Compositions and methods for treating mesothelin positive cancers

Abstract

To provide compositions and methods for treating mesothelin positive cancers.SOLUTION: The disclosure provides immune cells comprising a first activator receptor specific to mesothelin and a second inhibitory receptor specific to a ligand that has been lost in a mesothelin-positive cancer cell, and methods of making and using same for the treatment of cancer. Provided herein are compositions and methods related to treatment of MSLN(+) cancers. Advantageously, the compositions and methods disclosed herein may exploit loss of heterozygosity (LOH) to address MSLN(+) cancer.SELECTED DRAWING: Figure 15B

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 085,971, filed September 30, 2020, and U.S. Provisional Patent Application No. 63 / 068,245, filed August 20, 2020, each of which is incorporated by reference in its entirety.

[0002] The present disclosure relates to the fields of adoptive cell therapy and cancer treatment.

[0003] Incorporation by reference of sequence listing The application for the Sequence Listing Item contains a Sequence Listing that has been submitted in ASCII format via EFS-WEB and is incorporated by reference in its entirety. The ASCII copy, created on August 17, 2021, is named A2BI_019_03WO_SeqList_ST25.txt and is 1.08 MB in size. [Background technology]

[0004] Cell therapy is a powerful tool for the treatment of various diseases, especially cancer. In conventional adoptive cell therapy, immune cells are engineered to express specific receptors, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which induce the activation of immune cells against cellular targets through receptor interaction with ligands expressed by target cells. Identification of suitable target molecules remains difficult, since many targets are expressed in normal tissues. This expression may result in toxicity when transplanted cells target normal tissues that express the target molecule. Therefore, there is a need in the art for compositions and methods useful for the treatment of 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 are still no viable therapies that utilize MSLN. It is expressed in most mesotheliomas, as well as in many subsets 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, and this tissue itself may not be essential. Several investigational therapeutics directed against MSLN are being tested, including immunotoxin conjugates, antibody-drug conjugates, bispecific antibodies, CAR-T, and hybrid TCR-scFv. All effective therapeutic agents administered systemically have been toxic. Thus, there is a need in the art for compositions and methods relating to the treatment of MSLN(+) cancers. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Chang et al. Cancer Res 52:181-86 [Non-Patent Document 2] Hassan et al. J Clin Oncol 34:4171-79 Summary of the Invention [Means for solving the problem]

[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 address MSLN(+) cancer. The compositions and methods disclosed herein can, in some cases, avoid systemic toxicity to normal tissues by pairing MSLN-targeting activator receptors with blocker receptors. Without being bound by theory, the difference in blocker antigen expression in tumor tissues versus 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 a non-target antigen lost in MSLN+ cancer cells, wherein the first receptor is an activator receptor that responds to MSLN and the second receptor is an inhibitory receptor that responds to the non-target antigen.

[0009] In some embodiments of the immune cells of the present disclosure, the non-target antigen is lost in the MSLN+ cancer cells by loss of heterozygosity.

[0010] In some embodiments of the immune cells of the present disclosure, the extracellular ligand binding domain of the second receptor specifically binds to an allelic variant of a major histocompatibility complex (MHC) protein. 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. 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 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or CDR sequences with up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs of Table 6 or Table 7. In some embodiments, the extracellular ligand binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 42-47 or SEQ ID NOs: 48-53, or CDR sequences with up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs of SEQ ID NOs: 42-47 or SEQ ID NOs: 48-53. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises a polypeptide sequence selected from a polypeptide sequence 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: 30-41, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[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 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 up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs in Table 2. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising a sequence shown in Table 3, and a light chain variable (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 heavy chain variable (VH) portion comprising 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 a light chain variable (VL) portion comprising SEQ ID NO: 279, 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 first receptor comprises a sequence selected from the group consisting of 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. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an 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.

[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 having 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 having 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, or 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, or 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, or 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 thereof, 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 that is at least 90%, at least 95%, at least 97%, at least 99% identical to or identical to SEQ ID NO: 71. In some embodiments, the second receptor comprises 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.

[0014] In some embodiments of the immune cells of the present disclosure, the MSLN+ cancer cell is a mesothelioma cancer cell, an ovarian cancer cell, a cervical cancer cell, a colorectal cancer cell, an esophageal cancer cell, a head and neck cancer cell, a renal cancer cell, a uterine cancer cell, a gastric cancer cell, a pancreatic cancer cell, a lung cancer cell, a colorectal cancer cell, or a cholangiocarcinoma cell, or any cancer cell that expresses MSLN. In some embodiments, the MSLN+ cancer cell is a mesothelioma cancer cell, an ovarian cancer cell, a cervical cell, a uterine cancer cell, a gastric cancer cell, a pancreatic cancer cell, or a lung adenocarcinoma cell.

[0015] In some embodiments, the MSLN+ cancer cells are epithelial cancer cells. Epithelial cancers are cancers that originate from epithelial cells. 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 cells are MSLN+ / HLA-A*02- cancer cells that do not express HLA-A*02. In some embodiments, the MSLN+ / HLA-A*02- cancer cells are derived from MSLN+ / HLA-A*02+ cells by loss of heterozygosity at HLA-A, resulting in loss of HLA-A*02. In some embodiments, the first receptor and the second receptor together specifically activate the immune cells in the presence of MSLN+ / HLA-A*02- cancer cells that have loss of heterozygosity. In some embodiments, the first receptor and the second receptor together do not specifically activate the immune cells in the presence of MSLN+ cells that have not lost HLA-A*02 by loss of heterozygosity.

[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.

[0018] In some embodiments of the immune cells of the present disclosure, the expression and / or function of an MHC class I gene is reduced or eliminated. In some embodiments, the MHC class I gene is beta-2-microglobulin (B2M). In some embodiments, the immune cells further comprise an interfering RNA, the interfering RNA comprises a sequence complementary to a sequence of B2M mRNA. In some embodiments, the interfering RNA comprises a sequence selected from the group of sequences shown in Table 13, or a sequence having up to 1, 2, 3, or 4 substitutions, insertions, or deletions thereto. In some embodiments, the interfering RNA can induce RNAi-mediated degradation of B2M mRNA. In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises (a) a first sequence having a sequence complementary to a sequence of B2M mRNA from its 5'-end to its 3'-end, and (b) a second sequence having a sequence complementary to the first sequence from its 5'-end to its 3'-end, the first sequence and the second sequence forming an shRNA. In some embodiments, the shRNA is encoded by a sequence comprising the 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.

[0019] In some embodiments of the immune cells of the present disclosure, the expression and / or function of the MHC class I gene is reduced or eliminated. In some embodiments, the MHC class I gene is beta-2-microglobulin (B2M). In some embodiments, the immune cells comprise one or more modifications to a sequence encoding B2M, the one or more modifications reducing expression and / or eliminating function of B2M. In some embodiments, the one or more modifications comprise one or more inactivating mutations of an endogenous gene encoding B2M. In some embodiments, the one or more inactivating mutations comprise a deletion, an insertion, a substitution, or a frameshift mutation. In some embodiments, the one or more inactivating mutations are introduced with a nucleic acid-guided endonuclease in a complex with at least one guide nucleic acid (gNA) that specifically targets a sequence of the endogenous gene encoding B2M. In some embodiments, the gNA comprises a sequence selected from the group of sequence sets shown in Table 12, or a sequence having up to 1, 2, 3, or 4 substitutions, insertions, or deletions thereto.

[0020] In some embodiments of the immune cells of the present disclosure, the expression and / or function of an MHC class I gene is reduced or eliminated. In some embodiments, the MHC class I gene is HLA-A*02. In some embodiments, the immune cells comprise a polynucleotide comprising an interfering RNA comprising a sequence complementary to a sequence of HLA-A*02 mRNA. In some embodiments, the interfering RNA can induce RNA interference (RNAi)-mediated degradation of HLA-A*02 mRNA. In some embodiments, the interfering RNA is a short hairpin RNA (shRNA) comprising: (a) a first sequence having a sequence complementary to a sequence of HLA-A*02 mRNA from its 5' to 3' end; and (b) a second sequence having a sequence complementary to the first sequence from its 5' to 3' end, the first sequence and the second sequence forming an shRNA. In some embodiments, the shRNA comprises a sequence as shown in 14. In some embodiments, the immune cells comprise one or more modifications to the sequence of the endogenous gene encoding HLA-A*02, the one or modifications reducing expression and / or eliminating function of HLA-A*02. In some embodiments, the one or more modifications comprise one or more inactivating mutations of the endogenous gene encoding HLA-A*02. In some embodiments, the one or more inactivating mutations are introduced with a nucleic acid-guided endonuclease in complex with at least one guide nucleic acid (gNA) that specifically targets the sequence of the endogenous gene encoding HLA-A*02. 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, and the second receptor comprises a sequence of SEQ ID NO: 52, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the immune cells comprise an shRNA encoded by a sequence comprising 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 the sequences encoding the first and second receptors are separated by a sequence encoding an autocleaving polypeptide. In some embodiments, the autocleaving polypeptide comprises a T2A autocleaving polypeptide comprising a sequence of 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 pharma- ceutically 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 in the treatment of MSLN+ cancer.

[0026] The present disclosure provides a polynucleotide or polynucleotide system comprising one or more polynucleotides comprising a polynucleotide sequence encoding (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 a non-target antigen that is lost in MSLN+ cancer cells, wherein the first receptor is an activator receptor that responds to MSLN on MSLN+ cancer cells and the second receptor is an inhibitory receptor that responds to the non-target antigen.

[0027] In some embodiments of the polynucleotide or polynucleotide system of the present disclosure, the polynucleotide or polynucleotide system comprises one or more polynucleotides comprising polynucleotide sequences encoding a first receptor and a second receptor for use in generating the immune cells of the present disclosure. In some embodiments, the polynucleotide or polynucleotide system comprises a sequence encoding an shRNA specific for B2M. In some embodiments, the sequences encoding the first receptor, the second receptor, and the shRNA specific for B2M are encoded by the same polynucleotide. In some embodiments, (a) the sequence encoding the B2M-specific shRNA comprises 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; (b) the sequence encoding the first receptor comprises a sequence encoding a polypeptide of 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 of SEQ ID NO: 348, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

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

[0029] The present disclosure provides a method of killing MSLN+ cancer cells having loss of heterozygosity at the MHC class I locus, comprising administering to a subject an effective amount of an immune cell or pharmaceutical composition of the disclosure.

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

[0031] The present disclosure provides a method of treating cancer in a subject, the method comprising: (a) determining the HLA-A genotype or expression of normal cells and a plurality of cancer cells of the subject; (b) optionally determining expression of MSLN in the plurality of cancer cells of the subject; and (c) if the normal cells express HLA-A*02, the plurality of cancer cells do not express HLA-A*02, and the plurality of cancer cells are MSLN positive, administering to the subject an effective amount of an immune cell or pharmaceutical composition of the present disclosure. In some embodiments of the methods of the present disclosure, the subject is a heterozygous HLA-A*02 patient with a malignant tumor that expresses MSLN (MSLN+) and has lost HLA-A*02 expression. In some embodiments, the subject is a heterozygous HLA-A*02 patient with a recurrent unresectable or metastatic solid tumor that expresses MSLN and has lost HLA-A*02 expression. In some embodiments, the cancer comprises mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, renal 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 cancer has recurred in the subject. In some embodiments, the cancer is refractory to one or more previously administered anti-cancer therapies. In some embodiments, the cancer is metastatic.

[0032] In some embodiments of the methods of the present disclosure, the cancer cells include MSLN+ / HLA-A*02 cancer cells that do not express HLA-A*02. In some embodiments, the MSLN+ / HLA-A*02- cancer cells are derived from MSLN+ / HLA-A*02+ cells by loss of heterozygosity at HLA-A, resulting in loss of HLA-A*02. In some embodiments, the first receptor and the second receptor together specifically activate immune cells in the presence of MSLN+ / HLA-A*02- cancer cells. In some embodiments, the first receptor and the second receptor together do not specifically activate immune cells in the presence of MSLN+ cells that have not lost HLA-A*02.

[0033] In some embodiments of the disclosed methods, administration of the immune cells or pharmaceutical composition reduces the size of a tumor in a subject. 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.

[0034] In some embodiments of the disclosed methods, administration of the immune cells or pharmaceutical composition inhibits tumor growth in the subject.

[0035] In some embodiments of the disclosed methods, administration of the immune cells or pharmaceutical composition reduces the number of tumors in the subject.

