptor consists essentially of a sequence selected from SEQ ID NOs: 3-6 or 80.

[0225] Table 2: Sequences of the MSLN complementarity determining regions (CDRs)

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] In Table 2, the left column identifies the light chain (LC) CDRs that pair with the indicated heavy chain (HC) CDRs.

[0232] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises HC CDR1, HC CDR2, and HC CDR3 shown in Table 2 (e.g., HC CDR1, HC CDR2, and HC CDR3 in row 1, row 2, row 3, etc. of Table 2), or a sequence having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs in Table 2. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises LC CDR1, LC CDR2, and LC CDR3 shown in Table 2 (e.g., LC CDR1, LC CDR2, and LC CDR3 in row A, row B, or row C of Table 2), or a sequence having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs in Table 2. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises HC CDR1, HC CDR2, and HC CDR3 shown in Table 2 (e.g., HC CDR1, HC CDR2, and HC CDR3 in row 1, row 2, row 3, etc. of Table 2). In some embodiments, the extracellular ligand-binding domain of the first receptor comprises LC CDR1, LC CDR2, and LC CDR3 shown in Table 2 (eg, LC CDR1, LC CDR2, and LC CDR3 of row A, row B, or row C of Table 2).

[0233] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 shown in Table 2 (e.g., HC CDR1, HC CDR2, and HC CDR3 shown in row 1, and LC CDR1, LC CDR2, and LC CDR3 shown in row A). In some embodiments, the extracellular ligand-binding domain of the first receptor comprises HC CDR1, HC CDR2, and HC CDR3 shown in Table 2 (e.g., HC CDR1, HC CDR2, and HC CDR3 in row 1, row 2, row 3, etc. of Table 2), or a sequence having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 2. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises LC CDR1, LC CDR2, and LC CDR3 shown in Table 2 (e.g., LC CDR1, LC CDR2, and LC CDR3 in row A, row B, or row C of Table 2) or a sequence having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs in Table 2. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises one or more HC CDRs shown in Table 2 and one or more LC CDRs shown in Table 2. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises (i) HC CDR1, HC CDR2, and HC CDR3 shown in a row of Table 2 (e.g., HC CDR1, HC CDR2, and HC CDR3 in row 1, row 2, row 3, etc. of Table 2), and (ii) LC CDR1, LC CDR2, and LC CDR3 shown in a row of Table 2 (e.g., LC CDR1, LC CDR2, and LC CDR3 in row A, row B, or row C of Table 2). In each case, the HC CDR can be paired with any LC CDR, and because of the similarity of the heavy and light chains, the desired expression and binding activity can be confirmed by routine testing; however, the right column of Table 2 indicates preferred pairings between heavy and light chains in some embodiments.

[0234] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a HC CDR1 comprising the sequence SGDYYWS (SEQ ID NO: 438), a HC CDR2 comprising the sequence YIYYSGSTYYNPSLKS (SEQ ID NO: 454), and a HC CDR3 comprising the sequence CAREDVVKGAFDIW (SEQ ID NO: 533), or a CDR sequence having at most 1, 2, or 3 amino acid substitutions, insertions, or deletions relative thereto. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a HC CDR1 comprising the sequence SGDYYWS (SEQ ID NO: 438), a HC CDR2 comprising the sequence YIYYSGSTYYNPSLKS (SEQ ID NO: 454), and a HC CDR3 comprising the sequence CAREDVVKGAFDIW (SEQ ID NO: 533). In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a LC CDR1 comprising the sequence RASQSISSYLN (SEQ ID NO: 535), a LC CDR2 comprising the sequence AASSLQS (SEQ ID NO: 539), a LC CDR3 comprising the sequence QQSYSTPLT (SEQ ID NO: 542), or a CDR sequence having at most 1, 2, or 3 amino acid substitutions, insertions, or deletions relative thereto. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a LC CDR1 comprising the sequence RASQSISSYLN (SEQ ID NO: 535), a LC CDR2 comprising the sequence AASSLQS (SEQ ID NO: 539), and a LC CDR3 comprising the sequence QQSYSTPLT (SEQ ID NO: 542). In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising the sequence SGDYYWS (SEQ ID NO:438), a HC CDR2 comprising the sequence YIYYSGSTYYNPSLKS (SEQ ID NO:454), a HC CDR3 comprising the sequence CAREDVVKGAFDIW (SEQ ID NO:533), a LC CDR1 comprising the sequence RASQSISSYLN (SEQID NO:535), a LC CDR2 comprising the sequence AASSLQS (SEQ ID NO:539), and a LC CDR3 comprising the sequence QQSYSTPLT (SEQ ID NO:542), or CDR sequences having up to 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto.In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a HC CDR1 comprising the sequence SGDYYWS (SEQ ID NO:438), a HC CDR2 comprising the sequence YIYYSGSTYYNPSLKS (SEQ ID NO:454), a HC CDR3 comprising the sequence CAREDVVKGAFDIW (SEQ ID NO:533), a LC CDR1 comprising the sequence RASQSISSYLN (SEQID NO:535), a LC CDR2 comprising the sequence AASSLQS (SEQ ID NO:539), and a LC CDR3 comprising the sequence QQSYSTPLT (SEQ ID NO:542).

[0235] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv. In some embodiments, the scFv comprises a heavy chain comprising a CDR of a sequence selected from GYTMN (SEQ ID NO: 448), LITPYNGASSYNQKFRG (SEQ ID NO: 470), and GGYDGRGFDY (SEQ ID NO: 534). In some embodiments, the heavy chain comprises the sequence GYTMN (SEQ ID NO: 448), LITPYNGASSYNQKFRG (SEQ ID NO: 470), and GGYDGRGFDY (SEQ ID NO: 534). In some embodiments, the scFv comprises a light chain comprising a CDR of a sequence selected from SASSSVSYMH (SEQ ID NO: 538), DTSKLAS (SEQ ID NO: 541), and QQWSGYPLT (SEQ ID NO: 545). In some embodiments, the light chain comprises the sequence of SASSVSYMH (SEQ ID NO:538), DTSKLAS (SEQ ID NO:541), and QQWSGYPLT (SEQ ID NO:545).

[0236] The sequences of exemplary heavy and light chains of antigen binding domains specific for MSLN are shown below in Tables 3 and 4. In a preferred embodiment, the light chain paired with the heavy chain is shown on the right side of Table 3.

[0237] Table 3: Sequences of heavy chain variable fragments (VH)

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] Table 4: Sequences of light chain variable fragments (VL)

[0244]

[0245] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy chain region (VH) sequence shown in Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the VH shown in Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable light chain region (VL) sequence shown in Table 4. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the VL shown in Table 4.

[0246] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a VH, which (i) comprises the HC CDR1, HC CDR2 and HC CDR3 sequences shown in Table 2 (e.g., the HC CDR1, HC CDR2 and HC CDR3 in row 1, row 2, row 3, etc. of Table 2), and (ii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the VH sequence shown in Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor (i) comprises the LC CDR1, LC CDR2 and LC CDR3 sequences shown in a row of Table 2 (e.g., LC CDR1, LC CDR2 and LC CDR3 in row A, row B or row C of Table 2), and the VL sequence shown in Table 4, and (ii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the VL shown in Table 4.

[0247] In some embodiments, the extracellular ligand binding domain of the first receptor comprises (i) a VH sequence as shown in Table 3, or a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VH as shown in Table 3, and (ii) a VL sequence as shown in Table 4, or a VL having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VL as shown in Table 4. In each case, due to the similarity of the heavy and light chains, the VH can be paired with any VL to confirm the desired expression and binding activity by routine testing; however, the preferred pairings between Table 3 and Table 4 are indicated in the "LC" column of Table 3, corresponding to the # column of Table 4.

[0248] In some embodiments, the extracellular ligand binding domain of the first receptor comprises the VH sequence of SEQ ID NO: 233, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the VH sequence of SEQ ID NO: 233.

[0249] In some embodiments, the extracellular ligand binding domain of the first receptor comprises the VL sequence of SEQ ID NO: 279, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the VL sequence of SEQ ID NO: 279. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the VH sequence of SEQ ID NO: 233 and the VL sequence of SEQ ID NO: 279, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, VH and VL are separated by a linker, e.g., a linker comprising the sequence GGGGSGGGGSGGGGSGG (SEQ ID NO: 152). VH and VL can be in any orientation, e.g., VH, linker, VL; or alternatively, VL, linker, VH.

[0250] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) amino acid residues in the CDR of the antigen binding domain provided herein are replaced by another amino acid. The substitution can be "conservative" in the sense of substitution within the same amino acid family. Naturally occurring amino acids can be divided into the following four families, and conservative substitutions will occur within these families: (1) amino acids with basic side chains: lysine, arginine, histidine; (2) amino acids with acidic side chains: aspartic acid, glutamic acid; (3) amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine; and (4) amino acids with non-polar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine. By adding, deleting or substituting amino acids to change the amino acid sequence of the CDR of an antibody, various effects can be obtained, such as increasing the binding affinity to the target antigen.

[0251] Chimeric Antigen Receptor (CAR)

[0252] The present disclosure provides a first activator receptor and an immune cell comprising the same. In some embodiments, the first receptor is a chimeric antigen receptor.

[0253] As used herein, the term "chimeric antigen receptor (CAR)" may refer to an artificial receptor derived from a T cell receptor, and includes an engineered receptor that is transplanted to a specific immune effector cell with artificial specificity. CAR can be used to confer monoclonal antibodies specificity to T cells, thereby allowing the generation of a large number of specific T cells, such as for adoptive cell therapy. In a specific embodiment, CAR instructs cells to specificity for, for example, tumor-associated antigens. Exemplary CARs include intracellular activation domains, transmembrane domains, and extracellular domains comprising tumor-associated antigen binding regions. In some embodiments, CAR further includes a hinge domain. In a particular aspect, CAR includes a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody and a CD3 transmembrane domain and an intracellular domain fused. The specificity of other CAR designs can be derived from a ligand (e.g., peptide) of a receptor. In some cases, CAR includes a domain for additional costimulatory signal transduction, such as CD3, 4-1BB, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally ablate T cells after the addition of a prodrug, homing receptors, cytokines, and cytokine receptors.

[0254] In some embodiments, the extracellular ligand binding domain of the first receptor is fused to the extracellular domain of the CAR.

[0255] In some embodiments, the CAR of the present disclosure comprises an extracellular hinge region. The incorporation of the hinge region can affect the cytokine production of CAR-T cells and improve the in vivo expansion of CAR-T cells. Exemplary hinges can be separated from or derived from IgD and CD8 domains, such as IgG1. In some embodiments, the hinge is separated from or derived from CD8α or CD28.

[0256] In some embodiments, the hinge is isolated or derived from CD8α or CD28. In some embodiments, the CD8α hinge comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 7). In some embodiments, the CD8α hinge comprises SEQ ID NO: 7. In some embodiments, the CD8α hinge consists essentially of SEQ ID NO: 7. In some embodiments, the CD8α hinge is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence:

[0257] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 8). In some embodiments, the CD8 alpha hinge is encoded by SEQ ID NO:8.

[0258] In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence: CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 9). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 73. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence: TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 10). In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 10.

[0259] The CAR of the present disclosure can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In some embodiments, a transmembrane domain naturally associated with one of the domains in CAR is used. For example, a CAR comprising a CD28 co-stimulatory domain can also use a CD28 transmembrane domain. In some cases, a transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0260] Transmembrane domain can be derived from natural or synthetic sources.When the source is a natural source, the domain can be derived from any membrane-bound protein or transmembrane protein.The transmembrane region can be separated from or derived from (i.e., at least including its transmembrane region (one or more)) α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or separated from or derived from immunoglobulin such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it will mainly include hydrophobic residues such as leucine and valine.In some embodiments, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.Optionally, a short nucleotide polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection between the transmembrane domain of CAR and the cytoplasmic signal transduction domain. Glycine-serine doublets provide particularly suitable linkers.

[0261] In some embodiments of the CAR of the present disclosure, CAR comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or the same amino acid sequence as the following sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 11). In some embodiments, the CD28 transmembrane domain comprises or is substantially composed of SEQ ID NO: 11. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or the same nucleotide sequence as the following sequence: TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 12). In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 12.

[0262] In some embodiments of the CAR of the present disclosure, the CAR comprises an IL-2Rβ transmembrane domain. In some embodiments, the IL-2Rβ transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or the same amino acid sequence as the following sequence: IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 13). In some embodiments, the IL-2Rβ transmembrane domain comprises or is substantially composed of SEQ ID NO: 13. In some embodiments, the IL-2Rβ transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or the same amino acid sequence as the following sequence: In some embodiments, the IL-2Rβ transmembrane domain is encoded by SEQ ID NO:14.

[0263] The cytoplasmic domain or other intracellular signal transduction domain of the CAR disclosed herein is responsible for activating at least one normal effector function of the immune cell in which the CAR is placed. The term "effector function" refers to a specific function of a cell. Therefore, the term "intracellular signal transduction domain" refers to a protein portion that transduces effector function signals and instructs cells to perform specific functions. Although the entire intracellular signal transduction domain can usually be used, it is not necessary to use the entire domain in many cases. In terms of using a truncated portion of an intracellular signal transduction domain, as long as it transduces an effector function signal, such a truncated portion can be used instead of a complete chain. In some cases, multiple intracellular domains can be combined to achieve the desired function of the CAR-T cell disclosed herein. Therefore, the term intracellular signal transduction domain means any truncated portion including one or more intracellular signal transduction domains sufficient to transduce effector function signals.

[0264] Examples of intracellular signaling domains for use in the CARs of the present disclosure include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability.

[0265] Therefore, the intracellular domain of the CAR disclosed herein includes at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures the presence of T cell receptor (TCR) signal transduction necessary for activating the effector function of CAR T cells. In some embodiments, at least one cytoplasmic activation is a CD247 molecule (CD3ζ) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a 12kDa DNAX-activating protein (DAP12) activation domain.

[0266] In some embodiments, the CD3 zeta activation domain comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to:

[0267]

[0268] In some embodiments, the CD3 zeta activation domain comprises or consists essentially of SEQ ID NO: 15. In some embodiments, the CD3 zeta activation domain is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to:

[0269] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGCGTAGAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGACTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCTCGC(SEQ ID NO:16). In some embodiments, the CD3ζ activation domain is encoded by SEQ ID NO:16.

[0270] It is known that signals generated through the TCR alone are generally insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Thus, T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0271] The primary cytoplasm signal transduction sequence regulates the primary activation of the TCR complex in a stimulating or inhibitory manner. The primary cytoplasm signal transduction sequence that works in a stimulating manner can contain a signal transduction motif, which is referred to as an activation motif or ITAM based on immunoreceptor tyrosine. In some embodiments, ITAM contains a tyrosine separated from leucine or isoleucine by any two other amino acids (YxxL / I (SEQ ID NO: 546). In some embodiments, the cytoplasm domain contains 1, 2, 3, 4 or 5 ITAMs. The exemplary ITAM containing the cytoplasm domain is the CD3 ζ activation domain. Other examples of ITAM containing primary cytoplasm signal transduction sequences that can be used in the CAR of the present disclosure include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD5, CD22, CD79a, CD79b and CD66d.

[0272] In some embodiments, the CD3 zeta activation domain comprising a single ITAM comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 17). In some embodiments, the CD3 zeta activation domain comprises SEQ ID NO: 17. In some embodiments, the CD3 zeta activation domain comprising a single ITAM consists essentially of the amino acid sequence In some embodiments, the CD3ζ activation domain comprising a single ITAM is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence: In some embodiments, the CD3 zeta activation domain is encoded by SEQ ID NO:18.

[0273] In some embodiments, the cytoplasmic domain of CAR can be designed to include the CD3 ζ signal transduction domain itself or in combination with any other desired cytoplasmic domain (one or more) useful in the context of the CAR disclosed herein. For example, the cytoplasmic domain of CAR can include a CD3 ζ chain portion and a costimulatory domain. The costimulatory domain refers to a part of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are required for the effective response of lymphocytes to antigens. Examples of such molecules include costimulatory domains selected from the group consisting of IL-2Rβ, Fc receptor gamma (FcRγ), Fc receptor beta (FcRβ), CD3g molecule gamma (CD3γ), CD3δ, CD3ε, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen-related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1 BB), TNF receptor superfamily member 4 (OX40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell co-stimulation (ICOS), lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin-like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domain, or its functional variant. In some embodiments, the intracellular domain of the CAR of the present disclosure includes at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is separated from or derived from CD28.

[0274] In some embodiments, the intracellular domain of the CAR of the present disclosure comprises at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is separated from or derived from CD28. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or the same amino acid sequence as the following sequence: In some embodiments, the CD28 costimulatory domain comprises or consists essentially of SEQ ID NO: 19. In some embodiments, the CD28 costimulatory domain is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to:

[0275] AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGAC

[0276] CCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC (SEQ ID NO: 20). In some embodiments, the CD28 costimulatory domain is encoded by SEQ ID NO:20.

[0277] In some embodiments, the co-stimulatory domain is separated from or derived from 4-1BB. In some embodiments, the 4-1BB co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or the same as the following sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 283). In some embodiments, the 4-1BB co-stimulatory domain comprises or is substantially composed of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 283). In some embodiments, the 4-1BB co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or the same as the following sequence:

[0278] AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGA

[0279] GGCCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 284).

[0280] In some embodiments, the intracellular domain of CAR comprises a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, and a CD3ζ activation domain. In some embodiments, the intracellular domain of CAR comprises the following sequence:

[0281] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLL

[0282] YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQG

[0283] QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 285), or a sequence at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical thereto.

[0284] The cytoplasmic domains in the cytoplasmic signal transduction portion of the CAR disclosed herein can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker (e.g., 2 to 10 amino acids in length) can form a connection. Glycine-serine doublets provide examples of suitable linkers. Exemplary linkers include sequence GGGGSGGGGSGGGGSGG (SEQ ID NO: 152).

[0285] The cytoplasmic domains within the cytoplasmic signal transduction portion of the CAR of the present disclosure can be connected to each other in a random or specified order. Optionally, a short oligonucleotide or polypeptide linker (e.g., a length between 2 and 10 amino acids) can form a connection. Glycine-serine doublets provide an example of a suitable linker. Exemplary full-length activator receptors disclosed herein are described in Table 20. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NOs shown in Table 20: 286-347, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NOs shown in Table 20: 286-347. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 288, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 297, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 301, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 302, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 303, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 314, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 335, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 340, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 344, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0286] The cytoplasmic domains in the cytoplasmic signal transduction part of the CAR disclosed herein can be connected to each other in a random or specified order. Optionally, a short oligonucleotide or polypeptide linker (e.g., a length of 2 to 10 amino acids) can form a connection. Glycine-serine doublets provide an example of a suitable linker.

[0287] T cell receptor (TCR)

[0288] The present disclosure provides a first activator receptor and an immune cell comprising the same. In some embodiments, the first receptor is a T cell receptor (TCR).

[0289] As used herein, "TCR" (sometimes also referred to as "TCR complex" or "TCR / CD3 complex") refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more invariant CD3 chains (ζ, γ, δ, and ε) (sometimes referred to as subunits). The TCR alpha and beta chains can be disulfide-linked to act as heterodimers to bind peptide-MHC complexes. Once the TCR alpha / β heterodimer binds to the peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which causes them to phosphorylate and associate with downstream proteins, thereby transducing primary stimulation signals. In an exemplary TCR complex, TCR alpha and TCR beta polypeptides form heterodimers, CD3 ε and CD3 δ form heterodimers, CD3 ε and CD3 γ form heterodimers, and two CD3 ζ form homodimers.

[0290] Any suitable ligand binding domain can be fused to the extracellular domain, hinge domain or transmembrane of TCR described herein.For example, the ligand binding domain can be the antigen binding domain of antibody or TCR, or comprise antibody fragment, only Vβ domain, linear antibody, single chain variable fragment (scFv) or single domain antibody (sdAb).

[0291] In some embodiments, the ligand binding domain is fused to one or more extracellular domains or transmembrane domains of one or more TCR subunits. The TCR subunit can be TCRα, TCRβ, CD3δ, CD3ε, CD3γ or CD3ζ. For example, the ligand binding domain can be fused to TCRα or TCRβ, or the ligand-binding portion can be fused to two subunits, such as a portion of the ligand binding domain can be fused to both TCRα and TCRβ.

[0292] TCR subunits include TCRα, TCRβ, CD3ζ, CD3δ, CD3γ and CD3ε. Any one or more of TCRα, TCRβ chain, CD3γ, CD3δ, CD3ε or CD3ζ or fragments or derivatives thereof can be fused to one or more domains capable of providing the stimulation signal of the present disclosure, thereby enhancing TCR function and activity.

[0293] TCR transmembrane domains isolated or derived from any source are considered within the scope of the present disclosure. Transmembrane domains can be derived from natural or recombinant sources. When the source is a natural source, the domain can be derived from any membrane-bound or transmembrane protein.

[0294] In some embodiments, whenever the TCR complex has been bound to a target, the transmembrane domain is capable of sending a signal to the intracellular domain. The transmembrane domain specifically used in the present disclosure may include at least, for example, the transmembrane region (one or more) of the α, β or ζ chain, CD3δ, CD3ε or CD3γ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 of TCR.

[0295] In some embodiments, the transmembrane domain can be attached to the extracellular region of the TCR polypeptide, such as the antigen binding domain of the TCR α or β chain, via a hinge (e.g., a hinge from a human protein). For example, the hinge can be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge or a CD8a hinge. In some embodiments, the hinge is separated from or derived from CD8α or CD28.

[0296] In some embodiments, the extracellular ligand binding domain is attached to one or more transmembrane domains of the TCR. In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain, a TCR beta transmembrane domain, or both. In some embodiments, the transmembrane comprises a CD3 zeta transmembrane domain.

[0297] The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids in the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids in the intracellular region).

[0298] In some embodiments, transmembrane domains can be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, for example to minimize interactions with other members of the receptor complex.

[0299] When present, the transmembrane domain can be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain can be a membrane anchoring domain. Without limitation, a natural or artificial transmembrane domain can include a hydrophobic α-helix of about 20 amino acids, usually with a positive charge on the side of the transmembrane segment. The transmembrane domain can have one transmembrane segment or more than one transmembrane segment. The prediction of transmembrane domains / segments can use publicly available prediction tools (e.g., TMHMM, Krogh et al. Journal of Molecular Biology 2001; 305 (3): 567-580; or TMpred, Hofmann & Stoffel Biol. Chem. Hoppe-Seyler 1993; 347: 166). Non-limiting examples of membrane anchoring systems include the platelet-derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (post-translationally added to the signal sequence), and the like.

[0300] In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain. In some embodiments, the TCR alpha transmembrane domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the following sequence: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 21). In some embodiments, the TCR alpha transmembrane domain comprises or consists essentially of SEQ ID NO: 21. In some embodiments, the TCR alpha transmembrane domain is encoded by the following sequence:

[0301] GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG (SEQ ID NO: 22).

[0302] In some embodiments, the transmembrane domain comprises a TCRβ transmembrane domain. In some embodiments, the TCRβ transmembrane domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the following sequence: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 23). In some embodiments, the TCRβ transmembrane domain comprises or consists essentially of SEQ ID NO: 23. In some embodiments, the TCRβ transmembrane domain is encoded by the following sequence:

[0303] ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO: 24).

[0304] The TCR of the present disclosure may include one or more intracellular domains. PCT / US2002 / 045250 filed on September 6, 2020 describes an exemplary TCR comprising an intracellular domain for the present disclosure, the contents of which are incorporated herein by reference. In some embodiments, the intracellular domain includes one or more domains capable of providing a stimulation signal to the transmembrane domain. In some embodiments, the intracellular domain includes a first intracellular domain capable of providing a stimulation signal and a second intracellular domain capable of providing a stimulation signal. In other embodiments, the intracellular domain includes a first, second, and third intracellular domain capable of providing a stimulation signal. The intracellular domain capable of providing a stimulatory signal is selected from the following group: a CD28 molecule (CD28) domain, an LCK proto-oncogene, a Src family tyrosine kinase (Lck) domain, a TNF receptor superfamily member 9 (4-1BB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, a Src family tyrosine kinase (Fyn) domain, a ζ chain (ZAP70) domain of T cell receptor-associated protein kinase 70, a linker (LAT) domain for activating T cells, a lymphocyte cytoplasmic protein 2 (SLP76) domain, (TCR) α, TCRβ, CD3δ, CD3γ and CD3ε intracellular domains.

[0305] In some embodiments, the intracellular domain includes at least one intracellular signal transduction domain. The intracellular signal transduction domain generates a signal that promotes cell function, such as an immune effector function of a cell containing a TCR (e.g., a T cell expressing a TCR). In some embodiments, the intracellular domain of the first receptor of the present disclosure includes at least one intracellular signal transduction domain. For example, the intracellular domain of CD3γ, δ or ε includes a signal transduction domain.

[0306] In some embodiments, the extracellular domain, transmembrane domain, and intracellular domain are isolated or derived from the same protein, such as T cell receptor (TCR) α, TCR β, CD3 δ, CD3 γ, CD3 ε, or CD3 ζ.

[0307] Examples of intracellular domains used in the activator receptors of the present disclosure include cytoplasmic sequences of TCRα, TCRβ, CD3ζ, and 4-1BB, as well as intracellular signal transduction co-receptors that act synergistically to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capabilities.

[0308] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain.Exemplary primary intracellular signaling domains include domains derived from proteins responsible for primary stimulation or antigen-dependent stimulation.

[0309] In some embodiments, the intracellular domain comprises a CD3δ intracellular domain, a CD3ε intracellular domain, a CD3γ intracellular domain, a CD3ζ intracellular domain, a TCRα intracellular domain, or a TCRβ intracellular domain.

[0310] In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain. In some embodiments, the TCR alpha intracellular domain comprises Ser-Ser. In some embodiments, the TCR alpha intracellular domain is encoded by the sequence TCCAGC.

[0311] In some embodiments, the intracellular domain comprises a TCRβ intracellular domain. In some embodiments, the TCRβ intracellular domain comprises an amino acid sequence that is at least 80% identical, at least 90% identical, or identical to the following sequence: MAMVKRKDSR (SEQ ID NO: 25). In some embodiments, the TCRβ intracellular domain comprises or consists essentially of SEQID NO: 25. In some embodiments, the TCRβ intracellular domain is encoded by the following sequence:

[0312] ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 26).

[0313] In some embodiments, the intracellular signal transduction domain comprises at least one stimulatory intracellular domain. In some embodiments, the intracellular signal transduction domain comprises a primary intracellular signal transduction domain such as CD3δ, CD3γ and CD3ε intracellular domain and an additional stimulatory intracellular domain (e.g., a costimulatory domain). In some embodiments, the intracellular signal transduction domain comprises a primary intracellular signal transduction domain such as CD3δ, CD3γ and CD3ε intracellular domain and two additional stimulatory intracellular domains.

[0314] Exemplary costimulatory intracellular signal transduction domains include domains derived from proteins responsible for costimulatory signals or antigen-independent stimulation.Costimulatory molecules include but are not limited to MHC class I molecules, BTLA, Toll ligand receptors, and DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18) 4-1BB (CD137, TNF receptor superfamily member 9) and CD28 molecules (CD28).Costimulatory proteins can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins) and activation of NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, ligands that specifically bind to CD83, CD4, etc. The co-stimulatory domain may comprise the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signal transduction domain, or a functional variant thereof.

[0315] In some embodiments, the stimulation domain includes a costimulatory domain. In some embodiments, the costimulatory domain includes CD28 or 4-1BB costimulatory domains. CD28 and 4-1BB are fully characterized costimulatory molecules required for complete T cell activation and known to enhance T cell effector functions. For example, CD28 and 4-1BB have been used to promote cytokine release, cytolytic function and persistence in chimeric antigen receptors (CARs), which are superior to first-generation CARs containing only CD3ζ signal transduction domains. Similarly, including a costimulatory domain (such as CD28 and 4-1BB domains) in TCR can increase T cell effector functions, and specifically allow costimulation in the absence of costimulatory ligands, which are usually down-regulated on tumor cell surfaces. In some embodiments, the stimulation domain includes a CD28 intracellular domain or a 4-1BB intracellular domain.

[0316] Inhibitory receptors

[0317] The present disclosure provides a second receptor, which comprises an extracellular ligand binding domain that is specific to a non-target antigen (such as an allelic variant of a gene) that has been lost in a cancer cell. Non-target allelic variants can be lost in cancer cells by any mechanism, such as but not limited to epigenetic changes that affect the expression of non-target allelic variants, mutations in genes encoding non-target allelic variants, destruction of cell signal transduction that regulates the expression of non-target allelic variants, chromosome loss, partial or complete deletion of genomic loci, gene silencing by modified nucleic acids or heterochromatin, or loss of expression by other mechanisms. In variations of the compositions and methods disclosed herein, the cells or subjects treated may show loss of expression of non-target allelic variants due to non-genetic changes. Therefore, the present disclosure provides compositions and methods for killing cells and / or treating subjects lacking non-target antigen expression for any reason (including but not limited to loss of heterozygosity).

[0318] The non-target antigen can be a protein or an antigenic peptide complexed with major histocompatibility complex class I (MHC-I), wherein the non-target antigen comprises a polymorphism. Because the non-target antigen is polymorphic, the loss of a single copy of the gene encoding the non-target antigen (which can occur through the loss of heterozygosity in the cancer cell) produces a cancer cell that retains other polymorphic variants of the gene but has lost the non-target antigen. For example, a subject having HLA-A*02 and HLA-A*01 alleles at the HLA locus may suffer from a cancer that only lacks the HLA-A*02 allele. As another example, a subject having HLA-A*03 and HLA-A*01 alleles at the HLA locus may suffer from a cancer that only loses the HLA-A*03 allele. In such a subject, because the inhibitor receptor is designed to be specific to HLA-A*02, HLA-A*03 or other non-target antigens, the HLA-A*01 protein is still present but is not recognized by the inhibitory receptors of the immune cells that encounter the cancer cell. In normal non-malignant cells, HLA-A*02, HLA-A*03 or other non-target antigens are present and inhibit the activation of engineered immune cells. In cancer cells with loss of heterozygosity, HLA-A*02, HLA-A*03 or other allelic variants are lost. Because inhibitory receptors only respond to HLA-A*02, HLA-A*03 or other non-target antigens that are not present on cancer cells, immune cells engineered to express inhibitory receptors do not receive inhibitory signals from inhibitory receptors. Through this mechanism, immune cells are selectively activated and selectively kill cancer cells that express MSLN but lose HLA-A*02 (or another non-target antigen) due to loss of heterozygosity. HLA-A is used as an example here. Similar polymorphic variations also occur in other MHC genes and other non-MHC genes in the population. Thus, the present disclosure provides a second receptor comprising an extracellular ligand binding domain that is specific to a non-target antigen selected from intercellular adhesion molecule 1 (ICAM1), catechol-O-methyltransferase (COMT), CXC motif chemokine ligand 16 (CXCL16), leucine-rich repeat neuron 4 (LRRN4) and uroplakin 3B (UPK3B), or an antigenic peptide thereof complexed with major histocompatibility complex class I (MHC-I), wherein the non-target antigen may comprise a non-synonymous extracellular domain (e.g., in the extracellular domain of ICAM1, COMT, CXCL16) polymorphism, and an immune cell comprising the non-synonymous extracellular domain polymorphism. In some embodiments, the second receptor is an inhibitory chimeric antigen receptor. Alternatively, the non-target antigen may comprise a protein whose expression is lost in a tumor but present in a critical MSLN expressing normal tissue (e.g., LRRN4, UPK3B).

[0319] Exemplary inhibitory receptors are described in PCT / US2020 / 045228, filed September 6, 2020, PCT / US2020 / 064607, filed December 11, 2020, PC / US2021 / 029907, filed April 29, 2021, and PCT / US2020 / 059856, filed November 10, 2020, the contents of each of which are incorporated herein by reference.

[0320] In some embodiments, the second receptor is humanized.

[0321] The present disclosure provides a second receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can distinguish a single amino acid variant allele of a non-target antigen. This ability to distinguish allelic variants of non-target antigens allows the second receptor to suppress the activation of immune cells comprising the second receptor in the presence of non-target cells expressing the alleles recognized by the ligand binding domain. However, in the presence of target cells that have lost alleles (e.g., cancer cells that have lost one allele of a gene by loss of heterozygosity), the activation of immune cells is not suppressed.

[0322] The present disclosure provides a second receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can distinguish between different expression levels of non-target antigens. This allows the second receptor to inhibit the activation of immune cells comprising the second receptor in the presence of non-target cells expressing the second receptor ligand, but allows the immune cells to be activated in the presence of cancer cells expressing low levels or no expression of the second receptor ligand.

[0323] Inhibitor ligand

[0324] In some embodiments, non-target antigens are not expressed by target cells, but are expressed by non-target cells. In some embodiments, non-target antigens are expressed by healthy cells, i.e., cells that are not cancer cells. In some embodiments, target cells are multiple cancer cells that lose expression of non-target antigens by loss of heterozygosity (LOH). In some embodiments, non-target cells are multiple healthy cells (i.e., non-cancerous cells) that express both target antigens and non-target antigens.

[0325] Any cell surface molecule expressed by non-target cells but not expressed by target cells can be a suitable non-target antigen for the extracellular ligand binding domain of the second receptor. For example, cell adhesion molecules, cell-cell signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane proteins, neurotransmitter receptors or voltage-gated ion channels can be used as non-target antigens.

[0326] In some embodiments, the target antigen is a peptide antigen that is a cancer cell-specific antigen complexed with major histocompatibility complex class I (MHC-I).

[0327] In some embodiments, the non-target antigen is lost in cancer cells due to loss of heterozygosity. Exemplary non-target antigens lost in cancer cells due to loss of heterozygosity include ICAM1, COMT, and CXCL16. In some embodiments, the non-target antigen is selected from the group consisting of polymorphic variants of ICAM1, COMT, and CXCL16. In some embodiments, the non-target antigen is an antigenic peptide comprising polymorphic residues of ICAM1, COMT, or CXCL16 complexed with major histocompatibility complex class I (MHC-I).

[0328] It is contemplated that a non-target major histocompatibility complex class I MHC-I (or pMHC-I) antigen comprising any one of HLA-A, HLA-B, HLA-C or HLA-E is within the scope of the present disclosure. In some embodiments, the non-target antigen comprises a major histocompatibility complex (MHC) protein. In some embodiments, the MHC is MHC class I. In some embodiments, the MHC class I protein comprises a human leukocyte antigen (HLA) protein. In some embodiments, the non-target antigen comprises an allele of an HLA class I protein selected from the group consisting of: HLA-A, HLA-B, HLA-C or HLA-E. In some embodiments, the HLA-A allele comprises HLA-A*01, HLA-A*02, HLA-A*03 or HLA-A*11. In some embodiments, the HLA-B allele comprises HLA-B*07. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0329] In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the non-target antigen comprises an allele of HLA-A. In some embodiments, the allele of HLA-A comprises HLA-A*01, HLA-A*02, HLA-A*03, or HLA-A*11. In some embodiments, the non-target antigen comprises HLA-A*69. In some embodiments, the non-target antigen comprises a human leukocyte antigen A*02 allele (HLA-A*02). In some embodiments, the non-target antigen comprises a human leukocyte antigen A*03 allele (HLA-A*03). In some embodiments, the non-target antigen comprises a human leukocyte antigen A*11 allele (HLA-A*11).

[0330] In some embodiments, the non-target antigen comprises an allele of HLA-B. In some embodiments, the allele of HLA-B comprises HLA-B*07.

[0331] In some embodiments, the non-target antigen comprises HLA-C. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0332] In some embodiments, the non-target antigen comprises ICAM1 or an antigenic peptide thereof complexed with MHC-I.Human ICAM1 is frequently lost by LOH in cancer cells.

[0333] Wild-type human ICAM1 is described in NCBI Accession No. NP_000192.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, ICAM1 comprises the following amino acid sequence: MAPSSPRPAL PALLVLLGALFPGPGNAQTS VSPSKVILPR GGSVLVTCST SCDQPKLLGI6 ETPLPKKELL LPGNNRKVYE LSNVQEDSQPMCYSNCPDGQ STAKTFLTVY WTPERVELAP12 LPSWQPVGKN LTLRCQVEGG APRANLTVVLLRGEKELKRE PAVGEPAEVT TTVLVRRDHH18 GANFSCRTEL DLRPQGLELF ENTSAPYQLQTFVLPATPPQ LVSPRVLEVD TQGTVVCSLD24 GLFPVSEAQV HLALGDQRLN PTVTYGNDSFSAKASVSVTA EDEGTQRLTC AVILGNQSQE30 TLQTVTIYSF PAPNVILTKP EVSEGTEVTVKCEAHPRAKV TLNGVPAQPL GPRAQLLLKA361TPEDNGRSFS CSATLEVAGQ LIHKNQTRELRVLYGPRLDE RDCPGNWTWP ENSQQTPMCQ421AWGNPLPELK CLKDGTFPLP IGESVTVTRDLEGTYLCRAR STQGEVTR K VTVNVLSPRYE481IVIITVVAAA VIMGTAGLST YLYNRQRKIKKYRLQQAQKG TPMKPNTQAT PP (SEQ ID NO: 27).