[0036] In some embodiments of the disclosed methods, administration of the immune cells or pharmaceutical composition results in selective killing of cancer cells but not normal cells in the subject. In some embodiments, at least about 60% of the dead cells are cancer cells, about 65% of the dead cells are cancer cells, about 70% of the dead cells are cancer cells, about 75% of the dead cells are cancer cells, about 80% of the dead cells are cancer cells, about 85% of the dead cells are cancer cells, about 90% of the dead cells are cancer cells, about 95% of the dead cells are cancer cells, or about 100% of the dead cells are cancer cells. In some embodiments, administration of the immune cells or pharmaceutical composition results in killing of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or all of the cancer cells in the subject.

[0037] In some embodiments of the methods of the present disclosure, administration of the immune cells or pharmaceutical compositions results in fewer side effects in a subject than administration of an otherwise equivalent immune cell that comprises the first activator receptor but does not comprise the second inhibitory receptor.

[0038] The present disclosure provides a method of generating a plurality of immune cells, the method comprising: (a) providing a plurality of immune cells; and (b) transforming the plurality of immune cells with a polynucleotide, polynucleotide system, or vector of the disclosure.

[0039] 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]

[0040] [Figure 1] 1 is a table showing expression of candidate blocking factor genes from the TCGA database. (*) indicates genes previously identified as being expressed in the mesothelium. [Diagram 2] 1 is a plot showing mesothelin (MSLN) expression in normal tissues. [Diagram 3] 4 is a plot showing expression of MSLN across TCGA cancers (with tumor and normal samples). Abbreviations: BLCA (bladder cancer), BRCA (breast cancer), CESC (cervical squamous cell carcinoma and adenocarcinoma), CHOL cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal cancer), GBM (glioblastoma multiforme), HNSC (head and neck squamous cell carcinoma), KICH (kidney chromophobe), KIRP (kidney papillary renal cell carcinoma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PAAD (pancreatic adenocarcinoma), PRAD (prostate adenocarcinoma), PCPG (pheochromocytoma and paraganglioma), READ (rectal adenocarcinoma), SARC (sarcoma), SKCM (cutaneous melanoma), THCA (thyroid cancer), THYM (thymoma), STAD (gastric adenocarcinoma), UCEC (uterine endometrial carcinoma). [Figure 4] 1 is a plot showing MSLN expression in CCLE cell lines. [Diagram 5] 1 is a plot showing LRRN4 expression in normal tissues. [Figure 6] A plot showing expression of LRRN4 across TCGA cancers (with tumor and normal samples). [Figure 7]1 is a plot showing expression of LRRN4 across TCGA tumors. [Figure 8] 1 is a plot showing expression of LRRN4 in CCLE cell lines. [Figure 9] FIG. 1 is a plot showing the distribution of the 10 leucine-rich repeats (LRRs) and fibronectin type III domains relative to the transmembrane domain of LRRN4, most of which are likely located on the cell surface. [Figure 10A] We show that pMHC HLA-A*02 scFv LIR-1-based inhibitory receptors can inhibit Jurkat cell activation in cis in a cell-free bead-based assay. [Figure 10B] pMHC HLA-A*02 scFv LIR-1-based inhibitory receptor can inhibit activation of Jurkat cells by MSLN scFv CAR using leukemia cell line K562 as target cells. [Figure 11] Figure (left) and graph (right) showing that pMHC HLA-A*02 scFv LIR-1 based inhibitory receptor can inhibit Jurkat cell activation as measured by fold induction of IFNγ by MSLN scFv CAR using pMHC HLA-A*02 scFv LIR-1 based inhibitory receptor and HLA-A*02+ HeLa and SiHa cells as target cells. [Figure 12] We show that pMHC HLA-A*02 scFv LIR-1-based inhibitory receptor inhibits MSLN CAR activator-induced killing using HLA-A*02+SiHa cells but not HLA-A*02-SiHa cells. [Figure 13] FIG. 1 is a diagram of the bioinformatics pipeline used to identify potential inhibitory receptor targets lost in cancer cells due to loss of heterozygosity. [Figure 14] This is one bioinformatics pipeline that was used to identify potential inhibitory receptor targets that are not expressed in cancer cells. [Figure 15A] 1 is a pair of plots showing that HLA-A*02 blocking agent inhibits MSLN CAR activator directed to the high density antigen MSLN. Jurkat cells transfected with MSLN LBD1-CAR or MSLN LBD1-CAR and A2-LIR-1 co-cultured with K562 cells expressing either MSLN or MSLN and HLA-A*02 show blocking of activation by high density antigen with A2-LIR-1 blocking agent only in the presence of HLA-A*02. [Figure 15B] 1 is a pair of plots showing that HLA-A*02 blocking agent inhibits MSLN CAR activator directed to MSLN, a high-density antigen. Killing of endogenous MSLN+HeLa cells by MSLN LBD1-CAR T cells is blocked in the presence of HLA-A*02 with A2-LIR-1 blocking agent. [Figure 15C] 1 is a pair of plots showing that HLA-A*02 blocker inhibits MSLN CAR activator directed to MSLN, a high-density antigen. Killing of endogenous MSLN+HeLa cells by MSLN LBD2-CAR T cells is shown. The effect of A2-LIR-1 blocker on T cell killing is controlled in part by the activator LBD, suggesting that further optimization of the blocker module or activator / blocker pair may be required. [Figure 16]

[0023] Figure 1 is a series of plots showing that the HLA-A*02 LIR1 inhibitory receptor (PA2.1, murine and humanized) effectively blocks killing by T cells expressing MSLN 3rd generation CARs in the presence of MSLN and HeLa cells expressing HLA-A*02. Top row: MSLN+ HLA-A*02+ HeLa target cells, bottom row: MSLN+ HLA-A*02- HeLa cells (control). The murine SS1 3rd generation CAR (top right, boxed) provides a better window than the humanized M5 and humanized SS1 CARs. [Figure 17]