[0334] In some embodiments, ICAM1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 27. The polymorphic residues of ICAM1 are marked in bold and underlined in SEQ ID NO: 27. For example, rs5498 is a polymorphism at position 469 of SEQ ID NO: 27, which can be either K or E.

[0335] In some embodiments, the non-target antigen comprises a polymorphism of ICAM1. For example, the non-target antigen comprises a peptide derived from ICAM1, the peptide comprising a polymorphic residue of ICAM1. The polymorphic residue of ICAM1 comprises amino acid residue 469 of SEQ ID NO:27. In some embodiments, the non-target antigen comprises a peptide of ICAM1, the peptide comprising amino acid 469 of SEQ ID NO:27. In some embodiments, the non-target antigen comprises a K at position 469 of SEQ ID NO:27. In some embodiments, the non-target antigen comprises an E at position 469 of SEQ ID NO:27.

[0336] In some embodiments, the non-target antigen comprises an ICAM1 polymorphism having a K at position 469 of SEQ ID NO:27, and the second receptor comprises a ligand binding domain having a higher affinity for an ICAM1 ligand having a K at position 469 of SEQ ID NO:27 than for an ICAM1 ligand having an E at position 469 of SEQ ID NO:27. In some embodiments, the non-target antigen comprises an ICAM1 polymorphism having an E at position 469 of SEQ ID NO:27, and the second receptor comprises a ligand binding domain having a higher affinity for an ICAM1 ligand having an E at position 469 of SEQ ID NO:27 than for an ICAM1 ligand having a K at position 469 of SEQ ID NO:27.

[0337] In some embodiments, the non-target antigen comprises OMT or an antigenic peptide thereof complexed with MHC-I.Human COMT is frequently lost by LOH in cancer cells.

[0338] Wild-type human COMT is described in NCBI Record No. NP_000192.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, COMT comprises the following amino acid sequence:

[0339]

[0340] In some embodiments, COMT comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 28. The polymorphic residues of COMT are marked in bold and underlined in SEQ ID NO: 28. For example, V158M is a polymorphism at position 158 of SEQ ID NO: 28, which can be either V or M.

[0341] In some embodiments, the non-target antigen comprises a polymorphism of COMT. For example, the non-target antigen comprises a peptide derived from COMT, the peptide comprising a polymorphic residue of COMT. The polymorphic residue of COMT 1 comprises amino acid residue 158 of SEQ ID NO: 28. In some embodiments, the non-target antigen comprises a peptide of COMT comprising amino acid residue 158 of SEQ ID NO: 28. In some embodiments, the non-target antigen comprises a V at position 158 of SEQ ID NO: 28. In some embodiments, the non-target antigen comprises an M at position 158 of SEQ ID NO: 28.

[0342] In some embodiments, the non-target antigen comprises a COMT polymorphism having a V at position 158 of SEQ ID NO:28, and the second receptor comprises a ligand binding domain having a higher affinity for a COMT ligand having a V at position 158 of SEQ ID NO:28 than for a COMT ligand having an M at position 158 of SEQ ID NO:28. In some embodiments, the non-target antigen comprises a COMT polymorphism having an M at position 158 of SEQ ID NO:28, and the second receptor comprises a ligand binding domain having a higher affinity for a COMT ligand having an M at position 158 of SEQ ID NO:28 than for a COMT ligand having a V at position 158 of SEQ ID NO:28.

[0343] In some embodiments, the non-target antigen comprises CXC motif chemokine ligand 16 (CXCL16) or an antigenic peptide thereof in complex with MHC-I. The human CXCL16 precursor is described in NCBI accession number NP_001094282.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, CXCL16 comprises the following amino acid sequence:

[0344] P I SQASEGASSDIHTPAQML LSTLQSTQRPTLPVGSLSSD181KELTRPNETT IHTAGHSLA AGPEAGENQKQ PEKNAGPTAR TSATVPVLCLLAIIFILTAA241LSYVLCKRRR GQSPQSSPDL PVHYIPVAPD SNT (SEQ ID NO: 29).

[0345] In some embodiments, the non-target antigen comprises a polymorphism of CXCL16. For example, the non-target antigen comprises a peptide derived from CXCL16, the peptide comprising a polymorphic residue of CXCL16. The polymorphic residues of CXCL16 include positions 142 and 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16, the peptide comprising amino acid 142 or 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16, the peptide comprising an A at amino acid 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16, the peptide comprising a V at amino acid 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16, the peptide comprising an I at amino acid 142 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16, the peptide comprising a T at amino acid 142 of SEQ ID NO:29.

[0346] In some embodiments, the non-target antigen comprises a polymorphism of CXCL16. In some embodiments, the non-target antigen comprises a peptide of CXCL16. The peptide comprises an A at amino acid 200 of SEQ ID NO: 29, and the second receptor comprises a ligand binding domain having a higher affinity for a CXCL16 ligand having an A at position 200 of SEQ ID NO: 29 than for a CXCL16 ligand having a V at position 200 of SEQ ID NO: 29. In some embodiments, the non-target antigen comprises a peptide of CXCL16, the peptide comprising a V at amino acid 200 of SEQ ID NO: 29, and the second receptor comprises a ligand binding domain having a higher affinity for a CXCL16 ligand having a V at position 200 of SEQ ID NO: 29 than for a CXCL16 ligand having an A at position 200 of SEQ ID NO: 29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 comprising an I at amino acid 142 of SEQ ID NO:29, and the second receptor comprises a ligand binding domain having a higher affinity for a CXCL16 ligand having an I at position 142 of SEQ ID NO:29 than for a CXCL16 ligand having a T at position 142 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 comprising a T at amino acid 142 of SEQ ID NO:29, and the second receptor comprises a ligand binding domain having a higher affinity for a CXCL16 ligand having a T at position 142 of SEQ ID NO:29 than for a CXCL16 ligand having an I at position 142 of SEQ ID NO:29.

[0347] In some embodiments, the non-target antigen comprises HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07. Various single variable domains that bind to or recognize specific HLA alleles for the embodiments described herein are described in Table 5. (Complementarity determining regions are underlined):

[0348] Table 5: HLA scFv binding domains

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371] In some embodiments, the ligand binding domain of the second inhibitory receptor comprises an scFv. In some embodiments, the scFv binds to HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07 and comprises a sequence selected from the group consisting of the sequences shown in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the scFv binds to HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07 and comprises a sequence selected from the group consisting of the sequences shown in Table 5. In some embodiments, the non-target antigen comprises HLA-A*01, and the non-target extracellular ligand binding domain of the second receptor comprises an HLA-A*01 scFv sequence shown in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-A*02, and the non-target extracellular ligand binding domain of the second receptor comprises an HLA-A*02 scFv sequence shown in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-A*03, and the non-target extracellular ligand binding domain of the second receptor comprises an HLA-A*03 scFv sequence shown in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-A*11, and the non-target extracellular ligand binding domain of the second receptor comprises an HLA-A*11 scFv sequence shown in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-B*07, and the non-target extracellular ligand binding domain of the second receptor comprises an HLA-B*07 scFv sequence shown in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-C*07, and the non-target extracellular ligand binding domain of the second receptor comprises an HLA-C*07 scFv sequence shown in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0372] Table 6 below shows exemplary heavy and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR-L1, CDR-L2 and CDR-L3, respectively) of HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07 and HLA-C*07 ligand binding domains.

[0373] Table 6: CDRs corresponding to HLA antigen binding domains

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384] In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the ligand binding domain of the second inhibitory receptor comprises an HLA-A*01, HLA-A*02, HLA-A*03, or HLA-A*11 ligand binding domain comprising a CDR sequence as shown in Table 6 or Table 7.

[0385] In some embodiments, the non-target antigen comprises HLA-B. In some embodiments, the ligand binding domain of the second inhibitory receptor comprises an HLA-B*07 ligand binding domain comprising the CDR sequences shown in Table 6.

[0386] In some embodiments, the non-target antigen comprises HLA-C. In some embodiments, the ligand binding domain of the second inhibitory receptor comprises an HLA-C*07 ligand binding domain comprising the CDR sequences shown in Table 6.

[0387] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to an allelic variant of an HLA-A, HLA-B, or HLA-C protein. In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07.

[0388] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-A*01. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the HLA-A*01 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 disclosed in Table 6; or a CDR sequence having at most 1, 2 or 3 substitutions, deletions or insertions relative to the HLA-A*01 CDRs of Table 6.

[0389] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-A*02. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the HLA-A*02 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 disclosed in Table 6; or a CDR sequence having at most 1, 2 or 3 substitutions, deletions or insertions relative to the HLA-A*02 CDRs of Table 6.

[0390] In some embodiments, the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 103-108 or SEQ ID NOs: 109-114; or CDR sequences having up to 1, 2 or 3 substitutions, deletions or insertions relative to the CDRs of SEQ ID NOs: 103-108 or SEQ ID NOs: 109-114.

[0391] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-A*03. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the HLA-A*03 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 disclosed in Table 6; or a CDR sequence having at most 1, 2 or 3 substitutions, deletions or insertions relative to the HLA-A*03 CDRs of Table 6.

[0392] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-A*11. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the HLA-A*11 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 disclosed in Table 7; or a CDR sequence having at most 1, 2 or 3 substitutions, deletions or insertions relative to the HLA-A*11 CDRs of Table 7.

[0393] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-B*07. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the HLA-B*07 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 disclosed in Table 6; or a CDR sequence having at most 1, 2 or 3 substitutions, deletions or insertions relative to the HLA-B*07 CDRs of Table 6.

[0394] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-C*07. In some embodiments, the extracellular ligand binding domain of the second receptor comprises the HLA-C*07 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 disclosed in Table 6; or a CDR sequence having at most 1, 2 or 3 substitutions, deletions or insertions relative to the HLA-C*07 CDRs of Table 6.

[0395] In further embodiments of any ligand binding domain, each CDR sequence may have 1, 2, 3 or more substitutions, insertions or deletions. CDR sequences can tolerate substitutions, deletions or insertions. Using sequence alignment tools, routine experiments and known assays, one skilled in the art can generate and test variant sequences having 1, 2, 3 or more substitutions, insertions or deletions in the CDR sequences without undue experimentation.

[0396] In some embodiments, the non-target antigen comprises HLA-A*02, and the ligand binding domain of the second receptor comprises an HLA-A*02 ligand binding domain. In some embodiments, the ligand binding domain binds HLA-A*02 independently of a peptide in a pMHC complex comprising HLA-A*02. In some embodiments, the HLA-A*02 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*02 ligand binding domain comprises the sequence of any one of SEQ ID NOs: 30-41. In some embodiments, the HLA-A*02 ligand binding domain comprises a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the sequence of any one of SEQ ID NOs: 30-41.

[0397] In some embodiments, the non-target antigen comprises HLA-A*02, and the extracellular ligand binding domain of the second receptor comprises SEQ ID NO: 30, or a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. In some embodiments, the non-target antigen comprises HLA-A*02, and the extracellular ligand binding domain of the second receptor comprises the sequence of SEQ ID NO: 30.

[0398] In some embodiments, the non-target antigen comprises HLA-A*02, and the extracellular ligand binding domain of the second receptor comprises a VL comprising the following sequence:

[0399] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK

[0400] LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTSGGGTKLEIK (SEQ ID NO: 762), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain of the second receptor comprises a VH comprising the following sequence: QVQLQQSGPELVKPGASVRISCKASGYTFTSYHIHWVKQRPGQGLEWIGWIYPGNVNTEYNEKFKGKATLTADKSSSTAYMHLSSLTSEDSAVYFCAREEITYAMDYWG QGTSVTVSS (SEQ ID NO: 763), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, VH and VL are separated by a linker, e.g., GGGGSGGGGSGGGGSGG (SEQ ID NO: 152). In some embodiments, the order of VH and VL from N-terminus to C-terminus is VH, linker, and VL. In some embodiments, the order of VH and VL from N-terminus to C-terminus is VL, linker, and VH.

[0401] In some embodiments, the HLA-A*02 scFv comprises a complementarity determining region (CDR) of any one of SEQ ID NOs: 42-53. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOs: 42-53. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOs: 42-53. In some embodiments, the heavy chain of the antigen binding domain comprises a heavy chain CDR of any one of SEQ ID NOs: 42-53, and wherein the light chain of the antigen binding domain comprises a light chain CDR of any one of SEQ ID NOs: 42-53. In some embodiments, the HLA-A*02 antigen binding domain comprises a heavy chain and a light chain, and the heavy chain comprises a CDR selected from SEQ ID NOs: 45-47 and 51-53, and the light chain comprises a CDR selected from SEQ ID NOs: 42-44 and 48-50.

[0402] In some embodiments, the HLA-A*02 antigen binding domain comprises a heavy chain and a light chain, and the heavy chain comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 30-41, and the light chain comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOs: 30-41.

[0403] In some embodiments, the heavy chain comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 30-41, and wherein the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 30-41.

[0404] In some embodiments, the non-target antigen comprises HLA-A*01, and the extracellular ligand binding domain of the second receptor comprises an HLA-A*01 ligand binding domain. In some embodiments, the HLA-A*01 ligand binding domain comprises an scFv domain comprising a sequence selected from the sequences shown in Table 5, or a sequence that is at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-A*01 scFv comprises the HLA-A*01 CDR sequences shown in Table 6.

[0405] In some embodiments, the non-target antigen comprises HLA-A*03, and the extracellular ligand binding domain of the second receptor comprises an HLA-A*03 ligand binding domain. In some embodiments, the HLA-A*03 ligand binding domain comprises an scFv domain comprising a sequence selected from the sequences shown in Table 5, or a sequence that is at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-A*03 scFv comprises an HLA-A*03 CDR sequence as shown in Table 6.

[0406] In some embodiments, the non-target antigen comprises HLA-A*03, and the ligand binding domain of the second receptor comprises an HLA-A*03 ligand binding domain. In some embodiments, the ligand binding domain binds HLA-A*03 independently of a peptide in a pMHC complex comprising HLA-A*03. In some embodiments, the HLA-A*03 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*03 ligand binding domain comprises a sequence of any one of SEQ ID NOs: 615-628 or SEQ ID NO: 1259. In some embodiments, the HLA-A*03 ligand binding domain comprises a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 615-628 or SEQ ID NO: 1259.

[0407] In some embodiments, the non-target antigen comprises HLA-A*03, and the extracellular ligand binding domain of the second receptor comprises a sequence of SEQ ID NOs: 615-628, or a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. In some embodiments, the non-target antigen comprises HLA-A*03, and the extracellular ligand binding domain of the second receptor comprises a sequence of SEQ ID NOs: 615-628.

[0408] In some embodiments, the non-target antigen comprises HLA-A*03, and the extracellular ligand binding domain of the second receptor comprises the sequence of SEQ ID NO: 1259, or a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. In some embodiments, the non-target antigen comprises HLA-A*03, and the extracellular ligand binding domain of the second receptor comprises the sequence of SEQ ID NO: 1259.

[0409] In some embodiments, the non-target antigen comprises HLA-A*03, and the extracellular ligand binding domain of the second receptor comprises a VL comprising the following sequence: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTTVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPPTFGGGTKLEIK (SEQ ID NO: 1266), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain of the second receptor comprises a VH comprising the following sequence: EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSTNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTTLITPDYWG QGTTLTVSS (SEQ ID NO: 1267), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, VH and VL are separated by a linker, e.g., GGGGSGGGGSGGGGSGG (SEQ ID NO: 152). In some embodiments, the order of VH and VL from N-terminus to C-terminus is VH, linker, and VL. In some embodiments, the order of VH and VL from N-terminus to C-terminus is VL, linker, and VH.

[0410] In some embodiments, the HLA-A*03 extracellular ligand binding domain comprises a complementarity determining region (CDR) of any one of SEQ ID NOs: 638-641, 645-648, 650-653, 657-664, 676-682, 693-706, or 1260-1265. In some embodiments, the extracellular ligand binding domain comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 638-641, 645-648, 650-653, 657-664, 676-682, 693-706, or 1260-1265. In some embodiments, the extracellular ligand binding domain comprises a sequence identical to any one of SEQ ID NOs: 638-641, 645-648, 650-653, 657-664, 676-682, 693-706, or 1260-1265. In some embodiments, the heavy chain of the antigen binding domain comprises the heavy chain CDRs of any one of SEQ ID NOs: 638-641, 645-648, 650-653, 657-664, 676-682, 693-706, or 1260-1265, and wherein the light chain of the antigen binding domain comprises the light chain CDRs of any one of SEQ ID NOs: 638-641, 645-648, 650-653, 657-664, 676-682, 693-706, or 1260-1265. In some embodiments, the HLA-A*03 antigen binding domain comprises a heavy chain and a light chain, and the heavy chain comprises a CDR selected from SEQ ID NOs: 657-664, 676-682, 693-706 and 1263-1265, and the light chain comprises a CDR selected from SEQ ID NOs: 638-641, 645-648, 650-653 and 1260-1262.In some embodiments, the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of the following sequences: (i) SEQ ID NOs: 638, 645, 650, 657, 676, and 693; (ii) SEQ ID NOs: 638, 645, 650, 658, 677, and 694; (iii) SEQ ID NOs: 638, 645, 650, 659, 678, and 695; (iv) SEQ ID NOs: 638, 645, 650, 660, 678, and 696; (v) SEQ ID NOs: 638, 645, 650, 661, 679, and 697; (vi) SEQ ID NOs: NO:639, 646, 651, 657, 676 and 698; (vii) SEQ ID NO:638, 645, 650, 657, 676 and 699; (viii) SEQ ID NO:639, 646, 651, 657, 676 and 700; (ix) SEQ ID NO:638, 645, 650, 662, 680 and 701; (x) SEQ ID NO:639, 646, 651, 657, 676 and 702; (xi) SEQ ID NO:638, 645, 650, 661, 679 and 703; (xii) SEQ ID NO:640, 647, 652, 657, 676 and 704; (xiii) SEQ ID NO:639, 646, 651, 657, 676 and 704 NO:641, 648, 653, 663, 681 and 705; (xiv) SEQ ID NO:638, 645, 650, 664, 682 and 706; or (xv) SEQ ID NO:1260-1265.

[0411] In some embodiments, the HLA-A*03 antigen binding domain comprises a heavy chain and a light chain, the heavy chain comprising a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 615-628 or 1259, and the light chain comprising a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOs: 615-628 or 1259. In some embodiments, the heavy chain comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 615-628 or 1259, and wherein the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 615-629 or 1259.

[0412] In some embodiments, the non-target antigen comprises HLA-A*11, and the extracellular ligand binding domain of the second receptor comprises an HLA-A*11 ligand binding domain. In some embodiments, the HLA-A*11 ligand binding domain comprises an scFv domain comprising a sequence selected from the sequence shown in Table 5, or a sequence that is at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-A*11 scFv comprises the HLA-A*11 CDR sequence shown in Table 7.

[0413] In some embodiments, the non-target antigen comprises HLA-B*07, and the extracellular ligand binding domain of the second receptor comprises an HLA-B*07 ligand binding domain. In some embodiments, the HLA-B*07 ligand binding domain comprises an scFv domain comprising a sequence selected from the sequences shown in Table 5, or a sequence that is at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-B*07 scFv comprises the HLA-B*07 CDR sequences shown in Table 6.

[0414] In some embodiments, the non-target antigen comprises HLA-C*07, and the extracellular ligand binding domain of the second receptor comprises an HLA-C*07 ligand binding domain. In some embodiments, the HLA-C*07 ligand binding domain comprises an scFv domain comprising a sequence selected from the sequences shown in Table 5, or a sequence that is at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-C*07 scFv comprises the HLA-C*07 CDR sequences shown in Table 6.

[0415] In some embodiments, the non-target antigen comprises HLA-A*11. Various single variable domains known in the art or disclosed herein that bind and recognize HLA-A*11 are suitable for use in embodiments. Such scFvs include, for example, but not limited to, the following mouse and humanized scFv antibodies that bind to HLA-A*11 in a peptide-independent manner as shown in Table 5 above.

[0416] Table 7 below shows exemplary heavy and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR-L1, CDR-L2 and CDR-L3, respectively) of HLA-A*11 ligand binding domains. Any VH CDR in Table 7 can be combined with a VLCDR disclosed in Table 7.

[0417] Table 7: Exemplary anti-HLA-A*11 CDR sequences

[0418]

[0419] In some embodiments, the non-target antigen comprises HLA-A*11, and the ligand binding domain of the second receptor comprises an HLA-A*11 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-A*11 independently of a peptide in a pMHC complex comprising HLA-A*11. In some embodiments, the HLA-A*11 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*11 ligand binding domain comprises the sequence of any one of SEQ ID NOs: 114-122. In some embodiments, the HLA-A*11 ligand binding domain comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to the sequence of any one of SEQ ID NOs: 114-122.

[0420] In some embodiments, the HLA-A*11 scFv comprises a complementarity determining region (CDR) of any one of SEQ ID NOs: 114-122. In some embodiments, the scFv comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 114-122. In some embodiments, the scFv comprises a sequence that is identical to any one of SEQ ID NOs: 114-122. In some embodiments, the heavy chain of the antigen binding domain comprises a heavy chain CDR of any one of SEQ ID NOs: 132-140, and wherein the light chain of the antigen binding domain comprises a light chain CDR of SEQ ID NO: 141. In some embodiments, the HLA-A*11 antigen binding domain comprises a heavy chain and a light chain, and the heavy chain comprises one, two or three CDRs selected from SEQ ID NOs: 92-110, and the light chain comprises one, two or three CDRs selected from SEQ ID NOs: 111-113.

[0421] Table 8 below provides exemplary heavy and light chain sequences of HLA-A*11 antigen binding domains. In some embodiments, the HLA-A*11 antigen binding domain comprises a heavy chain and a light chain, the heavy chain comprising a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 132-140, and the light chain comprising a sequence at least 95% identical to the light chain portion of SEQ ID NO: 141.

[0422] In some embodiments, the heavy chain comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 114-122, and wherein the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 114-122.

[0423] Table 8: Exemplary anti-HLA-A*11 heavy and light chain sequences

[0424]

[0425]

[0426]

[0427] Differentially expressed inhibitor ligands

[0428] The present disclosure provides inhibitor ligands (non-target antigens) that are differentially expressed between cancer cells and normal cells.

[0429] The activation of inhibitory receptors is mediated by the presence of non-target antigens on the cell surface. Cells expressing non-target antigens will activate inhibitory receptors depending on the expression level of the non-target antigen. In some embodiments, both target cells and non-target cells express non-target antigens. However, in these embodiments, the level of non-target antigens expressed by non-target cells is higher than the level of non-target antigens expressed by target cells. The higher level of non-target antigens expressed by non-target cells activates inhibitory receptors, thereby preventing the activation of immune cells. In contrast, the level of non-target antigens expressed by the target is low and insufficient to activate inhibitory receptors, resulting in the activation of immune cells.

[0430] In alternative embodiments, the non-target antigen is expressed by the non-target cell but not by the target cell. When the non-target antigen is not expressed, the target cell activates the target receptor, thereby activating the immune cell.

[0431] Differential expression can be determined by any technique known in the art for measuring expression. These techniques especially include the technique of measuring mRNA and / or protein level of target gene in cells. The method of measuring protein level in sample includes immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and analytical method such as liquid chromatography-mass spectrometry (LC-MS). The method of measuring mRNA level includes real-time quantitative reverse transcription PCR (qRT-PCR) and high-throughput sequencing. For example, expression difference can be observed between normal cells and diseased cells (e.g. cancer cells).

[0432] The activation of inhibitory receptors by non-target antigens can be performed in various ways known in the art. The activation of inhibitory receptors by non-target antigens can be determined by methods known in the art. For example, the level of downstream intracellular signal transduction in cells expressing inhibitory receptors can be measured using reporter genes.

[0433] Without wishing to be bound by theory, whether the expression of non-target antigens inhibits the activation of immune cells by activating inhibitory receptors can occur based on the ratio of non-target antigens to inhibitory receptors. The expression levels of non-target antigens and inhibitory receptors and their ratios can be determined by methods known in the art, including immunohistochemistry and fluorescence activated cell sorting (FACS). Analysis of the expression levels of non-target antigens on target cells and non-target cells can be used to predict the selective targeting of immune cells expressing inhibitory receptors. For example, low or no expression of non-target antigens on target cells or non-target cells can indicate that inhibitory receptors will not be activated in the immune cells of the present disclosure.

[0434] Alternatively or additionally, and not wishing to be bound by theory, inhibition of immune cell activation by non-target antigens through activation of inhibitory receptors may depend on the affinity of the non-target antigens for the inhibitory receptors. Methods for measuring affinity are known in the art and include, among others, enzyme-linked immunosorbent assays or radioimmunoassays.

[0435] Alternatively or additionally, and not wishing to be bound by theory, based on crosstalk between inhibitory receptors and activator receptors, activation of immune cells by non-target antigens may be inhibited by activating inhibitory receptors, thereby leading to downregulation of activator receptor activity. For example, activation of inhibitory receptors by non-target antigens may lead to reduced expression of activator receptors on the surface of immune cells.

[0436] In some embodiments, the expression level of non-target antigen in target cells is lower than the expression level in normal cells. In some embodiments, non-target antigens are expressed by healthy cells (i.e., cells of non-cancerous cells). In some embodiments, the non-target antigen expression level in target cells is at least about 10 times, at least about 30 times, at least about 50 times, at least about 70 times, at least about 90 times, at least about 100 times, at least about 110 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, or at least about 1000 times lower than the non-target antigen expression level in non-target cells. In some embodiments, the non-target antigen expression level is about 10 times, about 30 times, about 50 times, about 70 times, about 90 times, about 100 times, or about 110 times lower than a plurality of healthy cells. In some embodiments, the non-target antigen expression level in a plurality of cancer cells is at least about 5 times lower than the non-target antigen expression level in a plurality of healthy cells. In some embodiments, the non-target antigen expression level in the target cells is at least about 5 times lower than the non-target antigen expression level in the non-target cells. In some embodiments, the target cells are a plurality of cancer cells that have low or no non-target antigen expression.

[0437] Any cell surface molecule expressed by non-target cells that is not expressed by target cells (or expressed at a low level) can be a suitable non-target antigen for the extracellular ligand binding domain of the second receptor. For example, cell adhesion molecules, cell-cell signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane proteins, neurotransmitter receptors or voltage-gated ion channels can be used as non-target antigens.

[0438] In some embodiments, the non-target antigen is selected from leucine-rich repeat neuronal 4 (LRRN4) and uroplaque protein B3 (UPKB3), or a peptide antigen of any of these complexed with major histocompatibility complex class I (MHC-I). In some embodiments, the non-target antigen is LRRN4 or a peptide antigen thereof complexed with MHC-I. In some embodiments, the non-target antigen is UPKB3 or a peptide antigen thereof complexed with MHC-I.

[0439] In some embodiments, the target antigen is a peptide antigen that is a cancer cell-specific antigen complexed with major histocompatibility complex class I (MHC-I).

[0440] Non-target MHC-I (pMHC) antigens comprising any of HLA-A, HLA-B, or HLA-C are considered to be within the scope of the present disclosure. In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the non-target antigen comprises HLA-B. In some embodiments, the non-target antigen comprises HLA-C.

[0441] Non-target antigens include proteins that are lowly expressed or not expressed in cancer cells (eg, lung cancer cells) but are expressed in normal tissues (eg, normal lung tissue).

[0442] In some embodiments, the non-target antigen comprises LRRN4 or an antigenic peptide thereof complexed with MHC-I. Human LRRN4 is described in NCBI Record No. NP_689824.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, LRRN4 comprises the following amino acid sequence:

[0443]

[0444] In some embodiments, LRRN4 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 75. In some embodiments, LRRN4 comprises a sequence that is identical to SEQ ID NO: 75.

[0445] In some embodiments, the non-target antigen comprises UPK3B or an antigenic peptide thereof in complex with MHC-I. All isoforms of UPK3B are considered to be within the scope of the present disclosure. Human UPK3B isoform a precursor is described in NCBI Record No. NP_085047.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the UPK3B isoform a precursor comprises the following amino acid sequence:

[0446]

[0447] The human UPK3B isoform b precursor is described in NCBI Record No. NP_872625.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the UPK3B isoform b precursor comprises the following amino acid sequence:

[0448]

[0449] Human UPK3B isoform c precursor is described in NCBI Accession No. NP_872624.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, UPK3B isoform c precursor comprises the following amino acid sequence:

[0450]

[0451] Human UPK3B isoform d precursor is described in NCBI Accession No. NP_001334613.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the UPK3B isoform d precursor comprises the following amino acid sequence:

[0452]

[0453] In some embodiments, UPKB3 comprises a sequence or subsequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 76-79. In some embodiments, UPKB3 comprises a sequence or subsequence that is identical to SEQ ID NOs: 76-79.

[0454] Inhibitory chimeric antigen receptor

[0455] The present disclosure provides a second receptor that is an inhibitory chimeric antigen receptor. The inhibitory receptor may comprise an extracellular ligand binding domain that binds and recognizes a non-target antigen or a peptide derivative thereof in an MHC-I complex.

[0456] As used herein, the term "inhibitory receptor" refers to a ligand binding domain fused to an intracellular signal transduction domain that can transduce an inhibitory signal that inhibits or prevents the immune activity of an immune cell. Inhibitory receptors have an immune cell inhibitory potential, which is distinct from receptor CARs that have an immune cell activation potential. For example, CARs are activating receptors because they contain intracellular stimulatory and / or costimulatory domains. Inhibitory receptors are inhibitory receptors containing intracellular inhibitory domains.

[0457] As used herein, "inhibitory signal" refers to a signal transduction or a change in protein expression in an immune cell that results in an inhibition of an immune response (e.g., a decrease in cytokine production or a decrease in immune cell activation). The inhibition or prevention of immune cells can be selective and / or reversible, or non-selective and / or reversible.

[0458] The inhibitory receptors of the present disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that can modulate receptor activity in a ligand-dependent manner is considered to be within the scope of the present disclosure. The inhibitory receptor responds to non-target antigens (e.g., HLA-A*02). For example, when a non-target antigen (e.g., HLA-A*02) binds or contacts an inhibitory receptor, the inhibitory receptor responds, and when the extracellular ligand binding domain of the inhibitory receptor binds to the non-target antigen, the inhibitory receptor activates an inhibitory signal in an immune cell expressing the inhibitory receptor.

[0459] The inhibitory receptors of the present disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that is capable of modulating receptor activity in a ligand-dependent manner is considered within the scope of the present disclosure.

[0460] In some embodiments, the ligand binding domain is an antigen binding domain.Exemplary antigen binding domains include, among others, scFv, SdAb, Vβ domain only, and TCR antigen binding domains derived from TCR α and β chain variable domains.

[0461] Any type of antigen binding domain is considered to be within the scope of the present disclosure.

[0462] In some embodiments, the extracellular ligand binding domain of the second receptor is a scFv.

[0463] In some embodiments, the extracellular ligand binding domain of the second receptor binds and recognizes intercellular adhesion molecule 1 (ICAM1), catechol-O-methyltransferase (COMT), CXC motif chemokine ligand 16 (CXCL16), leucine-rich repeat neuronal 4 (LRRN4) and polymorphic variants of uroplakin 3B UPK3B, or an antigenic peptide thereof in complex with major histocompatibility complex class I (MHC-I) or HLA-A*02. In some embodiments, the extracellular ligand binding domain of the second receptor is a scFv.

[0464] In some embodiments, the extracellular ligand binding domain of the second receptor is fused to the extracellular domain of the inhibitory receptor.

[0465] In some embodiments, the inhibitory receptors of the present disclosure comprise an extracellular hinge region. Exemplary hinges can be isolated or derived from IgD and CD8 domains, such as IgG1. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0466] The inhibitory receptors of the present disclosure can be designed to include a transmembrane domain fused to the extracellular domain of the inhibitory receptor. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0467] The membrane-spanning domain can be derived from a natural source or a synthetic source. When the source is natural, the domain can be derived from any membrane-bound protein or a membrane-spanning protein. The membrane-spanning region can be separated from or derived from (i.e., at least comprising its membrane-spanning region (one or more)) α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or separated from or derived from an immunoglobulin such as IgG4. Alternatively, the membrane-spanning domain can be synthetic, in which case it will mainly comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine can be found at each end of the synthetic membrane-spanning domain. Optionally, a short oligonucleotide or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection between the membrane-spanning domain and the intracellular domain of an inhibitory receptor. Glycine-serine doublets provide particularly suitable linkers.

[0468] The present disclosure provides an inhibitory receptor comprising an intracellular domain. The intracellular domain of the inhibitory receptor disclosed herein is responsible for inhibiting the activation of immune cells comprising inhibitory receptors, otherwise the immune cells will be activated in response to the activation signal of the first receptor. In some embodiments, the inhibitory intracellular domain comprises an inhibitory motif (ITIM) based on immunoreceptor tyrosine. In some embodiments, the inhibitory intracellular domain comprising ITIM can be separated from or derived from immune checkpoint inhibitors such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immunoinhibitory receptors expressed on the surface of T cells, which play a key role in weakening or terminating T cell responses.

[0469] In some embodiments, the inhibitory intracellular domain is isolated from human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and CD200 receptor 1. In some embodiments, the TRAIL receptor comprises TR10A, TR10B or TR10D.

[0470] In some embodiments, the inhibitory intracellular domain is isolated from phosphoprotein membrane anchor with glycosphingolipid microdomain 1 (PAG1). In some embodiments, the inhibitory intracellular domain is isolated from leukocyte immunoglobulin-like receptor B1 (LILRB1).

[0471] In some embodiments, the inhibitory domain is isolated or derived from a human protein, such as a human TRAIL receptor, CTLA-4, PD-1, PAG1, or LILRB1 protein.

[0472] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region, or a combination thereof.

[0473] In some embodiments, the inhibitory domain is isolated from or derived from killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin-like receptor B1 (LIR1, also known as LIR-1 and LILRB1), programmed cell death receptor 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin-like receptor K1 (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, ZAP70 SH2 domain (e.g., one or both of the N- and C-terminal SH2 domains), or ZAP70KI_K369A (kinase-inactive ZAP70).

[0474] In some embodiments, the inhibitory domain is isolated or derived from a human protein.

[0475] In some embodiments, the second inhibitory receptor comprises an inhibitory domain. In some embodiments, the second inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of the inhibitory receptor. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of the inhibitory receptor.

[0476] In some embodiments, the second inhibitory receptor comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, or a portion thereof isolated or derived from the same protein (e.g., an ITIM-containing protein). In some embodiments, the second inhibitory receptor comprises a hinge region isolated or derived from the same protein (e.g., an ITIM-containing protein) as the intracellular domain and / or transmembrane domain.

[0477] In some embodiments, the second receptor is a TCR (inhibitory TCR) comprising an inhibitory domain. In some embodiments, the inhibitory TCR comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of TCRα, TCRβ, CD3δ, CD3γ or CD3ε or a portion thereof. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of TCRα, TCRβ, CD3δ, CD3γ or CD3ε.

[0478] In some embodiments, the second receptor is a TCR comprising an inhibitory domain (inhibitory TCR). In some embodiments, the inhibitory domain is isolated or derived from LILRB1.

[0479] LILRB1 inhibitory receptor

[0480] The present disclosure provides a second inhibitory receptor comprising a LILRB1 inhibitory domain and optionally a LILRB1 transmembrane domain and / or a hinge domain or a functional variant thereof. Including a LILRB1 transmembrane domain and / or a LILRB1 hinge domain in an inhibitory receptor can increase the inhibitory signal generated by the inhibitory receptor compared to a reference inhibitory receptor having another transmembrane domain or another hinge domain. As described herein, the second inhibitory receptor comprising a LILRB1 inhibitory domain can be a CAR or a TCR. As described herein, any suitable ligand binding domain can be fused to a second inhibitory receptor based on LILRB1.

[0481] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), also known as leukocyte immunoglobulin-like receptor B1, as well as ILT2, LIR1, MIR7, PIRB, CD85J, ILT-2, LIR-1, MIR-7 and PIR-B, is a member of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1 protein belongs to the subfamily B class of LIR receptors. These receptors contain two to four extracellular immunoglobulin domains, a transmembrane domain and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen presenting cells and transduces negative signals that inhibit immune response stimulation. LILRB1 is believed to regulate inflammatory responses as well as cytotoxicity and plays a role in limiting autoresponsiveness. There are multiple transcript variants encoding different isoforms of LILRB1, all of which are considered to be within the scope of the present disclosure.