[0023] Figure 1 is a series of plots showing that HLA-A*02 LIR1 inhibitory receptor (PA2.1, murine and humanized) effectively blocks killing by T cells expressing MSLN 3rd generation CAR in the presence of MSLN+HLA-A*02+Capan-2 cells. [Figure 18] A pair of plots showing that killing of MSLN+HLA-A*02+ HeLa cells (left) or naturally MSLN+HLA-A*02+ HCT116 wild type (WT) cells by T cells expressing a second generation CAR with the murine SS1 scFv is effectively blocked by the HLA-A*02 scFv LIR1 inhibitory receptor. [Figure 19] A series of fluorescence-activated cell sorting (FACS) plots showing expression of murine SS1 second generation CAR by T cells with and without co-transduction of HLA-A*02 scFv LIR-1 blocking agent. [Figure 20A] 20B is a plot showing the effect of the LIR-1 hinge on the ability of the HLA-A*02 inhibitory receptor to block activation of Jurkat cells by the KRAS TCR. H: hinge, T: transmembrane domain, ICD: intracellular domain, s: short. The LIR-1 constructs are described in more detail in FIG. 20B. Humanized PA2.1 and humanized BB7.2 with the shorter LIR-1 hinge block, as well as the original longer hinge. [Figure 20B] 1 is a plot and table showing EC50 shifts (+ / -HLA-A*02 target cells) for Jurkat cells expressing KRAS TCR activators and the HLA-A*02 scFv LIR-1 inhibitory receptor shown in the table below (SEQ ID NOs: 352-356). [Figure 21A] 21B is a plot showing the effect of LIR-1 hinge on the ability of HLA-A*02 inhibitory receptor to block activation of Jurkat cells by KRAS TCR. H: hinge, TM: transmembrane domain, ICD: intracellular domain, s: short, tr: truncated. LIR-1 constructs are described in more detail in FIG. 21B. Mouse PA2.1, which has a slightly longer hinge, functions similarly to the original LIR-1 hinge in the T2-Jurkat assay. [Figure 21B] A plot and pair of tables showing EC50 shifts (+ / -HLA-A*02 target cells) for Jurkat cells expressing KRAS TCR activators and the HLA-A*02 scFv LIR-1 inhibitory receptors shown in the table below (sequence numbers 357-361) with lengths shown in the table on the left. [Figure 22A] Figure 22A shows a Tmod approach to achieve selective cytotoxicity at two targets (Tmod refers to immune cells expressing a combination of activating and inhibitory receptors). Figure 22A shows that the lungs (and other vital organs) are surrounded by MSLN(+) mesothelial lining, creating a high risk of on-target, off-tumor toxicity for MSLN-targeted drugs. By selecting patients heterozygous for HLA-A*02 whose tumors have lost this allele via LOH, there is an opportunity to target MSLN-activated CAR-T cells to specifically kill tumor cells and spare normal mesothelium. Figure 22B shows the molecular composition of the MSLN-targeted Tmod construct (Tmod refers to paired activating and inhibitory receptors). The two receptors are co-expressed in a single construct, and the encoded fusion protein is cleaved intracellularly to generate the activator and blocker. [Figure 22B] Figure 22A shows a Tmod approach to achieve selective cytotoxicity at two targets (Tmod refers to immune cells expressing a combination of activating and inhibitory receptors). Figure 22A shows that the lungs (and other vital organs) are surrounded by MSLN(+) mesothelial lining, creating a high risk of on-target, off-tumor toxicity for MSLN-targeted drugs. By selecting patients heterozygous for HLA-A*02 whose tumors have lost this allele via LOH, there is an opportunity to target MSLN-activated CAR-T cells to specifically kill tumor cells and spare normal mesothelium. Figure 22B shows the molecular composition of the MSLN-targeted Tmod construct (Tmod refers to paired activating and inhibitory receptors). The two receptors are co-expressed in a single construct, and the encoded fusion protein is cleaved intracellularly to generate the activator and blocker. [Figure 23A] Isolation and characterization of selective MSLN binders are shown. The on-target probe was labeled soluble MSLN (Acro Bio) and the off-target probe used for counter-selection was a mixture of soluble CEA and EGFR proteins. Figure 23A shows the enrichment of the IgG library. Figure 23B shows the enrichment of the scFv library. Figure 23C shows the surface expression of MSLN CAR (Gen3) in Jurkat cells. Cells were transfected with CAR constructs and stained with protein L or monomeric soluble MSLN (see Methods). Benchmark and CAR1-6 expression histograms are indicated. PE, phycoerythrin; NA, neutravidin; SA, streptavidin. "On-target NGS" corresponds to cell populations collected and subjected to DNA sequencing to determine the enrichment of individual idiotypes. Figure 23D shows the characterization of MSLN binders in a solid-state Jurkat cell assay using surface-attached MSLN protein (see Hamburger et al., 2020). Sixty-two CAR constructs (Gen3) with different scFvs were transiently transfected into Jurkat cells to express CAR, and functional responses to surface-bound recombinant human sMSLN (Acro Bio) were evaluated 6 hours later. Most produced some degree of response. [Figure 23B]Isolation and characterization of selective MSLN binders are shown. The on-target probe was labeled soluble MSLN (Acro Bio) and the off-target probe used for counter-selection was a mixture of soluble CEA and EGFR proteins. Figure 23A shows the enrichment of the IgG library. Figure 23B shows the enrichment of the scFv library. Figure 23C shows the surface expression of MSLN CAR (Gen3) in Jurkat cells. Cells were transfected with CAR constructs and stained with protein L or monomeric soluble MSLN (see Methods). Benchmark and CAR1-6 expression histograms are indicated. PE, phycoerythrin; NA, neutravidin; SA, streptavidin. "On-target NGS" corresponds to cell populations collected and subjected to DNA sequencing to determine the enrichment of individual idiotypes. Figure 23D shows the characterization of MSLN binders in a solid-state Jurkat cell assay using surface-attached MSLN protein (see Hamburger et al., 2020). Sixty-two CAR constructs (Gen3) with different scFvs were transiently transfected into Jurkat cells to express CAR, and functional responses to surface-bound recombinant human sMSLN (Acro Bio) were evaluated 6 hours later. Most produced some degree of response. [Figure 23C]Isolation and characterization of selective MSLN binders are shown. The on-target probe was labeled soluble MSLN (Acro Bio) and the off-target probe used for counter-selection was a mixture of soluble CEA and EGFR proteins. Figure 23A shows the enrichment of the IgG library. Figure 23B shows the enrichment of the scFv library. Figure 23C shows the surface expression of MSLN CAR (Gen3) in Jurkat cells. Cells were transfected with CAR constructs and stained with protein L or monomeric soluble MSLN (see Methods). Benchmark and CAR1-6 expression histograms are indicated. PE, phycoerythrin; NA, neutravidin; SA, streptavidin. "On-target NGS" corresponds to cell populations collected and subjected to DNA sequencing to determine the enrichment of individual idiotypes. Figure 23D shows the characterization of MSLN binders in a solid-state Jurkat cell assay using surface-attached MSLN protein (see Hamburger et al., 2020). Sixty-two CAR constructs (Gen3) with different scFvs were transiently transfected into Jurkat cells to express CAR, and functional responses to surface-bound recombinant human sMSLN (Acro Bio) were evaluated 6 hours later. Most produced some degree of response. [Figure 23D]Isolation and characterization of selective MSLN binders are shown. The on-target probe was labeled soluble MSLN (Acro Bio) and the off-target probe used for counter-selection was a mixture of soluble CEA and EGFR proteins. Figure 23A shows the enrichment of the IgG library. Figure 23B shows the enrichment of the scFv library. Figure 23C shows the surface expression of MSLN CAR (Gen3) in Jurkat cells. Cells were transfected with CAR constructs and stained with protein L or monomeric soluble MSLN (see Methods). Benchmark and CAR1-6 expression histograms are indicated. PE, phycoerythrin; NA, neutravidin; SA, streptavidin. "On-target NGS" corresponds to cell populations collected and subjected to DNA sequencing to determine the enrichment of individual idiotypes. Figure 23D shows the characterization of MSLN binders in a solid-state Jurkat cell assay using surface-attached MSLN protein (see Hamburger et al., 2020). Sixty-two CAR constructs (Gen3) with different scFvs were transiently transfected into Jurkat cells to express CAR, and functional responses to surface-bound recombinant human sMSLN (Acro Bio) were evaluated 6 hours later. Most produced some degree of response. [Figure 24A]Sensitivity of MSLN CARs versus benchmark CARs M5, SS1 and m912 is shown. All constructs were Gen3 except for SS1 (Gen2). Dose response (RLU) in Jurkat cells was measured to assess sensitivity in a 6-h co-culture assay. (1) Titrated MSLN-encoding mRNA was used to transfect HEK293 cells, (2) flow cytometry-based surface expression was converted to MSLN molecules / cell using QIFIKIT (Quantitative Analysis Kit, Agilent), and (3) the molecule / cell sensitivity (EC50) of six novel and three benchmark CARs was calculated by fitting the dose-response curves. For those CARs with sensitivity below the detection limit of the assay, EC50 was reported as less than 3000 MSLN molecules / cell. Maximum signal (Emax) for each construct is also shown. Experiments were repeated 1-4 times. [Figure 24B] CAR3 selectivity is shown. Example of MSLN CAR3 selectivity benchmarked against M5 CAR. Activation of CAR was measured in Jurkat cell functional assay with MSLN(+) or MSLN(-) cell lines. For MSLN(+) cell lines, variant MSLN(-) versions were generated by MSLN knockout for comparison. For more detailed off-target characterization, see Figure 32B. [Figure 25A] Figure 1 shows expression of MSLN in human cell lines assessed by staining with MSLN mAb and flow cytometry. K562 showed some cross-reactivity to anti-MSLN antibodies, but no functional reactivity was observed to CAR3 or M5 benchmark CARs. [Figure 25B] Plotted levels of MSLN and A*02 mRNA (CCLE) and protein (QIFIKIT) are shown to indicate correlation. Conversion between protein and mRNA levels was calculated using a standard curve (see Methods for Example 8 below). [Figure 26A]An overview 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). QIFIKIT (Quantitative Analysis Kit, Agilent) was used to quantify surface MSLN and A*02 in engineered and wild-type tumor cell lines. If the cell line HLA-A haplotype was heterozygous for A*02, the TPM value was divided by 2. Note that in some cases, the HLA-A allele copy number was unknown. The TPM value of MS751+ transduced A*02 (438 TPM) was estimated from the measurement of its surface A*02 protein level using a standard curve. Cell lines transduced with HLA-A*02 better mimic the A*02:MSLN ratio of normal lung tissue than cell lines expressing endogenous levels of the protein (bold black box). TPM, transcripts per million; na, not applicable; A*02: HLA-A*02. [Figure 26B] Quantification of MSLN molecules / cells using QIFIKIT. 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 both the cells and the QIFI beads. The number of MSLN molecules on the surface was quantified using the QIFI antigen standard curve. [Figure 27A] Characterization of MSLN CAR Tmod constructs in Jurkat cell functional assay. 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 control (open circles). Jurkat NFAT luciferase cells expressing CAR+ / - blockers were co-cultured with wild-type endogenous MSLN(+) HeLa cells transfected with titrations of A*02:01 mRNA. Functional responses (RLU) were assessed after 6 hours of co-culture. Titrated antigen molecules on the surface were quantified using QIFIKIT. IC50 (molecules / cell) values ​​are shown in the figure. CAR1-6 are Gen3, CAR M5 and SS1 are Gen2. [Figure 27B] Figure 1 shows a two-dimensional titration of MSLN and A*02 mRNA in MSLN(-) HeLa target cells to establish the EC50 for MSLN CAR3 Tmod constructs in Jurkat cells. MSLN(-) HeLa target cells were transfected with serial dilutions of MSLN mRNA and steady A*02 mRNA and Jurkat cells were transiently transfected to express MSLN CAR3 and A*02 blockers. Functional responses (RLU) were assessed after 6 hours of co-culture. [Figure 27C] Shown is transfection of MSLN(-) HeLa target cells with serial dilutions of A*02 mRNA and steady-state MSLN mRNA to establish the IC50 for the MSLN CAR3 Tmod construct in Jurkat cells. [Figure 28A] Plotted levels of MSLN and A*02 mRNA and protein are shown. EC50 and IC50 of constructs relative to MSLN and HLA-A expression levels in normal (GTEx database) and tumor tissues and cell lines (TCGA, CCLE database). Conversion between protein and mRNA levels was calculated using standard curves shown in FIG. 25B; Methods). The HeLa and MS751 A*02 transgenic cell line variants shown in the graphs better mimic the activator and blocker target ratios in normal tissues. [Figure 28B] Shows MSLN CAR and CAR3 Tmod cytotoxicity in primary T cells. Primary T cells transduced with CAR or Tmod were co-cultured with either tumor or normal target cells at effector:target (E:T) = 1:1 as indicated for 48 hours. A*02:MSLN (B:A) target antigen ratios ranged from 2 to 27:1. M5 was Gen2 CAR, all others were Gen3. Tumor = MSLN(+)A*02(-) target cells; normal = MSLN(+)A*02(+) target cells. [Figure 29A]Comparison of lead CAR3 receptor paired with A*02 blocker against benchmark CAR in cytotoxicity assay. SS1 CAR is Gen2 construct, others are Gen3. Primary T cells transduced with various CAR+ / -A*02 blockers using two separate lentiviral vectors were cultured with endogenous MSLN(+)A*02(-) tumor 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 dilution with untransduced T cells for a final effective effector:target (E:T) ratio of 0.6:1 or 0.3:1. Both E:T ratios resulted in selective killing in the presence of A*02 antigen when T cells also expressed A*02 blocker. [Figure 29B] Secreted IFN-g after 48 hours of co-culture with tumor or normal target cells is shown. [Figure 30A] MSLN CAR3 Tmod cells selectively kill RFP(+) tumor cells and spare GFP(+) normal cells in mixed tumor and normal cell co-cultures. Due to the adhesive nature of the HeLa cell line, dead targets tend to remain as clusters on the surface. White arrows point to some examples of dead RFP(+) tumor cells. Examples of E:T 0.6:1 and normal:tumor = 1:1. [Figure 30B] Cytotoxicity in mixed normal and tumor co-cultures at E:T=1:1 and normal:tumor=9:1 (see FIG. 31B for other ratios). Primary T cells transduced with CAR3 or CAR3 Tmod constructs were co-cultured with HeLa target cells for 48 hours and imaged using GFP and RFP expressed in MSLN(+)A*02(+) normal and MSLN(+)A*02(-) tumor cell lines, respectively. [Figure 31A]We show that MSLN CAR3 Tmod cells selectively kill RFP(+) tumor cells in mixed tumor and normal cell co-cultures. Primary T cells transduced with CAR3 Tmod constructs were co-cultured with HeLa cells for 48 hours and imaged using GFP and RFP expressed in MSLN(+)A*02(+) normal and MSLN(+)A*02(-) tumor cell lines, respectively. [Figure 31B] Shows cytotoxicity of CAR3 and CAR3 Tmod in mixed normal and tumor co-cultures with normal:tumor ranging from 9:1 to 1:9; E:T=0.6:1. [Figure 32A] We show that MSLN CAR3 Tmod constructs mediate selective, durable and reversible cytotoxicity. Primary T cells transduced with CAR3 or CAR3 Tmod were co-cultured with either tumor or normal target cells for 48 h at E:T=1.2:1. T cells were then harvested, depleted of dead or non-adherent target cells and re-seeded on fresh tumor or normal target cells for an additional 48 h. RACA, repeated antigen challenge assay. R1, round 1; R2, round 2. [Figure 32B] MSLN CAR3 Tmod construct selectivity in Jurkat cell functional assays on subsets of MSLN(+) and MSLN(-) control target cells shows no off-target activity (see Methods for Example 8 below). Bar heights correspond to the average from technical replicates. [Figure 33A] Schematic diagram of RACA (repeated antigen challenge assay) and reversibility assay. Primary T cells transduced with CAR3 or CAR3 Tmod were co-cultured with either tumor or normal targets for 48 hours at E:T=1.2:1. T cells were then harvested, depleted of any dead or elevated target cells, and re-seeded on fresh tumor or normal targets for another 48 hours. [Figure 33B]We show that soluble circulating MSLN (sMSLN) does not affect CAR-T activity. Acute cytotoxicity of tumor or normal target cells by M5 benchmark CAR or CAR3 was not affected by the presence of 500 ng / mL sMSLN (Acro Bio). [Figure 33C] 1 shows staining of transiently transfected CAR(+) Jurkat cells with labeled sMSLN monomer or tetramer analyzed by flow cytometry, indicating that sMSLN is structurally intact and capable of binding to the receptor. [Figure 34A] Figure 34 shows Tmod constructs mediating selective killing of tumor cells in xenograft models. Figure 34A shows a schematic of the dual adjacent tumor and normal MS751 xenograft model. Figure 34B shows bioluminescence values ​​are on the right of the color scale in flux units of photons / sec / cm2 / sr. Day 0 = before T cell injection, days 8 and 15 = after T cell injection. Figure 34C shows graft size assessed by caliper measurement (see results for Example 8 below). [Figure 34B] Figure 34 shows Tmod constructs mediating selective killing of tumor cells in xenograft models. Figure 34A shows a schematic of the dual adjacent tumor and normal MS751 xenograft model. Figure 34B shows bioluminescence values ​​are on the right of the color scale in flux units of photons / sec / cm2 / sr. Day 0 = before T cell injection, days 8 and 15 = after T cell injection. Figure 34C shows graft size assessed by caliper measurement (see results for Example 8 below). [Figure 34C] Figure 34 shows Tmod constructs mediating selective killing of tumor cells in xenograft models. Figure 34A shows a schematic of the dual adjacent tumor and normal MS751 xenograft model. Figure 34B shows bioluminescence values ​​are on the right of the color scale in flux units of photons / sec / cm2 / sr. Day 0 = before T cell injection, days 8 and 15 = after T cell injection. Figure 34C shows graft size assessed by caliper measurement (see results for Example 8 below). [Figure 35A]MSLN CAR Tmods selectively kill tumors in xenograft models. Figure 35A shows primary T cells transduced with MSLN CAR or CAR3 Tmods were co-cultured with either HLA-A KO tumor or A*02-transgenic normal MS751 target cells in vitro at E:T=1.4:1 for 48 hours. M5 was Gen2 CAR, all others were Gen3. Tumor=MSLN(+)A*02(-) target cells; Normal=MSLN(+)A*02(+) target cells. Figure 35B shows individual mouse xenograft growth curves for the data shown in Figure 34C. Figure 35C shows BLI quantification of normal and tumor cells after T cell injection. [Figure 35B] MSLN CAR Tmods selectively kill tumors in xenograft models. Figure 35A shows primary T cells transduced with MSLN CAR or CAR3 Tmods were co-cultured with either HLA-A KO tumor or A*02-transgenic normal MS751 target cells in vitro at E:T=1.4:1 for 48 hours. M5 was Gen2 CAR, all others were Gen3. Tumor=MSLN(+)A*02(-) target cells; Normal=MSLN(+)A*02(+) target cells. Figure 35B shows individual mouse xenograft growth curves for the data shown in Figure 34C. Figure 35C shows BLI quantification of normal and tumor cells after T cell injection. [Figure 35C] MSLN CAR Tmods selectively kill tumors in xenograft models. Figure 35A shows primary T cells transduced with MSLN CAR or CAR3 Tmods were co-cultured with either HLA-A KO tumor or A*02-transgenic normal MS751 target cells in vitro at E:T=1.4:1 for 48 hours. M5 was Gen2 CAR, all others were Gen3. Tumor=MSLN(+)A*02(-) target cells; Normal=MSLN(+)A*02(+) target cells. Figure 35B shows individual mouse xenograft growth curves for the data shown in Figure 34C. Figure 35C shows BLI quantification of normal and tumor cells after T cell injection. [Diagram 36]We show that cis-binding of A*02 blocker in A*02(+) or (-) T cells abrogates function. Blocker binding in A*02(+) Jurkat cells and primary T cells by A*02 tetramer was significantly reduced due to cis-binding of autologous A*02. Reduced binding (due to reduced availability of blocker) correlated with reduced blocker activity. Cytotoxicity assays show E:T=0.5:1. [Figure 37A] We show that the MSLN Tmod system can be expanded to autologous T cells. Figure 37A shows that cis-binding of autologous A*02 in A*02(+) donors eliminates CRISPR-mediated binding to A*02 tetramer B2M knockout (KO) and restores availability of blocking factor, as shown by binding to A*02 tetramers, similar to the levels observed in A*02(-) donors. Figure 37B shows a cytotoxicity assay showing activator-only and MSLN SS1 CAR Tmod primary T cells cultured with tumor (black) or normal (white) target cells. The MSLN SS1 CAR Tmod construct kills MSLN(+)A*02(-) tumor HeLa target cells, but no longer blocks in the presence of autologous A*02 as a result of cis-binding. In the case of A*02(+) donors, blocking is achieved only via B2M CRISPR KO. E:T=1.2:1. FIG. 37C shows representative images at 48 hours. [Figure 37B]We show that the MSLN Tmod system can be expanded to autologous T cells. Figure 37A shows that cis-binding of autologous A*02 in A*02(+) donors eliminates CRISPR-mediated binding to A*02 tetramer B2M knockout (KO) and restores availability of blocking factor, as shown by binding to A*02 tetramers, similar to the levels observed in A*02(-) donors. Figure 37B shows a cytotoxicity assay showing activator-only and MSLN SS1 CAR Tmod primary T cells cultured with tumor (black) or normal (white) target cells. The MSLN SS1 CAR Tmod construct kills MSLN(+)A*02(-) tumor HeLa target cells, but no longer blocks in the presence of autologous A*02 as a result of cis-binding. In the case of A*02(+) donors, blocking is achieved only via B2M CRISPR KO. E:T=1.2:1. FIG. 37C shows representative images at 48 hours. [Figure 37C] We show that the MSLN Tmod system can be expanded to autologous T cells. Figure 37A shows that cis-binding of autologous A*02 in A*02(+) donors eliminates CRISPR-mediated binding to A*02 tetramer B2M knockout (KO) and restores availability of blocking factor, as shown by binding to A*02 tetramers, similar to the levels observed in A*02(-) donors. Figure 37B shows a cytotoxicity assay showing activator-only and MSLN SS1 CAR Tmod primary T cells cultured with tumor (black) or normal (white) target cells. The MSLN SS1 CAR Tmod construct kills MSLN(+)A*02(-) tumor HeLa target cells, but no longer blocks in the presence of autologous A*02 as a result of cis-binding. In the case of A*02(+) donors, blocking is achieved only via B2M CRISPR KO. E:T=1.2:1. FIG. 37C shows representative images at 48 hours. [Figure 38A] Figure 1 shows the enrichment of anti-HLA-A*11 binders through multiple rounds of cell sorting from an scFv library. The on-target probe was a labeled HLA-A*11 tetramer and the off-target protein was a mixture of irrelevant MHC tetramers. [Figure 38B] Jurkat cell activation in an mRNA titration assay. HeLa target cells were transfected with serially diluted HLA-A*11 mRNA and Jurkat cells were transiently transfected to express HLA-A*11 CAR4. Functional responses (RLU) were assessed after 6 h of co-culture. PE, phycoerythrin. [Figure 39A] 1 shows that Jurkat cells expressing MSLN CAR3 and A*03, A*11 or B*07 blocker constructs were blocked in the presence of blocker antigen that is increased on endogenous MSLN(+) HeLa target cells. [Figure 39B] Figure 39 shows the cytotoxicity assay of primary T cells of MSLN CAR3 + A*11 blocker. Primary T cells transduced with CAR3 and A*11:01-directed blocker efficiently block HeLa target cells with A*11:01 and kill 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 both tumor and normal target cells used here expressed GFP. Figure 39C shows a representative co-culture image at 48 hours for Figure 39B. [Figure 39C] Figure 39 shows the cytotoxicity assay of primary T cells of MSLN CAR3 + A*11 blocker. Primary T cells transduced with CAR3 and A*11:01-directed blocker efficiently block HeLa target cells with A*11:01 and kill 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 both tumor and normal target cells used here expressed GFP. Figure 39C shows a representative co-culture image at 48 hours for Figure 39B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Provided herein are compositions and methods for treating cancer using immune cells that contain two receptor systems that respond to differences in gene expression of ligands between cancer and normal (i.e., healthy or wild-type) cells. These expression differences can be due to loss of heterozygosity in cancer cells. Alternatively, the expression differences can be because gene expression is not expressed in cancer cells or is expressed at a lower level in cancer cells than normal cells. The two-receptor system is expressed in immune cells, such as immune cells used in adoptive cell therapy, to target the activity of these immune cells to cancer cells that exhibit loss of heterozygosity or expression differences. In this two-receptor system, the first receptor (activator receptor, sometimes referred to herein as the A module) activates or promotes the activation of the immune cell, while the second receptor (inhibitory receptor, sometimes referred to herein as the blocker, inhibitor receptor, or B module) acts to inhibit the activation of the immune cell by the first receptor. Each receptor contains a ligand binding domain (LBD) that binds to a specific ligand. Signals from the two receptors upon ligand binding are integrated by immune cells. Differential expression of ligands for the first and second receptors in cancer and normal cells, for example due to loss of heterozygosity or differences in transcription levels of a gene 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.