[0482] In some embodiments of the inhibitory receptors described herein, the inhibitory receptor comprises one or more domains isolated or derived from LILRB1. In some embodiments of the receptor having one or more domains isolated or derived from LILRB1, the one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence or subsequence of SEQ ID NO:54, or identical to the sequence or subsequence of SEQ ID NO:54. In some embodiments, the one or more domains of LILRB1 comprise an amino acid sequence that is identical to the sequence or subsequence of SEQ ID NO:54. In some embodiments, the one or more domains of LILRB1 consist of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence or subsequence of SEQ ID NO:54, or identical to the sequence or subsequence of SEQ ID NO:54. In some embodiments, one or more domains of LILRB1 consist of an amino acid sequence identical to a sequence or a subsequence of SEQ ID NO:54.

[0483] In some embodiments of the receptor having one or more domains isolated or derived from LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or identical to, the sequence or a subsequence of SEQ ID NO:55.

[0484] In some embodiments of the receptor having one or more domains of LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is identical to the sequence or a subsequence of SEQ ID NO:55.

[0485] In various embodiments, an inhibitory receptor comprising a polypeptide is provided, wherein the polypeptide comprises one or more of the following: a LILRB1 hinge domain or a functional variant thereof; a LILRB1 transmembrane domain or a functional variant thereof; and a LILRB1 intracellular domain or an intracellular domain comprising at least one or at least two immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58) and SIYATL (SEQ ID NO: 59).

[0486] As used herein, "immunoreceptor tyrosine-based inhibitory motif" or "ITIM" refers to a conserved amino acid sequence with a consensus sequence of S / I / V / LxYxxI / V / L (SEQ ID NO: 547), etc., which is present in the cytoplasmic tail of many inhibitory receptors of the immune system. After an inhibitory receptor with an ITIM interacts with its ligand, the ITIM motif is phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or the inositol phosphatase known as SHIP.

[0487] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibition motif (ITIM), at least two ITIMs, at least 3 ITIMs, at least 4 ITIMs, at least 5 ITIMs, or at least 6 ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0488] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one ITIM selected from the group consisting of NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and SIYATL (SEQ ID NO:59).

[0489] In a further specific embodiment, the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58) and SIYATL (SEQ ID NO:59).

[0490] In some embodiments, the intracellular domain comprises ITIM NLYAAV (SEQ ID NO: 56) and ITIM VTYAEV (SEQ ID NO: 57). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 60. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 60.

[0491] In some embodiments, the intracellular domain comprises ITIM VTYAEV (SEQ ID NO: 57) and ITIM VTYAQL (SEQ ID NO: 58). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 61. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 61.

[0492] In some embodiments, the intracellular domain comprises ITIM VTYAQL (SEQ ID NO: 58) and ITIM SIYATL (SEQ ID NO: 59). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 62. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 62.

[0493] In some embodiments, the intracellular domain comprises ITIM NLYAAV (SEQ ID NO: 56), ITIM VTYAEV (SEQ ID NO: 57), and ITIM VTYAQL (SEQ ID NO: 58). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 63. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 63.

[0494] In some embodiments, the intracellular domain comprises ITIM VTYAEV (SEQ ID NO: 57), ITIM VTYAQL (SEQ ID NO: 58), and ITIM SIYATL (SEQ ID NO: 59). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 64. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 64.

[0495] In some embodiments, the intracellular domain comprises ITIM NLYAAV (SEQ ID NO: 56), ITIM VTYAEV (SEQ ID NO: 57), ITIM VTYAQL (SEQ ID NO: 58), and ITIMSIYATL (SEQ ID NO: 59). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 65. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 65.

[0496] In some embodiments, the intracellular domain comprises a sequence at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 70).In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to the LILRB1 intracellular domain (SEQ ID NO: 70).

[0497] The LILRB1 intracellular domain or a functional variant thereof disclosed herein may have at least 1, at least 2, at least 4, at least 4, at least 5, at least 6, at least 7 or at least 8 ITIMs. In some embodiments, the LILRB1 intracellular domain or a functional variant thereof has 2, 3, 4, 5 or 6 ITIMs.

[0498] In specific embodiments, the intracellular domain comprises two, three, four, five or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58) and SIYATL (SEQ ID NO: 59).

[0499] In a specific embodiment, the intracellular domain comprises at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and SIYATL (SEQ ID NO:59).

[0500] In a specific embodiment, the intracellular domain comprises three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0501] In a specific embodiment, the intracellular domain comprises four immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and SIYATL (SEQ ID NO:59).

[0502] In a specific embodiment, the intracellular domain comprises five immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and SIYATL (SEQ ID NO:59).

[0503] In a specific embodiment, the intracellular domain comprises six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0504] In a specific embodiment, the intracellular domain comprises at least seven immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and SIYATL (SEQ ID NO:59).

[0505] The LILRB1 protein has four immunoglobulin (Ig)-like domains, referred to as D1, D2, D3, and D4. In some embodiments, the LILRB1 hinge domain comprises the LILRB1D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1D3D4 domain comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 66, or identical to SEQ ID NO: 66. In some embodiments, the LILRB1D3D4 domain comprises or consists essentially of SEQ ID NO: 66.

[0506] In some embodiments, the polypeptide comprises a LILRB1 hinge domain or a functional variant thereof. In embodiments, the LILRB1 hinge domain or a functional variant thereof comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 73, SEQ ID NO: 66 or SEQ ID NO: 67, or identical to SEQ ID NO: 73, SEQ ID NO: 66 or SEQ ID NO: 67. In embodiments, the LILRB1 hinge domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO: 73, SEQ ID NO: 66 or SEQ ID NO: 67.

[0507] In some embodiments, the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO:73, SEQ ID NO:66, or SEQ ID NO:67.

[0508] In some embodiments, the LILRB1 hinge domain consists essentially of a sequence identical to SEQ ID NO:73, SEQ ID NO:66, or SEQ ID NO:67.

[0509] In some embodiments, the transmembrane domain is a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:74. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO:74. In some embodiments, the LILRB1 transmembrane domain comprises a sequence that is identical to SEQ ID NO:74. In an embodiment, the LILRB1 transmembrane domain consists essentially of a sequence that is identical to SEQ ID NO:74.

[0510] In some embodiments, the transmembrane domain can be connected to the extracellular region of the second inhibitory receptor, such as an antigen binding domain or a ligand binding domain, by a hinge (e.g., a hinge from a human protein). For example, in some embodiments, the hinge can be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge, a CD8a hinge, or a LILRB1 hinge.

[0511] In some embodiments, the second inhibitory receptor comprises an inhibitory domain. In some embodiments, the second inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory domain is isolated or derived from LILR1B.

[0512] Inhibitory receptors containing LILRB1 domain combinations

[0513] In some embodiments, the LILRB1-based inhibitory receptors of the present disclosure comprise more than one LILRB1 domain or a functional equivalent thereof. For example, in some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and an intracellular domain, or a LILRB1 hinge domain, a transmembrane domain, and an intracellular domain.

[0514] In specific embodiments, the inhibitory receptor comprises a LILRB1 hinge domain or a functional variant thereof, and a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:68, at least 96% identical to SEQ ID NO:68, at least 97% identical to SEQ ID NO:68, at least 98% identical to SEQ ID NO:68, or at least 99% identical to SEQ ID NO:68. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:68. In some embodiments, the polypeptide comprises a sequence that is identical to SEQ ID NO:68.

[0515] In other embodiments, the inhibitory receptor comprises: a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58) and SIYATL (SEQ ID NO: 59). In some embodiments, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least two ITIMs, wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58) and SIYATL (SEQ ID NO: 59).

[0516] In some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and an intracellular domain. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:69, at least 96% identical to SEQ ID NO:69, at least 97% identical to SEQ ID NO:69, at least 98% identical to SEQ ID NO:69, or at least 99% identical to SEQ ID NO:69. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:69. In some embodiments, the polypeptide comprises a sequence that is identical to SEQ ID NO:69.

[0517] In a preferred embodiment, the inhibitory receptor comprises: a LILRB1 hinge domain or a functional variant thereof; a LILRB1 transmembrane domain or a functional variant thereof; and a LILRB1 intracellular domain and / or an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from LYAAV (SEQ ID NO: 56), VTYAE (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58) and SIYATL (SEQ ID NO: 59).

[0518] In some embodiments, the inhibitory receptor comprises a sequence that is at least 95% identical to SEQ ID NO:71 or SEQ ID NO:72, or at least 99% identical to SEQ ID NO:71 or SEQ ID NO:72, or identical to SEQ ID NO:71 or SEQ ID NO:72.

[0519] In some embodiments, the polypeptide comprises a sequence that is at least 99% identical to SEQ ID NO:68, or at least 99% identical to SEQ ID NO:68, or identical to SEQ ID NO:68.

[0520] In some embodiments, the polypeptide comprises a sequence that is at least 99% identical to SEQ ID NO:69, or at least 99% identical to SEQ ID NO:69, or identical to SEQ ID NO:69.

[0521] Table 9: Polypeptide sequences of exemplary LILRB1-based inhibitory receptors

[0522]

[0523]

[0524]

[0525]

[0526] Exemplary inhibitory receptors of the present disclosure comprise a scFv specific for any HLA-A, HLA-B or HLA-C non-target antigen, the sequences of which are listed in Table 5, fused to the N-terminus of the LILRB1 hinge domain, transmembrane domain, and intracellular domain. In some embodiments, the LILRB1 hinge comprises the sequence of SEQ ID NO: 73, the LILRB1 transmembrane domain comprises the sequence of SEQ ID NO: 74, and the LILRB1 intracellular domain comprises the sequence of SEQ ID NO: 70. For example, the second inhibitory receptor comprises the scFv sequence of Table 5 fused to the N-terminus of SEQ ID NO: 71.

[0527] As a further example, the non-target antigen comprises HLA-A*02 and the second inhibitory receptor comprises the following sequence:

[0528]

[0529]

[0530] or a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical thereto.In some embodiments, the non-target antigen comprises HLA-A*02, and the second inhibitory receptor comprises the sequence of SEQ ID NO:348.

[0531] As another example, the non-target antigen comprises HLA-A*03, and the second inhibitory receptor comprises the following sequence:

[0532]

[0533] or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the non-target antigen comprises HLA-A*03, and the second inhibitory receptor comprises the sequence of SEQ ID NO:1268.

[0534] The corresponding nucleotide sequence contains the following sequence:

[0535]

[0536]

[0537] As a further example, the non-target antigen comprises HLA-A*11, and the second inhibitory receptor comprises the following sequence:

[0538]

[0539] or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the non-target antigen comprises HLA-A*11, and the second inhibitory receptor comprises the sequence of SEQ ID NO:1269.

[0540] The corresponding nucleotide sequence contains the following sequence:

[0541]

[0542]

[0543] As a further example, the non-target antigen comprises HLA-B*07, and the second inhibitory receptor comprises the following sequence:

[0544]

[0545] or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the non-target antigen comprises HLA-B*07 and the second inhibitory receptor comprises the sequence of SEQ ID NO:1270.

[0546] The corresponding nucleotide sequence contains the following sequence:

[0547]

[0548] Polynucleotides and vectors

[0549] The present disclosure provides polynucleotides (one or more) encoding the sequences of the first receptor and the second receptor of the present disclosure. The present disclosure provides immune cells comprising the polynucleotides and vectors described herein.

[0550] In some embodiments, the sequence of the first receptor and / or the second receptor is operably linked to a promoter. In some embodiments, the sequence encoding the first receptor is operably linked to a first promoter and the sequence encoding the second receptor is operably linked to a second promoter.

[0551] The present disclosure provides vectors comprising the polynucleotides described herein.

[0552] In some embodiments, the first receptor is encoded by a first vector and the second receptor is encoded by a second vector. In some embodiments, both receptors are encoded by a single vector. In some embodiments, the first vector and / or the second vector comprises an shRNA, such as a B2M shRNA.

[0553] In some embodiments, both receptors are encoded by a single vector.In some embodiments, the vector comprises a shRNA, such as a B2M shRNA.

[0554] In some embodiments, the first receptor and the second receptor are encoded by a single vector. Methods for encoding multiple polypeptides using a single vector are known to those of ordinary skill in the art, particularly including encoding multiple polypeptides under the control of different promoters, or if a single promoter is used to control the transcription of multiple polypeptides, using sequences encoding internal ribosome entry sites (IRES) and / or self-cleaving peptides. Exemplary self-cleaving peptides include T2A, P2A, E2A, and F2A self-cleaving peptides. In some embodiments, the T2A self-cleaving peptide comprises the sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 764). In some embodiments, the P2A self-cleaving peptide comprises the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 765). In some embodiments, the E2A self-cleaving peptide comprises the sequence QCTNYALLKLAGDVESNPGP (SEQID NO: 766). In some embodiments, the F2A self-cleaving peptide comprises the sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 767). In some embodiments, the T2A self-cleaving peptide comprises the sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 764). Any of the foregoing may also include an N-terminal GSG linker. For example, the T2A self-cleaving peptide may also comprise the sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351), which may be encoded by the sequence GGATCCGGAGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCC (SEQ ID NO: 768).

[0555] In some embodiments, the vector is an expression vector, ie, a vector used to express the first receptor and / or the second receptor in a suitable cell.

[0556] Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncoretroviruses, such as murine leukemia virus, because they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0557] The expression of natural or synthetic nucleic acids encoding receptors is usually achieved by operably linking the nucleic acid encoding the receptor or a portion thereof to a promoter and integrating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotic organisms. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.

[0558] The polynucleotide encoding the receptor can be cloned into various types of vectors. For example, the polynucleotide can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0559] In addition, expression vectors can be provided to cells such as immune cells in the form of viral vectors. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Typically, suitable vectors contain a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site and one or more selective markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193) in at least one organism.

[0560] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0561] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located in a 30 to 110 base pair (bp) region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so that promoter function is maintained when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it seems that individual elements can work collaboratively or independently to activate transcription.

[0562] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongated growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, U6 promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked thereto when such expression is desired, or can turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0563] In order to evaluate the expression of the receptor, the expression vector to be introduced into the cell may also contain a selective marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population sought to be transfected or infected by a viral vector. In other aspects, a selective marker may be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selective marker and the reporter gene may be flanked by suitable regulatory sequences to enable expression in the host cell. Useful selective markers include, for example, antibiotic resistance genes such as neo, etc.

[0564] Reporter gene is used to identify cells that may be transfected or transduced, and is used to assess the function of regulatory sequences. Generally, reporter gene is a gene that is not present in or is not expressed by a receptor organism or tissue, and the expression of its encoded polypeptide is demonstrated by some easily detectable characteristics (e.g., enzyme activity). The expression of reporter gene is measured at the appropriate time after DNA is introduced into the receptor cell. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well-known and can be prepared or commercially available using known techniques. Generally, the construct with the minimum 5' flanking region of the highest level of display reporter gene expression is identified as a promoter. Such promoter region can be connected to a reporter gene and used to assess the ability of a reagent to regulate promoter-driven transcription.

[0565] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical or biological methods.

[0566] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0567] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used methods for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0568] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0569] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to the inhibitors of the present disclosure, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, such as by immunological methods (ELISA and Western blots) or by assays described herein to identify agents that fall within the scope of the present disclosure.

[0570] Immune cells

[0571] The present disclosure provides immune cells comprising the receptors, vectors and polynucleotides described herein.

[0572] In some embodiments, the immune cell comprises: (a) a first receptor comprising a first extracellular ligand binding domain, the first extracellular ligand binding domain being specific for a target antigen selected from: (i) a cancer cell specific antigen, or a peptide antigen thereof in complex with major histocompatibility complex class I (MHC-I); or (ii) MSLN, or a peptide antigen thereof in complex with major histocompatibility complex class I (MHC-I); and (b) a second receptor comprising a second extracellular ligand binding domain, the second extracellular ligand binding domain being specific for a non-target antigen selected from: intercellular adhesion molecule 1 (ICAM1), catechol-O-methyltransferase (COMT), CXC motif chemokine ligand 16 (CXCL16), leucine-rich repeat neuronal 4 (LRRN4) and uroplakin 3B UPK3B, or a peptide antigen thereof in complex with major histocompatibility complex class I (MHC-I), or HLA-A*02. In some embodiments, the first receptor is a CAR or a TCR. In some embodiments, the second receptor is an inhibitory receptor, such as an inhibitory chimeric antigen receptor or TCR.

[0573] The present disclosure provides an immune cell comprising a first receptor and a second receptor, the first receptor comprising a sequence of SEQ ID NO: 303, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and the second receptor comprising a sequence of SEQ ID NO: 348, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the immune cell comprises an shRNA encoded by a sequence comprising SEQ ID NO: 349 or 350, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the immune cell comprises a first receptor, a second receptor, and a sequence encoding an shRNA, the first receptor comprising a sequence of SEQ ID NO: 303, the second receptor comprising a sequence of SEQ ID NO: 348, and the shRNA comprising a sequence of SEQ ID NO: 349 or 350. In some embodiments, the first receptor and the second receptor are encoded by a single polynucleotide, and wherein the sequences encoding the first receptor and the second receptor are separated by a sequence encoding a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351).

[0574] The present disclosure also provides an immune cell comprising a first receptor and a second receptor, the first receptor comprising a sequence of SEQ ID NO: 303, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto, and the second receptor comprising a sequence of SEQ ID NO: 1268, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the immune cell comprises an shRNA encoded by a sequence comprising SEQ ID NO: 349 or 350, or a sequence having at least 80%, at least 90% or at least 95% identity thereto. In some embodiments, the immune cell comprises a first receptor, a second receptor, and a sequence encoding shRNA, the first receptor comprising a sequence of SEQ ID NO: 303, the second receptor comprising a sequence of SEQ ID NO: 1268, and the shRNA comprising a sequence of SEQ ID NO: 349 or 350. In some embodiments, the first receptor and the second receptor are encoded by a single polynucleotide, and wherein the sequences encoding the first receptor and the second receptor are separated by a sequence encoding a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351).

[0575] As used herein, the term "immune cell" refers to a cell that participates in the innate or adaptive (acquired) immune system. Exemplary innate immune cells include phagocytes (such as neutrophils, monocytes and macrophages), natural killer (NK) cells, polymorphonuclear leukocytes (such as neutrophils, eosinophils and basophils), and mononuclear cells (such as monocytes, macrophages and mast cells). Immune cells that play a role in acquired immunity include lymphocytes, such as T cells and B cells.

[0576] As used herein, "T cell" refers to a type of lymphocyte derived from bone marrow precursors and developed in the thymus. After migrating to the thymus, several different types of T cells are produced, including helper CD4+T cells, cytotoxic CD8+T cells, memory T cells, regulatory CD4+T cells, and stem memory T cells. Ordinary technicians can distinguish different types of T cells based on the expression of markers. The method of distinguishing T cell types is obvious to ordinary technicians.

[0577] In some embodiments, the first receptor and the second receptor together specifically activate an immune cell in the presence of a target cell.

[0578] In some embodiments, immune cells are CD4+, CD8+, γδT cells, invariant T cells, iNK cells, NK cells, macrophages, or a combination thereof. In some embodiments, immune cells are γδT cells. In some embodiments, immune cells are constant T cells. In some embodiments, immune cells are constant natural killer T cells (iNKT cells). In some embodiments, immune cells are T cells. In some embodiments, immune cells are B cells. In some embodiments, immune cells are natural killer (NK) cells. In some embodiments, immune cells are CD8-. In some embodiments, immune cells are CD8+. In some embodiments, immune cells are CD4+. In some embodiments, immune cells are CD4-. In some embodiments, immune cells are CD8- / CD4+. In some embodiments, immune cells are CD8+CD4-T cells.

[0579] In some embodiments, the immune cell is non-natural. In some embodiments, the immune cell is isolated.

[0580] The method of transforming immune cell groups (such as T cells) with the carrier of the present disclosure is obvious to those of ordinary skill in the art.For example, CD3+T cell negative separation kit (Miltenyi) can be used to separate CD3+T cells from PBMC according to the manufacturer's instructions.In the presence of CD3 / 28Dynabead (1:1 cell to bead ratio) and 300 units / mL of IL-2 (Miltenyi), T cells are cultured at a density of 1x 10^6 cells / mL in X-Vivo 15 culture medium supplemented with 5% human A / B serum and 1% Pen / strep.After 2 days, T cells can be transduced with viral vectors (such as lentiviral vectors) using methods known in the art.In some embodiments, viral vectors are transduced with a multiplicity of infection (MOI) of 5.Then cells are cultured for another 5 days in IL-2 or other cytokines (such as a combination of IL-7 / 15 / 21) before enrichment.It is obvious to those of ordinary skill in the art to separate and culture other immune cell groups (such as B cells) or other T cell groups. Although this method outlines potential approaches, it should be noted that these methods are rapidly evolving. For example, excellent viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of growth to generate a highly transduced cell population that is >99% CD3+.

[0581] Methods of activating and culturing T cell populations comprising a TCR, CAR, inhibitory receptor, receptor, or vector encoding the same will be apparent to those of ordinary skill in the art.

[0582] Whether before or after the T cells are genetically modified to express a TCR, T cells can generally be activated and expanded using methods such as those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; 10040846; and U.S. Patent Application Publication No. 2006 / 0121005.

[0583] In some embodiments, the T cells of the present disclosure are expanded and activated in vitro. Generally, the T cells of the present disclosure are expanded in vitro by contact with a surface to which reagents that stimulate CD3 / TCR complex-related signals and ligands that stimulate co-stimulatory molecules on the surface of T cells are attached. In particular, a T cell population can be stimulated, for example, by contact with an anti-CD3 antibody as described herein. In order to co-stimulate auxiliary molecules on the surface of T cells, a ligand that binds to the auxiliary molecule is used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. In order to stimulate the proliferation of CD4+T cells or CD8+T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, France) can be used according to other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haan et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0584] In some embodiments, the primary stimulation signal and the costimulatory signal of T cell can be provided by different schemes.For example, the reagent providing each signal can be in solution or coupled to the surface.When coupled to the surface, the reagent can be coupled to the same surface (that is, in "cis" form) or to different surfaces (that is, in "trans" form).Or, a kind of reagent can be coupled to the surface, and another kind of reagent is in solution.In some embodiments, the reagent providing the costimulatory signal is combined with the cell surface, and the reagent providing the primary activation signal is in solution or coupled to the surface.In certain embodiments, both reagents can be in solution.In another embodiment, the reagent can be a soluble form, which is then cross-linked to the surface, such as expressing Fc receptors or antibodies or other binding agents that will be bound to the reagent.Regarding this, see, for example, the artificial antigen presenting cells (aAPC) of U.S. Patent Application Publication Nos. 20040101519 and 20060034810, which are intended to be used for activating and amplifying the T cells in the present disclosure.

[0585] In some embodiments, the two agents are immobilized on the beads, either on the same bead, i.e., "cis", or on separate beads, i.e., "trans". For example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and the two agents are co-immobilized on the same beads with equal molecular weights. In one embodiment, for CD4+T cell expansion and T cell growth, each antibody is used to bind to the beads at a ratio of 1:1. In some embodiments, the ratio of CD3:CD28 antibodies bound to the beads ranges from 100:1 to 1:100 and all integer values ​​therebetween. In one aspect of the present disclosure, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the ratio of CD3:CD28 is less than 1. In certain embodiments of the present disclosure, the ratio of anti-CD28 antibodies bound to the beads and anti-CD3 antibodies bound to the beads is greater than 2:1.

[0586] The ratio of particles to cells is 1:500 to 500:1 and any integer value therebetween can be used to stimulate T cells or other target cells. As can be readily appreciated by those of ordinary skill in the art, the ratio of particles to cells can depend on the particle size relative to the target cells. For example, beads of small size can only bind to a few cells, while larger beads can bind to many cells. In certain embodiments, the ratio of cells to particles ranges from 1:100 to 100:1 and any integer value therebetween, and in other embodiments, the ratio comprises 1:9 to 9:1 and any integer value therebetween, which can also be used to stimulate T cells. In some embodiments, a ratio of cells to beads of 1:1 is used. It will be appreciated by those skilled in the art that a variety of other ratios may be applicable to the present disclosure. In particular, the ratio will vary according to particle size and cell size and type.

[0587] In other embodiments of the present disclosure, cells (such as T cells) are combined with reagent-coated beads, beads and cells are subsequently separated, and cells are then cultured. In another embodiment, before culture, the reagent-coated beads and cells are not separated, but cultured together. In other embodiments, beads and cells are first concentrated by applying force (such as magnetic force), resulting in an increase in the connection of cell surface markers, thereby inducing cell stimulation.

[0588] For example, cell surface proteins can be connected by contacting paramagnetic beads attached with anti-CD3 and anti-CD28 with T cells. In one embodiment, cells (e.g., CD4+T cells) and beads (e.g., DYNABEADSCD3 / CD28T paramagnetic beads with a ratio of 1:1) are combined in a buffer. Similarly, it will be readily understood by those of ordinary skill in the art that any cell concentration can be used. In certain embodiments, it is necessary to significantly reduce the volume in which particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In other embodiments, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In other embodiments, a concentration of 125 or 150 million cells / ml may be used. In some embodiments, a concentration of 1 x 10 6 The cells were cultured at a density of 10 cells / mL.

[0589] In some embodiments, the mixture can be cultivated for several hours (about 3 hours) to about 14 days or any hour integer value therebetween. In another embodiment, beads and T cells are cultivated together for 2 to 3 days. Suitable conditions for T cell culture include suitable culture medium (for example, minimum essential medium or RPMI culture medium 1640 or X-vivo 15 (Lonza (Lonza))), which can contain factors necessary for proliferation and vigor, including serum (for example, fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include but are not limited to surfactants, human plasma protein powder and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with the addition of amino acids, sodium pyruvate, and vitamins, without serum or supplemented with an appropriate amount of serum (or plasma) or a defined hormone group, and / or a certain amount of cytokines (one or more) sufficient to grow and amplify T cells. In some embodiments, the culture medium comprises X-VIVO-15 culture medium supplemented with 5% human A / B serum, 1% penicillin / strep, and 300 units / ml of IL-2 (Miltenyi).

[0590] T cells are maintained under conditions necessary to support growth, such as an appropriate temperature (eg, 37° C.) and atmosphere (eg, air plus 5% CO 2 ).

[0591] In some embodiments, the T cells comprising TCR, CAR and inhibitory receptors of the present disclosure are autologous. Prior to amplification and genetic modification, a T cell source is obtained from a subject. Immune cells (such as T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments of the present disclosure, any number of T cell lines available in the art can be used. In certain embodiments of the present disclosure, any number of techniques known to those skilled in the art, such as Ficoll can be used. TM Isolate and obtain T cells from a unit of blood collected from a subject.

[0592] In some embodiments, cells from individual circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma portion and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution lacks calcium and can lack magnesium or can lack many (if not all) divalent cations. As will be readily understood by those of ordinary skill in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as PBS, PlasmaLyte A, or other saline solutions containing or not containing buffers without Ca2+, Mg2+. Alternatively, the apheresis sample can be freed of unwanted components and the cells resuspended directly in culture medium.

[0593] In some embodiments, the monocytes are removed by lysing the red blood cells, for example by PERCOLL TM Immune cells, such as T cells, are separated from peripheral blood lymphocytes by gradient centrifugation or by countercurrent centrifugal elutriation. Specific subsets of immune cells, such as T cells, B cells, or CD4+ T cells, can be further separated by positive or negative selection techniques. For example, in one embodiment, T cells are separated by incubation with anti-CD4-conjugated beads for a period of time sufficient to positively select the desired T cells.

[0594] Enrichment of immune cell populations such as T cell populations by negative selection can be achieved by a combination of antibodies against surface markers specific to negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, in order to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD 14, CD20, CD 11b, CD 16, HLA-DR and CD8.

[0595] In order to separate the desired immune cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be changed. In certain embodiments, it is necessary to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between cells and beads.

[0596] In some embodiments, cells can be incubated on a rotator at different speeds for different lengths of time at 2 to 10°C or at room temperature.

[0597] PBMCs for stimulating T cells or for isolating immune cells (such as T cells) therefrom can also be frozen after the washing steps. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and, to a certain extent, monocytes from the cell population. After the washing steps to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful herein, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or medium containing 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% glucose 5%, 0.45% NaCl, 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1° / minute and storing in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0598] The present disclosure provides immune cells expressing activator and / or blocker receptors described herein, wherein the immune cells have reduced expression and / or function of major histocompatibility (MHC) class I complex.

[0599] In some embodiments, the immune cells are autologous. For example, the immune cells are isolated from or derived from the same subject who receives the cells as part of a treatment regimen. It is advantageous to modify the autologous immune cells with a blocker receptor specific for MHC class I antigens to reduce the expression and / or function of MHC class I. Without wishing to be bound by theory, modifying the autologous immune cells to reduce the expression and / or function of MHC class I reduces the binding of the blocker receptor to the MHC class I expressed by the immune cells, whether in cis or trans.

[0600] In some embodiments, the immune cells are allogeneic. The allogeneic immune cells may be derived from a donor other than the subject to whom the immune cells are administered. Allogeneic immune cells are often referred to as "off-the-shelf" or "universal" in cell therapy because allogeneic cells may be prepared and stored for use with subjects of multiple genotypes.

[0601] Any suitable method of reducing the expression and / or function of the MHC class I complex is considered to be within the scope of the present disclosure, and particularly includes knocking down the expression of one or more interfering RNAs encoding MHC class I components, or modification of genes encoding MHC class I components.

[0602] The major histocompatibility complex (MHC) is a locus on the vertebrate genome that encodes a group of polypeptides required for the adaptive immune system. Among them are MHC class I polypeptides, which include HLA-A, HLA-B and HLA-C and their alleles. MHC class I alleles are highly polymorphic and expressed in all nucleated cells. MHC class I polypeptides encoded by HLA-A, HLA-B and HLA-C and their alleles form heterodimers with β2 microglobulin (B2M) and exist in complex with antigens on the cell surface. As described herein, MHC class I genes or polypeptides may refer to any polypeptide found in MHC or the corresponding gene encoding the polypeptide. In some embodiments, the immune cells of the present disclosure are inactivated by inhibitor ligands, which include MHC class I polypeptides, such as HLA-A, HLA-B and HLA-C and their alleles. The HLA-A allele can be, for example but not limited to, HLA-A*02, HLA-A*02:01, HLA-A*02:01:01, HLA-A*02:01:01:01, and / or any gene encoding a protein identical or similar to the HLA-A*02 protein. Therefore, in order to prevent autocrine signal transduction / binding as described herein, it is necessary to eliminate or reduce the expression of polypeptides encoded by HLA-A, HLA-B and HLA-C and their alleles in immune cells.

[0603] Immune cells with reduced expression of MHC class I peptides

[0604] In some embodiments, the immune cells described herein are modified to inactivate or reduce or eliminate the expression or function of endogenous genes encoding alleles of endogenous MHC class I polypeptides. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A, HLA-B and / or HLA-C. HLA-A, HLA-B and HLA-C are encoded by HLA-A, HLA-B and HLA-C loci. Each of HLA-A, HLA-B and HLA-C includes many variant alleles, all of which are considered to be within the scope of the present disclosure. In some embodiments, the gene encoding MHC class I polypeptides is HLA-A. In some embodiments, the gene encoding MHC class I polypeptides is HLA-A*02. In some embodiments, the gene encoding MHC class I polypeptides is HLA-A*02:01. In some embodiments, the gene encoding MHC class I polypeptides is HLA-A*02:01:01. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A*02:01:01:01.

[0605] In some embodiments, the genetically engineered immune cells described herein are modified to reduce or eliminate the expression of the B2M gene product. The β-2 microglobulin (B2M) gene encodes a protein associated with the major histocompatibility complex (MHC) class I (i.e., the MHC-I complex). The MHC-I complex is required for cell surface antigen presentation. When B2M is missing, the MHC-I complex is destroyed and does not work (Wang D et al. Stem Cells Transl Med. 4: 1234-1245 (2015)). In addition, gene editing techniques known in the art can be used to efficiently destroy the B2M gene (Ren et al. Clin. Cancer Res. 23: 2255-2266 (2017)). Reducing or eliminating B2M can reduce or eliminate functional MHC I on the surface of immune cells.

[0606] The present disclosure provides a gene editing system for editing endogenous target genes in immune cells. The present disclosure provides interfering RNA specific to target gene sequences. Gene editing systems, such as CRISPR / Cas systems, TALENs, and zinc fingers, can be used to generate double-strand breaks, which can be used to introduce mutations through gene repair mechanisms such as homology-directed repair or non-homologous end joining (NHEJ). NHEJ after resection of the broken ends or inappropriate end joining can be used to introduce deletions. In some embodiments, the target gene comprises a gene encoding an MHC-I complex subunit.

[0607] The target gene sequence includes but is not limited to promoters, enhancers, introns, exons, intron / exon junctions, transcription products (pre-mRNA, mRNA and splice variants) and / or 3' and 5' untranslated regions (UTR). For the purpose of genetic editing in immune cells as described herein, any gene element or combination of gene elements can be targeted. The modification of the target gene can be completed using any method known in the art to edit the target gene, resulting in expression or functional changes or destruction of the target gene or gene product.

[0608] In some embodiments, modifying a gene encoding an MHC class I polypeptide comprises deleting all or part of the gene. In some embodiments, modifying a gene encoding an MHC class I polypeptide comprises introducing a mutation into the gene. In some embodiments, the mutation comprises a deletion, insertion, substitution, or frameshift mutation. In some embodiments, modifying a gene comprises using a nucleic acid-guided endonuclease.

[0609] The gene sequences of the target genes described herein are known in the art. These sequences can be found in public databases, such as the NCBI gene bank or the NCBI nucleotide database. The sequences can be found using gene identifiers, for example, the HLA-A gene has an NCBI gene ID: 3105, the HLA-B gene has an NCBI gene ID: 3106, the HLA-C gene has an NCBI gene ID: 3107, and the B2M gene has an NCBI gene ID: 567 and an NCBI reference sequence: NC_000015.10. Gene sequences can also be found by searching public databases using keywords. For example, HLA-A alleles can be found in the NCBI nucleotide database by searching for the keywords "HLA-A*02", "HLA-A*02:01", "HLA-A*02:01:01" or "HLA-A*02:01:01:01". These sequences can be used for targeting in various gene editing techniques known in the art. Table 10 provides non-limiting exemplary sequences of HLA-A alleles and B2M gene sequences targeted for modification described herein.

[0610] Table 10. Exemplary target gene sequences

[0611] B2M mRNA SEQ ID NO:769 B2M gene (GenBank:567) SEQ ID NO:770 HLA-A*02:01:01:01 sequence encoding mRNA SEQ ID NO:771 HLA-A*02 (GenBank: LK021978.1) SEQ ID NO:772

[0612] One of ordinary skill in the art will appreciate that T can be substituted for U to convert an RNA sequence to a DNA sequence, and vice versa, and both are contemplated as target gene sequences of the present disclosure.

[0613] In some embodiments, a nucleic acid-guided endonuclease is used to edit a target gene in an immune cell described herein. Exemplary nucleic acid-guided endonucleases include class II endonucleases, such as CRISPR / Cas9.

[0614] As used herein, "CRISPR" or "CRISPR gene editing" refers to a group of clustered regularly spaced short palindromic repeats, or a system comprising such a group of repeats. "Cas" as used herein refers to CRISPR-associated proteins. The "CRISPR / Cas" system refers to a system derived from CRISPR and Cas, which can be used to silence, knock out or mutate target genes. The system is a prokaryotic immune system that confers resistance to foreign genetic elements (such as plasmids and phages) and provides an acquired immunity. The CRISPR / Cas system has been modified for gene editing. This is achieved by introducing one or more specially designed guide nucleic acids (gNA), usually guide RNA (gRNA), and a suitable Cas endonuclease that forms a ribonucleoprotein complex with gNA into eukaryotic cells. GNA guides the gNA-endonuclease protein complex to the target genomic position, and the endonuclease introduces a chain break at the target genomic position. This chain break can be repaired by cellular mechanisms, such as non-homologous end joining (causing deletions) or homologous repair (which can generate insertions), thereby introducing genetic modifications into the host cell genome.

[0615] CRISPR / Cas systems are classified by class and by type. Class 2 systems currently represent single interfering proteins classified into three different types (Type II, Type V, and Type VI). Any Class 2 CRISPR / Cas system suitable for gene editing, such as a Type II, Type V, or Type VI system, is contemplated to be within the scope of the present disclosure. Exemplary Class 2 Type II CRISPR systems include Cas9, Csn2, and Cas4. Exemplary Class 2 Type V CRISPR systems include Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, and Cas12k (C2c5). Exemplary Class 2 Type VI systems include Cas13, Cas13a (C2c2) Cas13b, Cas13c, and Cas13d.