[0042] Loss of heterozygosity (LOH) due to large chromosomal deletions is a cause of genetic differences in tumors. LOH is a common event in tumor development affecting nearly every locus in the genome, with approximately 20% of genes exhibiting LOH in an average tumor. LOH provides a means to clearly distinguish tumors from normal tissues, since all malignant cells can be found in tumors lacking a particular germline allele. One locus that undergoes LOH is the human leukocyte antigen (HLA) locus, which encodes a polymorphic, abundant, and ubiquitous surface antigen. The two-receptor system described herein utilizes a first receptor to activate T cells (sometimes referred to as the "activator module") exposed to tumor antigen-positive tumor cells, and a second receptor to prevent immune cell activation in the presence of surface blocker antigens such as the HLA-A*02 protein. 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 mixtures of tumor and "normal" cells.

[0043] In certain embodiments of the compositions and methods provided herein, immune cells comprising the two-receptor system described herein are used to treat mesothelin (MSLN)-positive cancers, including mesothelioma, ovarian, cervical, colorectal, esophageal, head and neck, renal, uterine, gastric, pancreatic, lung, colorectal, or bile duct cancers. In some embodiments, the cancer has recurred in the subject. In some embodiments, the cancer is refractory to one or more previously administered anti-cancer therapies. In some embodiments, the cancer is metastatic. In the case of MSLN-positive cancers, the target antigen of the activator receptor is MSLN or its peptide antigen in complex with major histocompatibility complex class I (MHC-I). MSLN is expressed in normal fat, fallopian tube, lung, and salivary gland tissues, among others (Figure 2). Due to its expression in certain tumors, MSLN is an attractive tumor-specific antigen that can mediate the selective killing of MSLN+ tumors if these cancer cells can be specifically targeted with appropriate therapeutic agents.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 therapy and reduce the adverse effects associated with these therapies, such as dose-limiting toxicity.

[0044] In some embodiments, the ligand for 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, which increases the safety window of the activator by blocking its cytolytic effect on normal MSLN-positive tissue. However, because tumor cells do not express the inhibitor, or blocker, ligand for the receptor, the activator receptor still induces targeted killing of tumor cells by immune cells that contain a two-receptor system. A target for the second inhibitory receptor is expressed by MSLN-positive tissues such as lung, mesothelial and adipose tissues, but not in cancer cells, and the inhibitory receptor recognizes this "non-target antigen" as an inhibitory stimulus. An exemplary target for the second inhibitory receptor is expressed by lung tissue and is lost from MSLN-positive cancer cells by loss of heterozygosity (LOH) or other mechanisms, leaving a single allele form in the cancer cells that can be distinguished from other alleles via 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, and others, which can be lost from MSLN-positive cancer cells through 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 a common nonsynonymous variant form with amino acid changes in their extracellular domains that are accessible to antibodies, which can be used as inhibitory or blocker receptor targets for cellular integrators designed to safely treat patients with MSLN-positive cancers with engineered T cells activated by MSLN or activator receptors such as the MSLN pMHC-responsive activator receptor.

[0045] The compositions and methods of the present disclosure can reduce or eliminate dose-limiting toxicity (DLT) caused by expression of MSLN on normal tissues. The present disclosure provides a method of targeting MSLN in cancer cells to treat MSLN-positive cancers using adoptive cell therapy by adding a second inhibitory receptor that blocks the activation of adoptive immune cells in the presence of a second ligand (a ligand other than MSLN or alternatively, a blocker antigen, referred to as a non-target antigen). Using the compositions and methods described herein, tumor cells expressing MSLN are attacked by adoptive cells, such as immune cells, that express two receptors because these tumor cells express only MSLN, the activator ligand. In contrast, normal cells expressing MSLN and the non-target antigen are protected from the adoptive immune cells. The response of the inhibitory receptor to the non-target antigen on normal cells prevents the activation of immune cells by the MSLN-targeted activator receptor. This dual targeting approach creates a therapeutic window that allows MSLN-directed cell therapy to be safely and effectively administered in MSLN-positive cancer patients.

[0046] The present disclosure provides methods and compositions that enable the use of potent MSLN CARs and TCRs to induce on-target toxicity, making these MSLN-targeted receptors useful as therapeutic agents by mitigating their toxicity.

[0047] Alternatively, the compositions and methods described herein can be used to kill target cells and / or treat subjects in which expression of a non-target antigen is partially or completely reduced due to causes other than loss of heterozygosity, including, but not limited to, partial gene deletion, epigenetic silencing, and point or truncating mutations in the sequence encoding the non-target antigen.

[0048] definition Before describing the present disclosure in more detail, it may be helpful to an understanding of the present invention to provide definitions of certain terms that will be used herein.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used to carry out or test a particular embodiment, preferred embodiments of compositions, methods and materials are described herein. For the purposes of this disclosure, the following terms are defined below. Additional definitions are described throughout this disclosure.

[0050] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers to a range of an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0051] As used herein, the term "isolated" means material that is substantially or essentially free from components that normally accompany it in its natural state. In certain embodiments, the terms "obtained" or "derived" are used synonymously with isolated.

[0052] The terms "subject", "patient" and "individual" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Also encompassed are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro. As used herein, "subject", "patient" or "individual" includes 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 and preferably human patients are included.

[0053] As used herein, "treatment" or "treating" includes any beneficial or desired effect, and may include even a minimal amelioration of symptoms. "Treatment" does not necessarily indicate a complete eradication or cure of a disease or condition, or its associated symptoms.

[0054] As used herein, "prevent" and similar words such as "prevented," "preventing" refer to an approach to prevent, inhibit, or reduce the likelihood of a disease symptom. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of a disease symptom. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms, and / or burden of a disease prior to onset or recurrence.

[0055] As used herein, the term "amount" refers to an "effective amount" or an "effective amount" of a virus to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0056] A "therapeutically effective amount" of a virus or cell may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the virus or cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient).

[0057] An "increased" or "augmented" amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically 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 fold or more (e.g., 500, 1000 fold) (including all integers and decimals greater than 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the level of activity in untreated cells.

[0058] A "reduced" or "diminished" amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically 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 or more fold (e.g., 500, 1000 fold) (including all integers and decimals greater than 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) above the level of activity in untreated cells.

[0059] "Maintain" or "preserve" or "maintain" or "no change" or "no substantial change" or "no substantial reduction" generally refers to a physiological response that is equivalent to the response elicited by either a vehicle or a control molecule / composition. An equivalent response is one that is not significantly or measurably different from the reference response.

[0060] Generally, "sequence identity" or "sequence homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, an advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990), and is 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 (generally 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 proteins being compared. Default parameters are provided to optimize searches with short query sequences, for example with the blastp program.This program can also use a SEG filter to mask-off segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired range of sequence identity is approximately 80% to 100%, and integer values ​​therebetween. Typically, the percent identity between the disclosed and claimed sequences is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0061] As used herein, a "polynucleotide system" refers to one or more polynucleotides that can be designed to work together for a particular application or to produce a desired transformed cell.

[0062] The term "exogenous" is used herein to refer to any molecule, including nucleic acids, proteins or peptides, small molecules, etc., that originate outside of an organism. In contrast, the term "endogenous" refers to any molecule that originates within an organism (i.e., is naturally produced by the organism).

[0063] The term "MOI" is used herein to refer to the multiplicity of infection, which is the ratio of agent (e.g., viral particles) to infected targets (e.g., cells).

[0064] In this description, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value in the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated. The term "about," when immediately preceding a number or numerical value, means that the number or numerical value is within a range of plus or minus 10%.

[0065] As used herein, "target cell" refers to the cell that is targeted by adoptive cell therapy.For example, target cell can be cancer cell, which can be killed by the transplanted T cell of adoptive cell therapy.The target cell of the present disclosure expresses target antigen as described herein and does not express non-target antigen.

[0066] As used herein, "non-target cells" refers to cells that are not targeted by adoptive cell therapy. For example, in adoptive cell therapy targeting cancer cells, normal, healthy, non-cancerous cells are non-target cells. Some or all of the non-target cells in a subject may express both target and non-target antigens. Non-target cells in a subject may express non-target antigens regardless of whether these cells also express target antigens.

[0067] As used herein, "non-targeted allelic variant" refers to an allele of a gene whose product is expressed by non-targeted cells but not by targeted cells. For example, a non-targeted allelic variant is an allele of a gene that is expressed by normal non-cancer cells of a subject but not by cancer cells of the subject. Expression of a non-targeted 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-targeted allelic variant.

[0068] As used herein, when used in reference to a ligand-binding domain, such as an antigen-binding domain, "specific for" or "specifically binds to" refers to a ligand-binding domain that has high specificity for a named target. Antibody specificity can be considered a measure of the goodness of fit between the ligand-binding domain and the corresponding ligand, or the ability of the ligand-binding domain to discriminate between similar or even dissimilar ligands. In comparison with specificity, affinity is a measure of the strength of binding between the ligand-binding domain and the ligand, such that a low affinity ligand-binding domain binds weakly and a high affinity ligand-binding domain binds strongly. A ligand-binding domain that is specific for a target allele is one that can discriminate between different alleles of the gene. For example, a ligand-binding domain that is specific for HLA-A*02 does not bind or only weakly binds other HLA-A alleles, such as HLA-A*01 or HLA-A*03. One skilled in the art will understand that a ligand-binding domain can be said to be specific for a particular target, but still has low levels of binding to one or more additional targets that do not affect its function in the receptor system described herein.

[0069] As used herein, "target antigen" refers to an antigen expressed by a target cell, such as a cancer cell, whether referred to using the term antigen or the name of a specific antigen. Expression of a target antigen is not limited to the target cell. A target antigen can be expressed by both cancer cells and normal, non-cancer cells in a subject.

[0070] As used herein, a "non-target antigen" (or "blocker antigen") refers to an antigen that is expressed by normal, non-cancer cells and not expressed in cancer cells, whether referred to using the term antigen or the name of the specific antigen. This difference in expression allows an inhibitory receptor to inhibit immune cell activation in the presence of non-target cells, but not in the presence of target cells.

[0071] Polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population. Polymorphism can include one or more base changes, insertions, repeats, or deletions. Polymorphisms include, for example, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), which are mutations that occur when a single nucleotide, adenine (A), thymine (T), cytosine (C), or guanine (G), is changed.

[0072] As used herein, "affinity" refers to the strength of binding of a ligand to an antigen for a single ligand-binding site on a receptor, e.g., the antigen-binding domain of any of the receptors described herein. Ligand-binding domains can have a weaker interaction (low affinity) or a stronger interaction (high affinity) with their ligand.

[0073] Kd, or dissociation constant, is a type of equilibrium constant that measures the tendency of a larger entity to reversibly dissociate into smaller components, such as when a macromolecular complex containing a receptor and its cognate ligand dissociates into the ligand and receptor. If Kd is high, a high concentration of ligand is required to occupy the receptor, meaning that the affinity of the receptor for the ligand is low. Conversely, a low Kd means that the ligand has a high affinity for the receptor.

[0074] As used herein, a "responsive" or "responsive to" receptor refers to a receptor that includes an intracellular domain that, upon binding of a ligand (i.e., an antigen), generates a signal that corresponds 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 that expresses 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 that expresses the activator receptor. The responsiveness of receptors and their ability to activate or inhibit immune cells that express the receptor can be assayed by any means known in the art and described herein, including, but not limited to, reporter assays and cytotoxicity assays.

[0075] As used herein, "activation" of an immune cell or an "activated" immune cell refers to an immune cell that can perform one or more functions characteristic of an immune response. These functions include proliferation, release of cytokines, and cytotoxicity, i.e., killing of target cells. Activated immune cells express markers that are evident to one of skill in the art. For example, activated T cells can express one or more of CD69, CD71, CD25, and HLA-DR. Immune cells expressing an activator receptor (e.g., MSLN CAR) can be activated by an activator receptor when they become responsive to binding of the receptor to a target antigen (e.g., MSLN) expressed by a target cell. A "target antigen" can also be referred to as an "activator antigen" and can be isolated or expressed by a target cell. Activation of an immune cell expressing an inhibitory receptor can be prevented when the inhibitory receptor becomes responsive to a non-target antigen (e.g., HLA-A*02) even when the activator receptor is bound to a target activator ligand. "Non-target antigens" may also be referred to as "inhibitory ligands" or "blocking factors," and may be isolated or expressed by target cells.

[0076] Receptor expression on immune cells can be verified by assays that report the presence of activator and inhibitory receptors as described herein. For example, a population of immune cells can be stained with a labeling molecule (e.g., a fluorophore-labeled receptor-specific antibody or a fluorophore-labeled receptor-specific ligand) and quantified using fluorescence-activated cell sorting (FACS) flow cytometry. This method allows the percentage of immune cells in a population of immune cells to be characterized as expressing activator receptors, inhibitory receptors, or both receptors. The ratio of activator and inhibitory receptors expressed by immune cells described herein can be determined, for example, by digital droplet PCR. These approaches can be used to characterize populations of cells for the production and manufacture of immune cells, pharmaceutical compositions, and kits described herein. It is understood that for the immune cells, pharmaceutical compositions, and kits described herein, the suitable percentage of immune cells expressing both activator and inhibitory receptors is determined specifically for the methods described herein. For example, a suitable percentage of immune cells expressing both activator 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%. By way of further example, 50%-99%, 60%-95%, 65%-95%, 65%-90%, 70%-90%, 75%-90%, 75%-85%, 80%-99%, 85%-99%, 90%-99%, or 95%-99% of immune cells can express both activator and inhibitory receptors. For example, a suitable ratio of activator and inhibitory receptors in immune cells can 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 is understood that purification, enrichment, and / or depletion steps can be used on populations of immune cells to meet the preferred values ​​for the immune cells, pharmaceutical compositions, and kits described herein.

[0077] Responsive receptors expressed by immune cells described herein can be verified by assays that measure the generation of signals expected to be generated by the intracellular domain of the receptor. Responsive receptors can be characterized using reporter cell lines such as Jurkat-luciferase NFAT cells (Jurkat cells). 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 marker for T cell activation. Jurkat cells can be transduced or transfected with activator receptors and / or inhibitory receptors described herein. Activator receptors respond to ligand binding when Jurkat cells express a luciferase reporter gene, and the level of responsiveness can be determined by the level of reporter gene expression. The presence of luciferase can be determined using any known luciferase detection reagent, such as luciferin. Inhibitory receptors, when co-expressed with activator receptors in Jurkat cells, respond to ligand binding and prevent normally responsive immune cells from expressing luciferase in response 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 that: 1) Jurkat cells express luciferase in the presence of activator receptor ligands and in the absence of inhibitory receptor ligands, and 2) luciferase expression in Jurkat cells is reduced or eliminated in the presence of both activator receptor ligands and inhibitory receptor ligands. Using this approach, the sensitivity, potency, and selectivity of activator receptors and specific pairs of activator receptors and inhibitory receptors can be determined. Sensitivity, potency, and selectivity can be quantified by EC50 or IC50 values ​​using dose-response experiments, in which activator receptor ligands and / or inhibitory receptor ligands are titrated into cultures of Jurkat cells expressing a particular pair of activator receptors or activator and inhibitory receptors.Alternatively, EC50 and IC50 values ​​can be determined in co-culture of immune cells (e.g., Jurkat cells or primary immune cells) expressing an activator receptor or a particular pair of activator and inhibitory receptors with target cells expressing increased amounts of activator or inhibitory ligands. Increased amounts of activator or inhibitory ligands can be achieved in target cells, for example, by titrating mRNA-encoding activator or inhibitory ligands into the target cells, or by using target cells that naturally express different levels of the target ligand. Exemplary suitable EC50 and IC50 values ​​for activator and inhibitory receptors when determining the target cells used that express various amounts of target and non-target ligands include an EC50 of 10 or less transcripts per million (TPM) for activator receptors, for example, an EC50 of 2-10 TPM, and an IC50 of 25 or less TPM for inhibitory receptors, for example, an IC50 of 5-21 TPM.

[0078] Activation of immune cells described herein expressing an activator receptor or a particular pair of an activator receptor and an inhibitory receptor can be further determined by an assay that measures the viability of target cells after co-incubation with the immune cells. Immune cells, sometimes referred to as effector cells, are co-incubated with target cells expressing an activator receptor ligand, an inhibitory receptor ligand, or both an activator receptor and an inhibitory receptor ligand. After co-incubation, the viability of the target cells is measured using any method that measures viability in cell culture. For example, viability can be determined using a mitochondrial function assay that uses tetrazolium substrate to measure active mitochondrial enzymes. Viability can also be determined using imaging-based methods. The target cells can express a fluorescent protein, such as green fluorescent protein or red fluorescent protein. A decrease in total cell fluorescence indicates a decrease in the viability of the target cells. A decrease in the viability of the target cells after incubation with immune cells expressing an activator receptor or a particular pair of an activator receptor and an inhibitory receptor is interpreted as target cell-mediated activation of the immune cells. A measure of the selectivity of the immune cells can also be determined using this approach. Immune cells expressing a pair of activator and inhibitory receptors are selective if the following is observed: 1) there is a decrease in viability in target cells expressing the activator receptor ligand but not the inhibitory receptor ligand, and 2) there is no decrease in viability in target cells expressing both the activator receptor ligand and the inhibitory receptor ligand. From these measurements, a "specific killing" value can be derived that quantifies the percentage of immune cell activation based on the decrease in target cell viability as a percentage of the negative control (immune cells not expressing the activator receptor). Furthermore, from these measurements, a "selectivity ratio" value can be derived that represents the ratio of the specific killing observed in target cells expressing the activator receptor ligand in the absence of the inhibitory receptor ligand to the specific killing observed in target cells expressing both the activator receptor ligand and the inhibitory receptor ligand.This approach can be used to characterize populations of cells for the production and manufacture of the 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 criteria: 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 and target cells expressing an activator receptor ligand in the absence of an inhibitory receptor ligand, and 2) no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 3%, or no more than 1% specific killing of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand.