[0616] CRISPR sequences, sometimes referred to as CRISPR loci, include alternating repeat sequences and spacers. In naturally occurring CRISPRs, the spacers typically include sequences exogenous to bacteria, such as plasmid or phage sequences. As described herein, the spacer sequences may also be referred to as "targeting sequences." In CRISPR / Cas systems used for genetic engineering, the spacers are derived from target gene sequences (gNA).

[0617] An exemplary Class 2 Type II CRISPR system relies on the protein Cas9, which is a nuclease with two active cleavage sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in gene editing systems. Pennisi (2013) Science 341: 833-836. In some embodiments, the Cas protein used to modify immune cells is Cas9.

[0618] Thus, the CRISPR / Cas system can be used to edit a target gene, for example, by adding or deleting base pairs, or introducing premature terminations to reduce the expression of the target, thereby targeting a gene for editing in the immune cells described herein. The CRISPR / Cas system can also be used like RNA interference to turn off a target gene in a reversible manner. For example, in mammalian cells, RNA can guide the Cas protein to the target gene promoter, spatially blocking the RNA polymerase.

[0619] The Cas protein can be derived from any bacterial or archaeal Cas protein.Any suitable CRISPR / Cas system is considered to be within the scope of the present disclosure. In other aspects, the Cas protein comprises one or more of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a (Cpf1), Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, CasY, homologs thereof, or modified forms thereof. In some embodiments, the Cas protein is a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, Cas10, or a combination or complex of these proteins. In some embodiments, the Cas protein is a Cas9 protein.

[0620] Artificial CRISPR / Cas systems that inhibit target genes can be generated using techniques known in the art, such as those described in U.S. Publication No. 20140068797 and Cong (2013) Science 339:819-823. Other artificial CRISPR / Cas systems known in the art that inhibit target genes can also be generated, for example, as described in Tsai (2014) Nature Biotechnol., 32:6 569-576, U.S. Patent Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359. Methods for designing suitable gNAs for specific Cas proteins are known to those of ordinary skill in the art.

[0621] The present disclosure provides gene targeting guide nucleic acids (gNA) that can direct the activity of related polypeptides (e.g., nucleic acid-guided endonucleases) to specific target gene sequences within the target nucleic acid genome. Genome targeting nucleic acids can be RNA. Genome targeting RNA is referred to herein as "guide RNA" or "gRNA". Guide RNA may include at least a targeting sequence and a CRISPR repeat sequence that hybridizes with the target nucleic acid sequence of interest. In some type II systems, gRNA also includes a second RNA called a tracrRNA sequence, also referred to herein as a "scaffold" sequence. In type II guide RNA (gRNA), CRISPR repeat sequences and scaffold sequences hybridize with each other to form a duplex. In type V guide RNA (gRNA), crRNA forms a duplex. In both systems, the duplex can bind to a site-directed polypeptide so that the guide RNA and the site-directed polypeptide form a complex. Gene targeting nucleic acids can provide target specificity to the complex by its association with the site-directed polypeptide. Therefore, gene targeting nucleic acids can guide the activity of site-directed polypeptides.

[0622] In some embodiments, the present disclosure provides a guide RNA comprising a targeting sequence and a guide RNA scaffold sequence, wherein the targeting sequence is complementary to a sequence of a target gene.

[0623] Exemplary guide RNAs include a targeting sequence of about 15 to 20 bases. As will be appreciated by those of ordinary skill in the art, each gRNA can be designed to include a targeting sequence complementary to its genomic target sequence. For example, each targeting sequence (e.g., an RNA version of the DNA sequence presented in Tables 11 and 14, minus three 3' nucleotides representing a PAM site) can be placed in a single RNA chimera or crRNA.

[0624] The gene targeting nucleic acid can be a double-molecule guide RNA. The gene targeting nucleic acid can be a single-molecule guide RNA. The gene targeting nucleic acid can be a guide RNA of any known configuration known in the art, for example including paired gRNAs, or multiple gRNAs used in a single step. Although it is clear from the genomic sequence where the coding sequence and splice junctions are, other features required for gene expression may be specific and unclear.

[0625] The double-molecule guide RNA may comprise double-stranded RNA. The first strand comprises a sequence in the 5' to 3' direction, an optional spacer extension sequence, a targeting sequence, and a minimum CRISPR repeat sequence. The second strand may comprise a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3'tracrRNA sequence, and an optional tracrRNA extension sequence.

[0626] The single molecule guide RNA (sgRNA) in the type II system can include an optional spacer extension sequence, a targeting sequence, a minimum CRISPR repeat sequence, a single molecule guide joint, a minimum tracrRNA sequence, a 3'tracrRNA sequence, and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension can include an element that provides additional functions (e.g., stability) for the guide RNA. The single molecule guide joint can connect the minimum CRISPR repeat sequence and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can include one or more hairpins.

[0627] In some embodiments, the guide RNA or single-molecule guide RNA (sgRNA) may include a targeting sequence and a scaffold sequence. In some embodiments, the scaffold sequence is a Cas9gRNA sequence. In some embodiments, the scaffold sequence is encoded by a DNA sequence comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the following sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 773). In some embodiments, the scaffold sequence is encoded by a DNA sequence comprising GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 773).

[0628] In some embodiments, such as those in which the CRISPR / Cas system is a Cas9 system, the sgRNA can comprise a targeting sequence of 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a targeting sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a targeting sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a variable length targeting sequence having 17 to 30 nucleotides at the 5' end of the sgRNA sequence.

[0629] The arrangement of suitable scaffold sequences and scaffold targeting sequences will depend on the choice of endonuclease and are known to those skilled in the art.

[0630] Single-molecule guide RNA (sgRNA) in type II systems, such as Cas9, can contain a minimal CRISPR repeat sequence and a targeting sequence in the 5' to 3' direction.

[0631] As an illustration, the guide RNA used in the CRISPR / Cas9 or CRISPR / Cpf1 system, or other smaller RNAs, can be easily synthesized by chemical methods, as shown below and described in the art. Although chemical synthesis methods continue to expand, when the length of the polynucleotide increases significantly beyond a hundred or so nucleotides, it often becomes more challenging to purify such RNA by methods such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE). One method for generating longer RNAs is to produce two or more molecules linked together. Longer RNAs, such as RNAs encoding Cas9 or Cpf1 nuclease, are easier to generate enzymatically. Various types of RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic generation of RNA, such as modifications that enhance stability, reduce the likelihood or extent of an innate immune response, and / or enhance other properties, as described in the art.

[0632] The targeting sequence of gRNA hybridizes with the sequence in the target nucleic acid of interest. The targeting sequence of the genome targeting nucleic acid can interact with the target nucleic acid in a sequence-specific manner by hybridization (i.e., base pairing). The nucleotide sequence of the targeting sequence can vary according to the sequence of the target nucleic acid of interest.

[0633] In the Cas9 system described herein, the targeting sequence can be designed to hybridize with a target nucleic acid located at the 5' reverse complement of the PAM of the Cas9 enzyme used in the system. The targeting sequence can completely match the target sequence or can have a mismatch. Each CRISPR / Cas system protein can have a specific PAM sequence in a specific direction and position that it recognizes in the target DNA. For example, Streptococcus pyogenes (S.pyogenes) Cas9 recognizes a PAM containing the sequence 5'-NRG-3' in the target nucleic acid, wherein R contains A or G, wherein N is any nucleotide and N is adjacent to the 3' of the target nucleic acid sequence targeted by the targeting sequence. It is obvious to those of ordinary skill in the art to select a suitable PAM sequence.

[0634] The target sequence is complementary to and hybridizes with the targeting sequence of gRNA. The target nucleic acid sequence may include 20 nucleotides. The target nucleic acid may include less than 20 nucleotides. The target nucleic acid may include more than 20 nucleotides. The target nucleic acid may include at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, such as those in which the CRISPR / Cas system is a Cas9 system, the target nucleic acid sequence may include 20 nucleotides of 5' of the first nucleotide of the reverse complement of the PAM sequence next to it. The target nucleic acid sequence is generally referred to as a PAM strand or a target strand, while a complementary nucleic acid sequence is generally referred to as a non-PAM strand or a non-target strand. Those skilled in the art will recognize that the targeting sequence hybridizes with the non-PAM strand of the target nucleic acid, see, for example, US20190185849A1.

[0635] In some examples, the percent complementarity between the targeting sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some examples, the percent complementarity between the targeting sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some examples, the percent complementarity between the targeting sequence and the target nucleic acid is 100% over the six consecutive 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. The percent complementarity between the targeting sequence and the target nucleic acid can be at least 60% over about 20 consecutive nucleotides. The length of the targeting sequence and the target nucleic acid can differ by 1 to 6 nucleotides, which can be considered as one or more bulges.

[0636] Computer programs known to those of ordinary skill in the art can be used to design or select targeting sequences. Computer programs can use variables such as predicted melting temperatures, secondary structure formation, predicted annealing temperatures, sequence identity, genomic background, chromatin accessibility, %GC, the frequency of genomic occurrence (e.g., due to mismatches, insertions or deletions, the same or similar but different sequences at one or more sites), methylation status, the presence of SNPs, etc. Available computer programs can use NCBI gene IDs, official gene symbols, Ensembl gene IDs, genomic coordinates, or DNA sequences as inputs, and create output files containing sgRNA targeting the appropriate genomic regions designated as inputs. Computer programs can also provide a summary of statistics and scores, indicating that sgRNA targets and off-target binding to target genes (Doench et al. Nat Biotechnol. 34: 184-191 (2016)). The present disclosure provides guide RNAs comprising targeting sequences. In some embodiments, the guide RNA further comprises a guide RNA scaffold sequence. In some embodiments, the targeting sequence is complementary to the sequence of a target gene selected from the group consisting of HLA-A, HLA-B, HLA-C, B2M or its alleles. In some embodiments, the target gene is the HLA-A gene. In some embodiments, the target gene is the HLA-B gene. In some embodiments, the target gene is the HLA-C gene. In some embodiments, the target gene is HLA-A, HLA-B, HLA-C or a combination thereof. In some embodiments, the targeting sequence comprises a sequence having about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity or the same sequence as the sequences disclosed in Tables 11 and 14.

[0637] In some embodiments, the gNA specifically targets a sequence of the endogenous HLA-A locus. In some embodiments, the gNA specifically targeting the HLA-A locus sequence comprises a sequence having about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with a sequence selected from the sequences disclosed in Table 11. In some embodiments, the gNA specifically targeting the HLA-A locus sequence comprises a sequence selected from the sequences disclosed in Table 11.

[0638] In some embodiments, the gNA specifically targets the sequence of the HLA-A*02 allele. For example, the gRNA specifically targets and hybridizes to a sequence possessed by all HLA-A*02 alleles, but not the sequences possessed by the HLA-A*02 and HLA-A*03 alleles. In some embodiments, the gNA specifically targets the sequence of the HLA-A*02:01 allele. In some embodiments, the gNA specifically targets the sequence of the HLA-A*02:01:01 allele. In some embodiments, the gNA specifically targets the sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets the sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets the sequence of the HLA-A*02:01:01:01 allele.

[0639] In some embodiments, the gNA specifically targets the DNA sequence encoding HLA-A*02.

[0640] In some embodiments, the gNA specifically targets a coding DNA sequence possessed by more than 1000 HLA-A*02 alleles. In some embodiments, the gNA specifically targeting a coding DNA sequence in more than 1000 HLA-A*02 alleles comprises a sequence that is about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identical or identical to a sequence selected from the group consisting of those shown in Table 11.

[0641] The sequences in Tables 11-14 are represented as DNA sequences. Those skilled in the art will appreciate that thymine (T) can be replaced with uracil (U) in any DNA sequence, including those shown in Tables 11-14, to obtain the corresponding RNA sequence.

[0642] Table 11. Exemplary sequences targeting HLA-A alleles

[0643]

[0644]

[0645]

[0646] The sequences disclosed in Table 11 include the corresponding genomic sequences, including the PAM sequence. It will be understood by those skilled in the art that the targeting sequence of the gRNA does not include the three 3' terminal nucleotides of the sequence in Table 11, which represent the corresponding PAM site of the gRNA.

[0647] The present disclosure provides gNAs comprising targeting sequences specific to the B2M gene. In some embodiments, the gNA specifically targets the coding sequence (CDS) sequence of the B2M gene. In some embodiments, the gNA comprises a sequence targeting the promoter sequence of the B2M gene.

[0648] In some embodiments, the gNA comprises a targeting sequence and a gNA scaffold sequence. In some embodiments, the targeting sequence comprises a sequence shown in Table 12, or a sequence having about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity thereto.

[0649] In some embodiments, the targeting sequence is complementary to the sequence of the B2M gene. In some embodiments, the B2M gene comprises a sequence having about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity to the B2M sequence shown in Table 10.

[0650] Table 12. Exemplary sequences targeting B2M

[0651]

[0652]

[0653]

[0654] In some embodiments, TALEN gene editing is used to edit the immune cells described herein.

[0655] "TALEN" or "TALEN gene editing" refers to transcription activator-like effector nucleases, which are artificial nucleases used to edit target genes.

[0656] TALEN is artificially produced by fusing the TAL effector DNA binding domain with the DNA cleavage domain. Transcription activator-like effectors (TALEs) derived from Xanthomonas bacteria can be engineered to bind to any desired DNA sequence, including a portion of a target gene, such as a TCR subunit, an MHC class I complex component, or CD52. By combining engineered TALEs with DNA cleavage domains, restriction enzymes specific to any desired DNA sequence (including target gene sequences) can be produced. They can then be introduced into cells, where they can be used for genome editing.

[0657] To generate TALENs, the TALE protein is fused to a nuclease (N), which is a wild-type or mutant folded endonuclease. Several FokI mutations have been made for use in TALENs; for example, these increase cleavage specificity or activity.

[0658] The FokI domain functions as a dimer, requiring two constructs with distinct DNA binding domains for sites in the target genome with appropriate orientation and spacing. The number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain, as well as the number of bases between the two individual TALEN binding sites, appear to be important parameters for achieving high levels of activity.

[0659] TALENs specific for sequences in a target gene can be constructed using any method known in the art, including various schemes using modular components.

[0660] In some embodiments, ZFN gene editing is used to edit a target gene in an immune cell described herein.

[0661] “ZFN” or “zinc finger nuclease” or “ZFN gene editing” refers to zinc finger nuclease, an artificial nuclease that can be used to edit a target gene.

[0662] Like TALENs, ZFNs contain a folded nuclease domain (or a derivative thereof) fused to a DNA binding domain. In the case of ZFNs, the DNA binding domain contains one or more zinc fingers.

[0663] Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. Zinc fingers can include, for example, Cys2His2, and can recognize sequences of about 3-bp. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize sequences of about 6, 9, 12, 15 or 18-bp. Various selection and modular assembly techniques can be used to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0664] Like TALEN, ZFN must dimerize to cut DNA. Therefore, a pair of ZFN is needed to target non-palindromic DNA sites. Two individual ZFN must bind to opposite strands of DNA, with their nucleases appropriately spaced.

[0665] Also like TALENs, ZFNs can create double-strand breaks in DNA, which, if not repaired correctly, can produce frameshift mutations, leading to reduced expression and amount of the target gene or gene product in the cell. ZFNs can also be used in homologous recombination to create mutations in target genes.

[0666] Any method known in the art can be used to construct ZFNs specific for a sequence in a target gene.

[0667] In some embodiments, RNA interference is used to reduce the expression and function of one or more MCH-I components. "RNAi" or "RNA interference" refers to the process of sequence-specific post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA). Duplex RNA, such as siRNA (small interfering RNA), miRNA (microRNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi interaction RNA) or rasiRNA (repeat-associated siRNA) and modified forms thereof, can mediate RNA interference. These dsRNA molecules can be commercially available or can be designed and prepared based on known sequence information. The antisense strands of these molecules can include RNA, DNA, PNA or a combination thereof. DNA / RNA chimeric polynucleotides include but are not limited to double-stranded polynucleotides composed of DNA and RNA that inhibit the expression of a target gene. As described herein, a dsRNA molecule can also include one or more modified nucleotides, which can be integrated into one or two chains.

[0668] In RNAi gene silencing or knockdown, a dsRNA comprising a first (antisense) strand complementary to a portion of a target gene and a second (sense) strand fully or partially complementary to the first antisense strand is introduced into an organism. After introduction into the organism, the target gene-specific dsRNA is processed into relatively small fragments (siRNA), which can then be distributed throughout the organism, reducing the messenger RNA of the target gene, producing a phenotype very similar to that produced by complete or partial deletion of the target gene.

[0669] Some dsRNA in cells can undergo the action of Dicer enzyme (a ribonuclease III enzyme). Dicer can process dsRNA into shorter dsRNA fragments, i.e. siRNA. RNAi also involves a nuclease complex called RNA-induced silencing complex (RISC). After being cut by Dicer, siRNA enters the RISC complex and directly cuts a single-stranded RNA target with a sequence complementary to the antisense strand of the siRNA duplex. The other strand of siRNA is the passenger strand. The cutting of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex. Therefore, siRNA can downregulate or knock down gene expression by mediating RNA interference in a sequence-specific manner.

[0670] As used herein, with respect to RNA interference, "target gene" or "target sequence" refers to a gene or gene sequence whose corresponding RNA is targeted to be degraded by the RNAi approach using dsRNA or siRNA as described herein. Exemplary target gene sequences are shown in Table 10. In order to target a gene, for example, using siRNA, the siRNA comprises an antisense region complementary to or substantially complementary to at least a portion of the target gene or sequence, and a sense strand complementary to the antisense strand. Once introduced into a cell, the siRNA guides the RISC complex to cut the RNA comprising the target sequence, thereby degrading the RNA. The present disclosure provides interfering RNA. The double-stranded RNA molecules of the present disclosure can be in the form of any type of RNA interference molecules known in the art. In some embodiments, the double-stranded RNA molecule is a small interfering RNA (siRNA). In other embodiments, the double-stranded RNA molecule is a short hairpin RNA (shRNA) molecule. In other embodiments, the double-stranded RNA molecule is a Dicer substrate processed in a cell to produce siRNA. In other embodiments, the double-stranded RNA molecule is a part of a microRNA precursor molecule.

[0671] In some embodiments, the shRNA is of a length suitable for use as a Dicer substrate, which can be processed to generate a RISC-active siRNA molecule. See, e.g., Rossi et al., US 2005 / 0244858.

[0672] Dicer substrate double-stranded RNA (e.g., shRNA) can have a length sufficient to be processed by Dicer to produce an active siRNA, and can further include one or more of the following properties: (i) the Dicer substrate shRNA can be asymmetric, for example, having a 3' overhang on the antisense strand, (ii) the Dicer substrate shRNA can have a modified 3' end on the sense strand to direct the orientation of Dicer binding and processing of the dsRNA into an active siRNA, such as by incorporation of one or more DNA nucleotides, and (iii) the first and second strands of the Dicer substrate ds RNA can be 21 to 30 bp in length.

[0673] In some embodiments, the interfering RNA comprises a sequence complementary to the sequence of the B2M mRNA. In some embodiments, the interfering RNA is capable of inducing RNAi-mediated degradation of the B2M mRNA. In some embodiments, the B2M mRNA sequence comprises a coding sequence. In some embodiments, the B2M mRNA sequence comprises an untranslated region.

[0674] In some embodiments, the interfering RNA comprises a sequence complementary to the HLA-A*02 mRNA sequence. I...

Claims

1. An immune cell comprising: a. a first receptor comprising an extracellular ligand binding domain specific for mesothelin (MSLN); and b. a second receptor comprising an extracellular ligand binding domain specific for HLA-A*03, wherein the first receptor is an activator receptor responsive to MSLN; and wherein the second receptor is an inhibitory receptor responsive to HLA-A*03.

2. The immune cell of claim 1, wherein the HLA-A*03 is lost in MSLN+ cancer cells by loss of heterozygosity.

3. An immune cell according to any one of claims 1-2, wherein the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6; or a CDR sequence having at most 1, 2 or 3 substitutions, deletions or insertions relative to the CDRs in Table 6 or Table 7.

4. An immune cell according to any one of claims 1-2, wherein the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of the sequences disclosed in SEQ ID NO: 1260-1265 or Table 6; or a CDR sequence having a maximum of 1, 2 or 3 substitutions, deletions or insertions relative to the CDRs of the sequences disclosed in SEQ ID NO: 1260-1265 or Table 6.

5. An immune cell according to any one of claims 1-4, wherein the extracellular ligand binding domain of the second receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 1260-1265.

6. An immune cell according to any one of claims 1-5, wherein the extracellular ligand binding domain of the second receptor comprises SEQ ID NO:1259 or the sequence disclosed in Table 5, or any one of the sequences having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

7. An immune cell according to any one of claims 1-5, wherein the extracellular ligand binding domain of the second receptor comprises a variable heavy chain (VH) portion, which comprises SEQ ID NO: 1267 or a sequence that is at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical thereto; wherein the extracellular ligand binding domain of the second receptor comprises a variable light chain (VL) portion, which comprises SEQ ID NO: 1266 or a sequence that is at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical thereto; and / or wherein the extracellular ligand binding domain of the second receptor comprises SEQ ID NO: 1268, or a sequence that is at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical thereto.

8. The immune cell of any one of claims 1-7, wherein the first receptor is a chimeric antigen receptor (CAR).

9. An immune cell according to any one of claims 1-8, wherein the extracellular ligand binding domain of the first receptor comprises the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 2; or a CDR sequence having a maximum of 1, 2 or 3 substitutions, deletions or insertions relative to the CDRs in Table 2.

10. An immune cell according to any one of claims 1-8, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy chain (VH) portion and a variable light chain (VL) portion, the variable heavy chain (VH) portion comprises a sequence shown in Table 3, and the variable light chain (VL) portion comprises a sequence shown in Table 4; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

11. An immune cell according to any one of claims 1-10, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy chain (VH) portion and a variable light chain (VL) portion, the variable heavy chain (VH) portion comprises SEQ ID NO: 233 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto, and the variable light chain (VL) portion comprises SEQ ID NO: 279 or a sequence having 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

12. An immune cell according to any one of claims 1-10, wherein the extracellular ligand binding domain of the first receptor comprises a sequence selected from SEQ ID NOs: 3-6, 80 and 154-215, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

13. An immune cell according to any one of claims 1-10, wherein the extracellular ligand binding domain of the first receptor comprises the scFv sequence of SEQ ID NO: 171; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

14. The immune cell according to any one of claims 1-13, wherein the first receptor comprises a hinge domain, a transmembrane domain and an intracellular domain.

15. The immune cell of claim 14, wherein the hinge domain comprises a CD8α hinge domain.

16. The immune cell of claim 15, wherein the CD8α hinge domain comprises the sequence of SEQ ID NO: 7, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

17. The immune cell of any one of claims 8-16, wherein the transmembrane domain comprises a CD28 transmembrane domain.

18. The immune cell of claim 17, wherein the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 11, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

19. An immune cell according to any one of claims 8-18, wherein the intracellular domain comprises a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, and a CD3 ζ activation domain.

20. The immune cell of claim 19, wherein the intracellular domain comprises the sequence of SEQ ID NO: 285, or a sequence that is at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical thereto.

21. The immune cell of any one of claims 1-20, wherein the first receptor comprises the sequence of SEQ ID NO: 303, or a sequence that is at least 90%, at least 95%, at least 97% or at least 99% identical thereto.

22. The immune cell according to any one of claims 1-21, wherein the second receptor comprises the LILRB1 intracellular domain or a functional variant thereof.

23. The immune cell of claim 22, wherein the LILRB1 intracellular domain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO:70, or a sequence that is identical to SEQ ID NO:

70.

24. The immune cell according to any one of claims 1-23, wherein the second receptor comprises a LILRB1 transmembrane domain or a functional variant thereof.

25. The immune cell according to claim 24, wherein the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 74 or a sequence that is identical to SEQ ID NO:

74.

26. The immune cell according to any one of claims 1-25, wherein the second receptor comprises a LILRB1 hinge domain or a functional variant thereof.

27. The immune cell of claim 26, wherein the LILRB1 hinge domain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO:73, or a sequence that is identical to SEQ ID NO:

73.

28. The immune cell of any one of claims 1-27, wherein the second receptor comprises a LILRB1 intracellular domain, a LILRB1 transmembrane domain, a LILRB1 hinge domain, a functional variant of any of these domains, or a combination thereof.

29. The immune cell of claim 28, wherein the LILRB1 hinge domain, LILRB1 intracellular domain, and LILRB1 transmembrane domain comprise SEQ ID NO: 71, or a sequence that is at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 71, or a sequence that is identical to SEQ ID NO:

71.

30. The immune cell of any one of claims 1-29, wherein the second receptor comprises the sequence of SEQ ID NO: 1268, or a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical thereto.

31. The immune cell of any one of claims 1-30, wherein the MSLN+ cancer cells are mesothelioma cancer cells, ovarian cancer cells, cervical cancer cells, colorectal cancer cells, esophageal cancer cells, head and neck cancer cells, renal cancer cells, uterine cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, colorectal cancer cells or bile duct cancer cells.

32. The immune cell of claim 31, wherein the MSLN+ cancer cells are mesothelioma cancer cells, ovarian cancer cells, cervical cancer cells, uterine cancer cells, gastric cancer cells, pancreatic cancer cells, or lung adenocarcinoma cells.

33. The immune cell according to any one of claims 1-32, wherein the MSLN+ cancer cells are MSLN+ / HLA-A*03- cancer cells that do not express HLA-A*03.

34. The immune cell of claim 33, wherein the MSLN+ / HLA-A*03- cancer cells are derived from MSLN+ / HLA-A*03+ cells that have lost HLA-A*03 through loss of heterozygosity at HLA-A.

35. The immune cell of any one of claims 1-34, wherein the first receptor and the second receptor together specifically activate the immune cell in the presence of the MSLN+ / HLA-A*03- cancer cells having loss of heterozygosity.

36. An immune cell according to any one of claims 1-35, wherein in the presence of MSLN+ cells that have not lost HLA-A*03 through loss of heterozygosity, the first receptor and the second receptor together do not specifically activate the immune cell.

37. The immune cell of any one of claims 1-36, wherein the immune cell is a T cell.

38. The immune cell according to claim 37, wherein the T cell is a CD8+CD4-T cell or a CD8-CD4+T cell.

39. The immune cell of any one of claims 1-38, wherein the expression and / or function of MHC class I genes has been reduced or eliminated.

40. The immune cell of claim 39, wherein the MHC class I gene is beta-2-microglobulin (B2M).

41. The immune cell according to claim 40, further comprising a polynucleotide comprising an interfering RNA, wherein the interfering RNA comprises a sequence complementary to a sequence of B2M mRNA.

42. An immune cell according to claim 41, wherein the interfering RNA comprises a sequence selected from the sequences shown in Table 13, or a sequence having at most 1, 2, 3 or 4 substitutions, insertions or deletions relative thereto.

43. The immune cell according to claim 41 or 42, wherein the interfering RNA is capable of inducing RNAi-mediated degradation of the B2M mRNA.

44. The immune cell of claim 43, wherein the interfering RNA is a short hairpin RNA (shRNA).

45. The immune cell of claim 44, wherein the shRNA comprises: a. a first sequence having a sequence complementary to the B2M mRNA sequence from the 5' end to the 3' end; and b. a second sequence having a sequence complementary to the first sequence from the 5' end to the 3' end, The first sequence and the second sequence form shRNA.

46. ​​The immune cell of claim 44 or 45, wherein the shRNA is encoded by a sequence comprising GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGC TTTGAGTGC (SEQ ID NO: 349) or GTTAACTTCCAATTTACATACCGAA GTATGTAAATTGGAAGTTAAC (SEQ ID NO: 350), or a sequence having at least 80%, at least 90% or at least 95% identity thereto.

47. The immune cell of claim 40, comprising one or more modifications to a sequence encoding B2M, wherein the one or more modifications reduce expression of B2M and / or eliminate function of B2M.

48. The immune cell of claim 47, wherein the one or more modifications comprise one or more inactivating mutations of an endogenous gene encoding B2M.

49. The immune cell of claim 48, wherein the one or more inactivating mutations comprise a deletion, insertion, substitution, or frameshift mutation.

50. An immune cell according to any one of claims 48 or 49, wherein the one or more inactivating mutations are introduced by a nucleic acid-guided endonuclease complexed with at least one guide nucleic acid (gNA) that specifically targets a sequence of an endogenous gene encoding B2M.

51. The immune cell of claim 50, wherein the gNA comprises a sequence selected from the sequences shown in Table 12, or a sequence having at most 1, 2, 3 or 4 substitutions, insertions or deletions therefrom.

52. The immune cell of claim 42, wherein the MHC class I gene is HLA-A*03.

53. The immune cell according to claim 52, further comprising a polynucleotide containing an interfering RNA, wherein the interfering RNA comprises a sequence complementary to the sequence of HLA-A*03 mRNA.

54. An immune cell according to claim 53, wherein the interfering RNA is capable of inducing RNA interference (RNAi)-mediated degradation of the HLA-A*03 mRNA.

55. The immune cell of claim 54, wherein the interfering RNA is a short hairpin RNA (shRNA) comprising: a. a first sequence having a sequence complementary to the HLA-A*03 mRNA sequence from the 5' end to the 3' end; and b. a second sequence having a sequence complementary to the first sequence from the 5' end to the 3' end, The first sequence and the second sequence form shRNA.

56. The immune cell of claim 52, comprising one or more modifications to the sequence of an endogenous gene encoding HLA-A*03, wherein the one or more modifications reduce the expression of HLA-A*03 and / or eliminate the function of HLA-A*03.

57. An immune cell according to claim 56, wherein the one or more modifications comprise one or more inactivating mutations of an endogenous gene encoding HLA-A*03.

58. An immune cell according to claim 56 or 57, wherein the one or more inactivating mutations are introduced by a nucleic acid-guided endonuclease complexed with at least one guide nucleic acid (gNA) that specifically targets a sequence of an endogenous gene encoding HLA-A*03.

59. An immune cell according to any one of claims 1-58, wherein the first receptor comprises the sequence of SEQ ID NO: 164, or a sequence that is at least 90%, at least 95%, at least 97% or at least 99% identical thereto, and the second receptor comprises the sequence of SEQ ID NO: 1259, or a sequence that is at least 90%, at least 95%, at least 97% or at least 99% identical thereto.

60. An immune cell according to claim 59, comprising a shRNA, wherein the shRNA is encoded by a sequence comprising: GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAA GCTTTGAGTGC (SEQ ID NO: 349) or GTTAACTTCCAATTTACATACCG AAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 350), or a sequence having at least 80%, at least 90% or at least 95% identity thereto.

61. The immune cell of claim 59 or 60, wherein the first receptor and the second receptor are encoded by a single polynucleotide, and wherein the sequences encoding the first receptor and the second receptor are separated by a sequence encoding a self-cleaving polypeptide.

62. The immune cell of claim 61, wherein the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351).

63. The immune cell of any one of claims 1-62, wherein the immune cell is autologous.

64. The immune cell of any one of claims 1-62, wherein the immune cell is allogeneic.

65. A pharmaceutical composition comprising a therapeutically effective amount of the immune cells according to any one of claims 1-64.

66. The pharmaceutical composition of claim 65, further comprising a pharmaceutically acceptable carrier, diluent or excipient.

67. A pharmaceutical composition according to claim 65 or 66 for use as a medicament for the treatment of MSLN+ cancer.

68. A polynucleotide or polynucleotide system comprising one or more polynucleotides comprising a polynucleotide sequence encoding a receptor comprising: a. a first receptor comprising an extracellular ligand binding domain specific for mesothelin (MSLN); and b. a second receptor comprising an extracellular ligand binding domain specific for HLA-A*03, wherein the first receptor is an activator receptor responsive to MSLN; and wherein the second receptor is an inhibitory receptor responsive to HLA-A*03.

69. A polynucleotide or polynucleotide system comprising one or more polynucleotides for producing an immune cell according to any one of claims 1-64, wherein the polynucleotide comprises a polynucleotide sequence encoding the first receptor and the second receptor.

70. The polynucleotide or polynucleotide system of claim 68 or 69, comprising a sequence encoding a shRNA specific for B2M.

71. The polynucleotide or polynucleotide system of claim 70, wherein the sequences encoding the first receptor, the second receptor and the shRNA specific for B2M are encoded by the same polynucleotide.

72. The polynucleotide or polynucleotide system of claim 70 or 71, wherein a. a sequence encoding a shRNA specific to B2M comprises GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 349) or GTT AACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 350), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto; b. the sequence encoding the first receptor comprises a sequence encoding a polypeptide comprising SEQ ID NO: 303, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto; and c. The sequence encoding the second receptor comprises a sequence encoding a polypeptide comprising the sequence of SEQ ID NO: 1268, or a sequence having at least 80%, at least 90% or at least 95% identity thereto.

73. A vector comprising one or more polynucleotides according to any one of claims 68-72.

74. A method for killing MSLN+ cancer cells having loss of heterozygosity at the HLA-A*03 locus, comprising administering to a subject an effective amount of an immune cell according to any one of claims 1-64 or a pharmaceutical composition according to any one of claims 65-67.

75. A method for treating MSLN+ cancer in a subject having an MSLN+ tumor with loss of heterozygosity at the HLA-A*03 locus, comprising administering to the subject an effective amount of an immune cell according to any one of claims 1-64 or a pharmaceutical composition according to any one of claims 65-67.

76. A method of treating cancer in a subject, comprising: a. determining the HLA-A genotype or expression of normal cells and multiple cancer cells of the subject; b. Optionally, determining MSLN expression in a plurality of cancer cells of the subject; and c. If the normal cells express HLA-A*03 and the plurality of cancer cells do not express HLA-A*03, and the plurality of cancer cells are MSLN positive, administering to the subject an effective amount of the immune cell according to any one of claims 1-64 or the pharmaceutical composition according to any one of claims 65-67.

77. The method of claim 75, wherein the subject is a heterozygous HLA-A*03 patient having a malignancy that expresses MSLN (MSLN+) and has lost HLA-A*03 expression.

78. The method of claim 75, wherein the subject is a heterozygous HLA-A*03 patient with a recurrent unresectable solid tumor or a metastatic solid tumor that expresses MSLN and has lost HLA-A*03 expression.

79. The method of any one of claims 76-78, wherein the cancer comprises mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or bile duct cancer.

80. The method of claim 79, wherein the subject's cancer has relapsed, the cancer is refractory to one or more previously administered anti-cancer therapies, and / or the cancer is metastatic.

81. The method of any one of claims 76-80, wherein the cancer cells comprise MSLN+ / HLA-A*03- cancer cells that do not express HLA-A*03.

82. The method of claim 81, wherein the MSLN+ / HLA-A*03- cancer cells are derived from MSLN+ / HLA-A*03+ cells that have lost HLA-A*03 through loss of heterozygosity at HLA-A.

83. The method of any one of claims 76-82, wherein the first receptor and the second receptor together specifically activate the immune cell in the presence of MSLN+ / HLA-A*03- cancer cells.

84. The method of any one of claims 76-83, wherein the first receptor and the second receptor together do not specifically activate the immune cell in the presence of MSLN+ cells that have not lost HLA-A*03.

85. The method of any one of claims 76-84, wherein administration of the immune cell of any one of claims 1-58 or the pharmaceutical composition of any one of claims 59-61 reduces tumor size in the subject.

86. The method of claim 85, wherein the tumor is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

87. The method of claim 85, wherein the tumor is eliminated.

88. The method of claim 85 or 86, wherein administration of the immune cell or the pharmaceutical composition prevents growth of a tumor in the subject.

89. The method of any one of claims 75-88, wherein administration of the immune cell or the pharmaceutical composition reduces the number of tumors in the subject.

90. The method of any one of claims 75-88, wherein administration of the immune cell or the pharmaceutical composition results in selective killing of cancer cells but not normal cells of the subject.

91. The method of claim 88, wherein at least about 60% of the killed cells are cancer cells, about 65% of the killed cells are cancer cells, about 70% of the killed cells are cancer cells, about 75% of the killed cells are cancer cells, about 80% of the killed cells are cancer cells, about 85% of the killed cells are cancer cells, about 90% of the killed cells are cancer cells, about 95% of the killed cells are cancer cells, or about 100% of the killed cells are cancer cells.

92. The method of claim 90, wherein administration of the immune cells or the pharmaceutical composition results in killing of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or all of the subject's cancer cells.

93. The method of any one of claims 75-91, wherein administration of the immune cell or the pharmaceutical composition produces fewer side effects to the subject than administration of an otherwise equivalent immune cell comprising the first activator receptor but not the second inhibitory receptor.

94. A method for preparing a plurality of immune cells, comprising: a. Provide multiple immune cells, and b. Transforming the plurality of immune cells using the polynucleotide system according to any one of claims 68-72 or the vector according to claim 73.

95. A kit comprising the immune cell according to any one of claims 1-64 or the pharmaceutical composition according to any one of claims 65-67.

96. The kit of claim 95, further comprising instructions for use.

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