[0079] As further examples, suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits may be the following criteria: 1) 30%-99%, 40%-99%, 50%-99%, 55%-95%, 60%-95%, 60%-90%, 50%-80%, 50%-70%, or 50%-60% of target cells that express an activator ligand but not an inhibitory ligand are killed; and 2) 1%-40%, 3%-40%, 5%-40%, 5%-30%, 10%-30%, 15%-30%, or 5%-20% of target cells that express an activator ligand and an inhibitory ligand are killed. As still further examples, 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 and target cells expressing an activator receptor ligand in the absence of an inhibitory receptor ligand, and 2) no more than 20% specific killing of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand. As a further example, immune cells may be 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 that express an activator ligand and do not express an inhibitory ligand over a 6, 12, 18, 24, 30, 36, 42, 48, 54, or 60 hour period, 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 that express an activator ligand and an inhibitory ligand over the same period.

[0080] A suitable specific killing value of a target cell expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50% to at least about 95%. A suitable specific killing value of a target cell expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits 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%. A suitable specific killing value of a target cell expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, up to about 50%, up to about 55%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, or up to about 95%. Suitable specific killing values ​​of target cells expressing both activator and inhibitory receptor ligands for immune cells, pharmaceutical compositions, and kits may 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 may be determined after 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 of co-incubation of immune cells with target cells.

[0081] A suitable specific killing value of a target cell expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50% to at least about 95%. A suitable specific killing value of a target cell expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits 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%. A suitable specific killing value of a target cell expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, up to about 50%, up to about 55%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, or up to about 95%. Suitable specific killing values ​​of target cells expressing both activator and inhibitory receptor ligands for immune cells, pharmaceutical compositions, and kits may 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 may be determined after 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 of co-incubation of immune cells with target cells.

[0082] As used herein, the term "functional variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein and retains one or more desired activities of the parent protein. A functional variant can be a fragment of a protein (i.e., a variant having an N-terminal and / or C-terminal deletion) that retains one or more desired activities of the parent protein.

[0083] All publications and patents mentioned in this specification 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 case of conflict, this application, including any definitions herein, will control. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not and should not be considered as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0084] Activator Receptor The present disclosure provides a first receptor comprising a first extracellular ligand-binding domain specific for a target antigen, including a cancer cell-specific antigen, or a peptide antigen thereof, in complex with major histocompatibility complex class I (MHC-I). The first receptor is an activator receptor, and upon binding of the target antigen by the extracellular ligand-binding domain of the first receptor, mediates activation of an immune cell expressing the first receptor. The first receptor is responsive to the target antigen (i.e., an activator ligand). For example, when the target antigen binds to or contacts the first receptor, the first receptor is responsive and activates an immune cell expressing the first receptor upon binding of the target antigen by the extracellular ligand-binding domain of the first receptor. In some embodiments, the first receptor is a chimeric antigen receptor (CAR). In some embodiments, the first receptor is a T cell receptor (TCR).

[0085] In some embodiments, the first receptor is humanized. As used herein, "humanization" refers to replacing a sequence or subsequence in a transgene that is isolated or derived from a non-human species with a homologous or functionally equivalent human sequence. For example, a humanized antibody can be created by grafting mouse CDRs onto human framework sequences, followed by back-substitution of certain human framework residues for the corresponding mouse residues from the original antibody.

[0086] activator target In some embodiments, the target antigen for the first receptor is a cancer cell specific antigen.Any cell surface molecule expressed by target cancer cell can be suitable target antigen for the first receptor ligand binding domain.For example, cell adhesion molecule, intercellular signaling molecule, extracellular domain, molecule involved in chemotaxis, glycoprotein, G protein-coupled receptor, transmembrane, receptor for neurotransmitter, or voltage-dependent ion channel can be used as target antigen.

[0087] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in complex with major histocompatibility complex class I (MHC-I). Any molecule expressed by a target cancer cell and presented as a peptide antigen (pMHC) by major histocompatibility complex class I (MHC-I) on the cancer cell surface can be a suitable target antigen for the first receptor extracellular ligand-binding domain.

[0088] In some embodiments, the cancer cell-specific antigen is mesothelin (MSLN) in complex with major histocompatibility complex class I (MHC-I), or a peptide antigen thereof.

[0089] The major histocompatibility complex class I (MHC-I) is a protein complex that presents antigens to cells of the immune system and induces an immune response. The human leukocyte antigens (HLA) that correspond to MHC-I are HLA-A, HLA-B, and HLA-C.

[0090] Cancer cell specific pMHC antigens including any of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G are contemplated to be within the scope of this disclosure. In some embodiments, the cancer cell specific antigen comprises HLA-A. HLA-A receptors are heterodimers that include a heavy alpha chain and a smaller beta chain. The alpha chain is encoded by a variant of HLA-A, while the beta chain (beta2-microglobulin) is invariant. There are thousands of variant HLA-A genes, all of which fall within the scope of this disclosure. In some embodiments, the MHC-I comprises the human leukocyte antigen A*02 allele (HLA-A*02).

[0091] In some embodiments, the cancer cell-specific antigen comprises HLA-B. Several hundred versions (alleles) of the HLA-B gene are known, each of which is given a specific number (such as HLA-B*27).

[0092] In some embodiments, the cancer cell specific antigen comprises HLA-C. HLA-C belongs to the HLA class I heavy chain paralogs. This 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.

[0093] 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.

[0094] In some embodiments, the cancer cell specific antigen is MSLN or its peptide antigen in complex with major histocompatibility complex class I (MHC-I). MSLN is a 40KDa protein that is normally expressed in mesothelial cells, as well as lung, fallopian tube, salivary gland and adipose tissue (Figure 2). MSLN is expressed in multiple human tumor types, including mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, renal cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or bile duct cancer. In some embodiments, the cancer has recurred in the subject. In some embodiments, the cancer is refractory to one or more previously administered anti-cancer therapies. In some embodiments, the cancer is metastatic.

[0095] All isoforms of MSLN are contemplated as cancer cell-specific antigens of the present disclosure.MSLN isoform 1 preprotein is described in NCBI record number NP_005814.2, the contents of which are incorporated herein by reference.In some embodiments, MSLN comprises the following amino acid sequence: [ka]

[0096] In some embodiments, the MSLN comprises a sequence that shares 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% identity with SEQ ID NO:1.

[0097] MSLN isoform 2 preprotein is described in NCBI accession number NP_037536.2, the contents of which are incorporated herein by reference. In some embodiments, MSLN comprises the following amino acid sequence: [ka]

[0098] In some embodiments, the MSLN comprises a sequence that shares 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% identity with SEQ ID NO:2. In some embodiments, the cancer cell-specific antigen is a peptide antigen derived from MSLN. In some embodiments, the peptide antigen comprises a sequence having 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% identity to a partial sequence of SEQ ID NO:1 and / or SEQ ID NO:2. In some embodiments, the peptide antigen comprises a sequence identical to a partial sequence of SEQ ID NO:1 and / or SEQ ID NO:2.

[0099] Extracellular Ligand Binding Domain 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.

[0100] In some embodiments, the cancer cell-specific antigen is MSLN or an MSLN-derived peptide antigen complexed with MHC-I, and the ligand-binding domain of the first receptor recognizes and binds to the MSLN antigen.

[0101] Any type of ligand-binding domain capable of regulating the activity of a receptor in a ligand-dependent manner is contemplated within the scope of the present disclosure. In some embodiments, the ligand-binding domain is an antigen-binding domain. Exemplary antigen-binding domains include, inter alia, scFv, SdAb, Vβ-only domains, and TCR antigen-binding domains derived from TCR α and β chain variable domains.

[0102] Any type of antigen binding domain is contemplated within the scope of this disclosure.

[0103] For example, the first extracellular ligand binding domain can be part of a continuous polypeptide chain, including, for example, a Vβ-only 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 embodiments, the first extracellular ligand binding domain comprises an antigen binding domain, including an antibody fragment. In further embodiments, the first extracellular ligand binding domain comprises an antibody fragment, including an scFv or an sdAb.

[0104] The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins, or fragments thereof, of polyclonal or monoclonal origin, and can be derived from natural or recombinant sources.

[0105] The term "antibody fragment" or "antibody binding domain" refers to at least a portion of an antibody or a recombinant variant thereof that contains the antigen-binding domain, i.e., the antigen-determining variable region of an intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and a defined epitope thereof. 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") (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.

[0106] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, wherein the light and heavy chain variable regions are linked contiguously via a short flexible polypeptide linker and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0107] "Heavy chain variable region" or "VH" (or "VHH" in the case of single domain antibodies, e.g., nanobodies) with respect to antibodies refers to the fragment of a heavy chain containing the three CDRs interposed between adjacent stretches known as framework regions, which are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0108] As used herein, unless otherwise specified, an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and an scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0109] In some embodiments, the antigen binding domain of the activator and / or inhibitory receptor comprises an scFv. In some embodiments, the scFv comprises a VL and a VH region joined by a linker. In some embodiments, the linker comprises a glycine serine linker, e.g., 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).

[0110] 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.

[0111] The term "recombinant antibody" refers to an antibody produced using recombinant DNA techniques, e.g., an antibody expressed by a bacteriophage or yeast expression system. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence techniques that are available and well known in the art.

[0112] The term "Vβ domain", "Vβ-only domain", "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) beta variable domain that specifically binds to an antigen in the absence of a second TCR variable domain. A Vβ-only domain engages an antigen using complementarity determining regions (CDRs). Each Vβ-only domain contains three complement determining regions (CDR1, CDR2, and CDR3). Additional elements may be combined, provided that the Vβ domain is configured to bind an epitope in the absence of a second TCR variable domain.

[0113] 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 β chain variable domain (Vβ).

[0114] In some embodiments, the extracellular ligand binding domain of the first receptor is a TCR It contains an alpha chain variable domain and a TCR beta chain variable domain.

[0115] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv antigen binding domain. Exemplary MSLN scFvs are shown in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

Table 1-45

Table 1-46

Table 1-47

Table 1-48

Table 1-49

Table 1-50

Table 1-51

Table 1-52

Table 1-53

Table 1-54

Table 1-55

Table 1-56

Table 1-57

Table 1-58

Table 1-59

Table 1-60

Table 1-61

Table 1-62

Table 1-63

Table 1-64

Table 1-65

Table 1-66

[0116] In some embodiments, the extracellular ligand-binding domain of the first receptor is an 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, the underlines indicate CDR sequences.

[0117] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an 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 a sequence of SEQ ID NO: 3-6, 80, or 154-215, or a sequence set forth in Table 1. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an antigen binding domain comprising a sequence of SEQ ID NO: 3-6, 80, or 154-215, or a sequence set forth in Table 1.

[0118] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a 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 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.

[0119] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an 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 an scFv antigen binding domain comprising the 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 the group consisting of SEQ ID NOs: 3-6 or 80. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0120] In Table 2, the light chain (LC) CDRs paired with the indicated heavy chain (HC) CDRs are shown in the left column.

[0121] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an HC CDR1, an HC CDR2, and an HC CDR3 set forth in Table 2 (e.g., HC CDR1, HC CDR2, and HC CDR3 of line #1, line #2, line #3 of Table 2, etc.), or a sequence with up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs in Table 2. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an LC CDR1, LC CDR2, and LC CDR3 set forth in Table 2 (e.g., LC CDR1, LC CDR2, and LC CDR3 of line A, line B, or line C of Table 2), or a sequence with up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs in Table 2. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an HC CDR1, an HC CDR2, and an HC CDR3 set forth in Table 2 (e.g., an HC CDR1, an HC CDR2, and an HC CDR3 of line #1, line #2, line #3, etc., of Table 2). In some embodiments, the extracellular ligand binding domain of the first receptor comprises an LC CDR1, an LC CDR2, and an LC CDR3 set forth in Table 2 (e.g., an LC CDR1, an LC CDR2, and an LC CDR3 of line A, line B, or line C of Table 2).

[0122] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1, a HC CDR2, a HC CDR3, a LC CDR1, a LC CDR2, and a LC CDR3 set forth in Table 2 (e.g., the HC CDR1, the HC CDR2, and the HC CDR3 set forth in line 1, and the LC CDR1, the LC CDR2, and the LC CDR3 of line A). In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1, a HC CDR2, and a HC CDR3 set forth in Table 2 (e.g., the HC CDR1, the HC CDR2, and the HC CDR3 of line #1, line #2, line #3 of Table 2, etc.), or a sequence having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs of Table 2. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a LC CDR1, a LC CDR2, and a LC CDR3 set forth in Table 2 (e.g., a LC CDR1, a LC CDR2, and a LC CDR3 of line A, line B, or line C of Table 2), or a sequence having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs of Table 2. In some embodiments, the extracellular ligand binding domain of the first receptor comprises one or more HC CDRs set forth in Table 2 and one or more LC CDRs set forth in Table 2. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises (i) an HC CDR1, an HC CDR2, and an HC CDR3 set forth in one line of Table 2 (e.g., HC CDR1, HC CDR2, and HC CDR3 of line #1, line #2, line #3, etc. of Table 2), and (ii) an LC CDR1, an LC CDR2, and an LC CDR3 set forth in one line of Table 2 (e.g., LC CDR1, LC CDR2, and LC CDR3 of line A, line B, or line C of Table 2). In each case, the HC CDR can be paired with any of the LC CDRs, since the heavy and light chains share similarities, with routine testing to confirm the desired expression and binding activity. However, preferred pairings between the heavy and light chains of some embodiments are shown in the right column of Table 2.

[0123] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising the sequence of SGDYYWS (SEQ ID NO: 438), a HC CDR2 comprising the sequence of YIYYSGSTYYNPSLKS (SEQ ID NO: 454), and a HC CDR3 comprising the sequence of CAREDVVKGAFDIW (SEQ ID NO: 533), or a CDR sequence having up to 1, 2, or 3 amino acid substitutions, insertions, or deletions therein. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising the sequence of SGDYYWS (SEQ ID NO: 438), a HC CDR2 comprising the sequence of YIYYSGSTYYNPSLKS (SEQ ID NO: 454), and a HC CDR3 comprising the sequence of CAREDVVKGAFDIW (SEQ ID NO: 533). In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a LC CDR1 comprising the sequence of RASQSISSYLN (SEQ ID NO: 535), a LC CDR2 comprising the sequence of AASSLQS (SEQ ID NO: 539), and a LC CDR3 comprising the sequence of QQSYSTPLT (SEQ ID NO: 542), or a CDR sequence having up to 1, 2, or 3 amino acid substitutions, insertions, or deletions therein. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a LC CDR1 comprising the sequence of RASQSISSYLN (SEQ ID NO: 535), a LC CDR2 comprising the sequence of AASSLQS (SEQ ID NO: 539), and a LC CDR3 comprising the sequence of QQSYSTPLT (SEQ ID NO: 542). In some embodiments, the extracellular ligand binding domain of the first receptor comprises an HC CDR1 comprising the sequence of SGDYYWS (SEQ ID NO: 438), an HC CDR2 comprising the sequence of YIYYSGSTYYNPSLKS (SEQ ID NO: 454), an HC CDR3 comprising the sequence of CAREDVVKGAFDIW (SEQ ID NO: 533), an LC CDR1 comprising the sequence of RASQSISSYLN (SEQ ID NO: 535), an LC CDR2 comprising the sequence of AASSLQS (SEQ ID NO: 539), and an LC CDR3 comprising the sequence of QQSYSTPLT (SEQ ID NO: 542), or CDR sequences having up to 1, 2, or 3 amino acid substitutions, insertions, or deletions compared thereto.In some embodiments, the extracellular ligand binding domain of the first receptor comprises an HC CDR1 comprising the sequence of SGDYYWS (SEQ ID NO: 438), an HC CDR2 comprising the sequence of YIYYSGSTYYNPSLKS (SEQ ID NO: 454), an HC CDR3 comprising the sequence of CAREDVVKGAFDIW (SEQ ID NO: 533), an LC CDR1 comprising the sequence of RASQSISSYLN (SEQ ID NO: 535), an LC CDR2 comprising the sequence of AASSLQS (SEQ ID NO: 539), and an LC CDR3 comprising the sequence of QQSYSTPLT (SEQ ID NO: 542).

[0124] 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 CDRs selected from the sequences of GYTMN (SEQ ID NO: 448), LITPYNGASSYNQKFRG (SEQ ID NO: 470), and GGYDGRGFDY (SEQ ID NO: 534). In some embodiments, the heavy chain comprises the sequences of 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 CDRs selected from the sequences of SASSSVSYMH (SEQ ID NO: 538), DTSKLAS (SEQ ID NO: 541), and QQWSGYPLT (SEQ ID NO: 545). In some embodiments, the light chain comprises the sequences of SASSSVSYMH (SEQ ID NO: 538), DTSKLAS (SEQ ID NO: 541), and QQWSGYPLT (SEQ ID NO: 545).

[0125] Exemplary heavy and light chain sequences of antigen binding domains specific for MSLN are set forth 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. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 4]

[0126] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a heavy chain variable region (VH) sequence set forth 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 to a VH set forth in Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a light chain variable region (VL) sequence set forth 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 to a VL set forth in Table 4.

[0127] In some embodiments, the extracellular ligand binding domain of the first receptor (i) comprises an HC CDR1, HC CDR2, and HC CDR3 sequence set forth in Table 2 (e.g., the HC CDR1, HC CDR2, and HC CDR3 of line #1, line #2, line #3 of Table 2), and (ii) a VH 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 sequence set forth in Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor (i) comprises an LC CDR1, LC CDR2, and LC CDR3 sequence set forth in one line of Table 2 (e.g., the LC CDR1, LC CDR2, and LC CDR3 of line A, line B, or line C of Table 2), and a VL sequence set forth 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 to a VL sequence set forth in Table 4.

[0128] In some embodiments, the extracellular ligand binding domain of the first receptor comprises (i) a VH sequence set forth 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 sequence set forth in Table 3, and (ii) a VL sequence set forth in Table 4 or a VL sequence set forth in Table 4. In each case, the VH can be paired with any of the VLs using routine testing to confirm the desired expression and binding activity since the heavy and light chains share similarities, but the preferred pairings between Tables 3 and 4 are shown in the "LC" column of Table 3, which corresponds to the # column of Table 4.

[0129] 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.

[0130] 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, the VH and VL are separated by a linker, e.g., a linker comprising the sequence GGGGSGGGGSGGGGSGG (SEQ ID NO: 152). The VH and VL can be in any orientation, e.g., VH, linker, VL, or alternatively, VL, linker VH.

[0131] 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 with another amino acid. The substitutions can be "conservative" in the sense that they are substitutions within the same family of amino acids. Naturally occurring amino acids can be divided into four families, and conservative substitutions are made within those 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. The amino acid sequence of the CDR of an antibody can be altered by adding, deleting, or substituting amino acids to achieve various effects, such as increasing the binding affinity to the target antigen.

[0132] Chimeric antigen receptors (CARs) 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.

[0133] As used herein, the term "chimeric antigen receptor (CAR)" can refer to an artificial receptor derived from a T cell receptor, and encompasses engineered receptors that graft artificial specificity onto a particular immune effector cell. CARs may be used to confer the specificity of a monoclonal antibody to a T cell, thereby allowing large numbers of specific T cells to be generated, for example, for use in adoptive cell therapy. In certain embodiments, the CAR directs the specificity of a cell to, for example, a tumor-associated antigen. An exemplary CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain that includes a tumor-associated antigen binding region. In some embodiments, the CAR further comprises a hinge domain. In certain aspects, the CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3 transmembrane domain and endodomain. The specificity of other CAR designs can be derived from the receptor's ligand (e.g., a peptide). In certain cases, the CAR contains domains for additional costimulatory signaling, 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 costimulatory molecules, reporter genes for imaging, gene products that conditionally eliminate T cells upon addition of a prodrug, homing receptors, cytokines, and cytokine receptors.

[0134] In some embodiments, the extracellular ligand-binding domain of the first receptor is fused to the extracellular domain of the CAR.

[0135] In some embodiments, the CAR of the present disclosure comprises an extracellular hinge region. Incorporation of the hinge region can affect cytokine production from CAR-T cells and improve CAR-T cell expansion in vivo. Exemplary hinges can be isolated or derived from IgD and CD8 domains, e.g., IgG1. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0136] In some embodiments, the hinge is isolated or derived from CD8α or CD28. In some embodiments, the CD8α hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of 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 CD8 alpha hinge is encoded by a nucleotide sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 8). In some embodiments, the CD8 alpha hinge is encoded by SEQ ID NO: 8.

[0137] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 9). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 9. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 10). In some embodiments, the CD28 hinge is encoded by SEQ ID NO:10.

[0138] The CAR of the present disclosure can be designed to include a transmembrane domain that is fused to the extracellular domain of the CAR. In some embodiments, a transmembrane domain that naturally associates with one of the domains in the CAR is used. For example, a CAR that includes a CD28 costimulatory domain may also use a CD28 transmembrane domain. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or different surface membrane protein and to minimize interaction with other members of the receptor complex.

[0139] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region may be isolated or derived from (i.e., including at least the transmembrane region thereof) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or an immunoglobulin such as IgG4. Alternatively, the transmembrane domain may be synthetic, in which case it comprises mainly hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane and cytoplasmic signaling domains of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0140] In some embodiments of the CAR of the present disclosure, the 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 identical to the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 11). In some embodiments, the CD28 transmembrane domain comprises or consists essentially 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 identical to the sequence of TTCTGGGTGCTGGTCGTTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 12). In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 12.

[0141] In some embodiments of the CAR of the present disclosure, the CAR comprises an IL-2R beta transmembrane domain. In some embodiments, the IL-2R beta 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 is identical to the sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 13). In some embodiments, the IL-2R beta transmembrane domain comprises or consists essentially of SEQ ID NO: 13. In some embodiments, the IL-2R beta transmembrane domain comprises [ka] (SEQ ID NO: 14). In some embodiments, the IL-2R beta 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 identical to the sequence of (SEQ ID NO: 14).

[0142] The cytoplasmic domain, or otherwise, intracellular signaling domain, of the CAR of the present disclosure is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and causes the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion may be used in place of the intact chain, so long as it transduces an effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired function of the CAR-T cells of the present disclosure. Thus, the term intracellular signaling domain is meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce an effector function signal.

[0143] Examples of intracellular signaling domains for use in the CARs of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capability.

[0144] Thus, the intracellular domain of the CAR of the present disclosure comprises at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures that there is T cell receptor (TCR) signaling necessary to activate the effector function of the CAR T cell. In some embodiments, the at least one cytoplasmic activation domain is the CD247 molecule (CD3zeta) activation domain, the stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or the DNAX activation protein of 12 kDa (DAP12) activation domain.

[0145] In some embodiments, the CD3 zeta activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 15).

[0146] 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 has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of AGAGTGAAGTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGCGTAGAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGACTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 16). In some embodiments, the CD3 zeta activation domain is encoded by SEQ ID NO:16).

[0147] It is known that signals generated solely through the TCR are often insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0148] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. In some embodiments, the ITAM contains a tyrosine separated from a leucine or isoleucine by any two other amino acids (YxxL / I (SEQ ID NO:546)). In some embodiments, the cytoplasmic domain contains 1, 2, 3, 4, or 5 ITAMs. An exemplary ITAM-containing cytoplasmic domain is the CD3zeta activation domain. Further examples of ITAM-containing primary cytoplasmic signaling sequences that can be used in the CARs of the present disclosure include those derived from TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD5, CD22, CD79a, CD79b, and CD66d.

[0149] In some embodiments, a CD3 zeta activation domain comprising a single ITAM comprises an amino acid sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 17). In some embodiments, a CD3 zeta activation domain comprises SEQ ID NO: 17. In some embodiments, a CD3 zeta activation domain comprising a single ITAM consists essentially of the amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 17). In some embodiments, a CD3 zeta activation domain comprising a single ITAM comprises [ka] (SEQ ID NO: 18). In some embodiments, the CD3 zeta activation 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 identical to the sequence of SEQ ID NO: 18.

[0150] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise a CD3ζ signaling domain by itself or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3ζ chain portion and a costimulatory domain. The costimulatory domain refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. Examples of such molecules include the costimulatory domains 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 (4-1BB), TNF receptor The co-stimulatory domain is selected from the group consisting of OX40, 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-stimulatory (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 a functional variant thereof. 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 isolated or derived from CD28.

[0151] In some embodiments, the intracellular domain of a CAR of the present disclosure comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 19). 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 having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC (SEQ ID NO: 20). In some embodiments, the CD28 costimulatory domain is encoded by SEQ ID NO: 20.

[0152] In some embodiments, the costimulatory domain is isolated or derived from 4-1BB. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 283). In some embodiments, the 4-1BB costimulatory domain comprises or consists essentially of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 283). In some embodiments, the 4-1BB costimulatory 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 identical to the sequence of AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGGCCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 284).

[0153] In some embodiments, the intracellular domain of the CAR comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3zeta activation domain. In some embodiments, the intracellular domain of the CAR comprises a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 285), or a sequence with at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity thereto.

[0154] The cytoplasmic domains within the cytoplasmic signaling portion of the CAR of the present disclosure can be linked together in a random or specified order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, may form the linkage. A glycine-serine doublet provides one example of a suitable linker. An exemplary linker comprises the sequence GGGGSGGGGSGGGGSGG (SEQ ID NO: 152).

[0155] The cytoplasmic domains within the cytoplasmic signaling portion of a CAR of the present disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, may form the linkage. A glycine-serine doublet provides an example of a suitable linker. Exemplary full-length activator receptors of the present disclosure are set forth in Table 20. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO:286-347 set forth in Table 20, 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:286-347 set forth in Table 20. 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 the 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.

[0156] The cytoplasmic domains within the cytoplasmic signaling portion of the CAR of the present disclosure can be linked together in a random or specified order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, may form the linkage. A glycine-serine doublet provides one example of a suitable linker.

[0157] T cell receptor (TCR) 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).

[0158] As used herein, "TCR," sometimes referred to as "TCR complex" or "TCR / CD3 complex," refers to a protein complex that includes one or more of the TCR alpha chain, TCR beta chain, and invariant CD3 chain (zeta, gamma, delta, and epsilon), sometimes referred to as subunits. The TCR alpha and beta chains can be disulfide-linked to function as a heterodimer that binds to peptide-MHC complexes. When the TCR alpha / beta heterodimer engages peptide-MHC, a conformational change in the TCR complex within the associated invariant CD3 subunit is induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal. In an exemplary TCR complex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3 epsilon and CD3 delta form a heterodimer, and CD3 epsilon and CD3 gamma for the heterodimer, as well as two CD3 zetas form homodimers.

[0159] Any suitable ligand binding domain may be fused to the extracellular domain, hinge domain or transmembrane of a TCR as described herein. For example, the ligand binding domain may be an antibody or an antigen binding domain of a TCR, or may comprise an antibody fragment, a Vβ-only domain, a linear antibody, a single chain variable fragment (scFv), or a single domain antibody (sdAb).

[0160] In some embodiments, the ligand binding domain is fused to one or more extracellular or transmembrane domains of one or more TCR subunits. The TCR subunits may be TCR alpha, TCR beta, CD3 delta, CD3 epsilon, CD3 gamma, or CD3 zeta. For example, the ligand binding domain may be fused to TCR alpha, or TCR beta, or the ligand binding portion may be fused to two subunits, e.g., the ligand binding domain may be fused to both TCR alpha and TCR beta.

[0161] TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma, and CD3 epsilon. Any one or more of the TCR alpha, TCR beta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta, or fragments or derivatives thereof, may be fused to one or more domains capable of providing a stimulatory signal of the present disclosure, thereby enhancing TCR function and activity.

[0162] TCR transmembrane domains isolated or derived from any source are contemplated within the scope of this disclosure. The transmembrane domains may be derived from either natural or recombinant sources. If the source is natural, the domains may be derived from any membrane-bound or transmembrane protein.

[0163] In some embodiments, the transmembrane domain can transmit a signal to the intracellular domain whenever the TCR complex is bound to a target. A transmembrane domain of particular use in the present disclosure can include, for example, at least the transmembrane region of the alpha, beta, or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0164] In some embodiments, the transmembrane domain can be attached to the extracellular region of a polypeptide of a TCR, e.g., the antigen binding domain of a TCR alpha or beta chain, via a hinge, e.g., a hinge derived from a human protein. For example, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0165] In some embodiments, the extracellular ligand binding domain is linked 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.

[0166] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., 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 of 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 of the intracellular region).

[0167] In some embodiments, transmembrane domains may be selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0168] If present, the transmembrane domain may be a native TCR transmembrane domain, a native transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain may be a membrane anchor domain. Without being limited thereto, a native or artificial transmembrane domain may contain a hydrophobic a-helix of about 20 amino acids, often with a positive charge adjacent to the transmembrane segment. The transmembrane domain may have one transmembrane segment or two or more transmembrane segments. Predictions of transmembrane domains / segments may be made using published 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 anchor systems include the platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (post-translationally added to a signal sequence), and the like.

[0169] 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 having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or is identical to the 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 sequence GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG (SEQ ID NO: 22).

[0170] In some embodiments, the transmembrane domain comprises a TCR beta transmembrane domain. In some embodiments, the TCR beta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or is identical to the sequence of TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 23). In some embodiments, the TCR beta transmembrane domain comprises or consists essentially of SEQ ID NO: 23. In some embodiments, the TCR beta transmembrane domain is encoded by the sequence of ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO: 24).

[0171] The TCRs of the present disclosure can include one or more intracellular domains. Exemplary TCRs including intracellular domains for use in the present disclosure are described in PCT / US2020 / 045250, filed September 6, 2020, the contents of which are incorporated herein by reference. In some embodiments, the intracellular domain includes one or more domains capable of providing a stimulatory signal to the transmembrane domain. In some embodiments, the intracellular domain includes a first intracellular domain capable of providing a stimulatory signal, and a second intracellular domain capable of providing a stimulatory signal. In other embodiments, the intracellular domain includes a first, second, and third intracellular domain capable of providing a stimulatory signal. The intracellular domain capable of providing a stimulatory signal is selected from the group consisting of 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 zeta chain of T cell receptor-associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, a lymphocyte cytoplasmic protein 2 (SLP76) domain, (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, and CD3 epsilon intracellular domain.

[0172] In some embodiments, the intracellular domain comprises at least one intracellular signaling domain. The intracellular signaling domain generates a signal that promotes immune effector function of a functional cell, e.g., a TCR-containing cell, e.g., a TCR-expressing T cell. In some embodiments, the intracellular domain of the first receptor of the present disclosure comprises at least one intracellular signaling domain. For example, the intracellular domain of CD3 gamma, delta, or epsilon comprises a signaling domain.

[0173] In some embodiments, the extracellular domain, transmembrane domain and intracellular domain are isolated from or derived from the same protein, e.g., T cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, CD3 epsilon or CD3 zeta.

[0174] Examples of intracellular domains for use in the activator receptors of the present disclosure include the cytoplasmic sequences of TCR alpha, TCR beta, CD3 zeta, and 4-1BB, as well as intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences having the same functional capabilities.

[0175] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from proteins responsible for primary, or antigen-dependent, stimulation.

[0176] In some embodiments, the intracellular domain comprises a CD3 delta intracellular domain, a CD3 epsilon intracellular domain, a CD3 gamma intracellular domain, a CD3 zeta intracellular domain, a TCR alpha intracellular domain, or a TCR beta intracellular domain.

[0177] 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.

[0178] In some embodiments, the intracellular domain comprises a TCR beta intracellular domain. In some embodiments, the TCR beta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, or identical to the sequence of MAMVKRKDSR (SEQ ID NO: 25). In some embodiments, the TCR beta intracellular domain comprises or consists essentially of SEQ ID NO: 25. In some embodiments, the TCR beta intracellular domain is encoded by the sequence of ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 26).

[0179] In some embodiments, the intracellular signaling domain comprises at least one stimulatory intracellular domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as CD3 delta, CD3 gamma, and CD3 epsilon intracellular domains, and one additional stimulatory intracellular domain, e.g., a costimulatory domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as CD3 delta, CD3 gamma, and CD3 epsilon intracellular domains, and two additional stimulatory intracellular domains.

[0180] Exemplary costimulatory intracellular signaling domains include those 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, signaling lymphocyte activation molecules (SLAM proteins), and NK cell activating 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, CD83, a ligand that specifically binds to CD4, etc. A costimulatory domain can comprise the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional variant thereof.

[0181] In some embodiments, the stimulatory domain comprises a costimulatory domain. In some embodiments, the costimulatory domain comprises a CD28 or 4-1BB costimulatory domain. CD28 and 4-1BB are well-characterized costimulatory molecules required for full T cell activation and are known to enhance T cell effector function. For example, CD28 and 4-1BB have been utilized in chimeric antigen receptors (CARs) to enhance cytokine release, cytolytic function, and persistence over first generation CARs that contain only CD3 zeta signaling domains. Similarly, the inclusion of costimulatory domains, such as CD28 and 4-1BB domains, in TCRs can increase T cell effector function and specifically allow costimulation in the absence of costimulatory ligands that are typically downregulated on the surface of tumor cells. In some embodiments, the stimulatory domain comprises a CD28 intracellular domain or a 4-1BB intracellular domain.

[0182] Inhibitory receptors The present disclosure provides a second receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is lost in a cancer cell, for example, an allelic variant of a gene. The non-target allelic variant may be lost in the cancer cell through any mechanism, including, but not limited to, epigenetic changes that affect the expression of the non-target allelic variant, mutations to the gene encoding the non-target allelic variant, disruption of cell signaling that regulates the expression of the non-target allelic variant, chromosomal loss, partial or complete deletion of a genomic locus, gene silencing due to modification of nucleic acid or heterochromatin, or loss of expression due to other mechanisms. In a variation of the compositions and methods disclosed herein, the treated cells or subjects may show loss of expression of the non-target allelic variant due to non-genetic changes. Thus, the present disclosure provides compositions and methods for killing cells and / or treating subjects that lack expression of the non-target antigen from any cause, including, but not limited to, loss of heterozygosity.

[0183] The non-target antigen may be a protein in complex with major histocompatibility complex class I (MHC-I) or an antigenic peptide thereof, and the non-target antigen includes polymorphisms. Because the non-target antigen is polymorphic, loss of a single copy of the gene encoding the non-target antigen, which may occur by loss of heterozygosity in cancer cells, results in cancer cells that retain other polymorphic variants of the gene but have lost the non-target antigen. For example, a subject with HLA-A*02 and HLA-A*01 alleles at the HLA locus may have a cancer in which only the HLA-A*02 allele is lost. In such a subject, the HLA-A*01 protein is still present, but is not recognized by the inhibitory receptors of immune cells that encounter the cancer cells, because the inhibitory receptors are designed to be specific for HLA-A*02 (or other non-target antigens). In normal non-malignant cells, HLA-A*02 (or other non-target antigens) are present and inhibit the activation of the engineered immune cells. In cancer cells with loss of heterozygosity, HLA-A*02 allele variants (or other non-target antigens) are lost. Because the inhibitory receptor only responds to HLA-A*02 (or other non-target antigens) that are not present in cancer cells, immune cells engineered to express the inhibitory receptor do not receive inhibitory signals from the inhibitory receptor. By this mechanism, immune cells are selectively activated and selectively kill cancer cells that express MSLN but have lost HLA-A*02 (or another non-target antigen) due to loss of heterozygosity. Herein, HLA-A is used as an example. Similar polymorphic variations occur in other populations of MHC genes and in other non-MHC genes.Thus, the present disclosure provides a second receptor comprising an extracellular ligand-binding domain 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 an antigenic peptide thereof in complex with major histocompatibility complex class I (MHC-I), where the non-target antigen may comprise a non-synonymous extracellular domain polymorphism (e.g., in the extracellular domain of ICAM1, COMT, CXCL16), and immune cells comprising them. 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 the tumor but is present in significant MSLN-expressing normal tissues (e.g., LRRN4, UPK3B).

[0184] Exemplary inhibitory receptors are described in PCT / US2020 / 045228, filed September 6, 2020, PCT / US2020 / 064607, filed December 11, 2020, PCT / US2021 / 029907, filed April 29, 2021, and PCT / US2020 / 059856, filed November 10, 2020, the contents of each of which are incorporated by reference herein.

[0185] In some embodiments, the second receptor is humanized.

[0186] The present disclosure provides a second receptor that is an inhibitory receptor, comprising extracellular ligand binding that can distinguish single amino acid variant alleles of non-target antigens.This ability to distinguish allelic variants of non-target antigens allows the second receptor to suppress the activation of immune cells that comprise the second receptor in the presence of non-target cells that express the allele recognized by the ligand binding domain.However, the activation of immune cells is not suppressed in the presence of target cells that have lost the allele, for example, cancer cells that have lost one allele of a gene due to loss of heterozygosity.

[0187] The present disclosure provides a second receptor that is an inhibitory receptor that comprises an extracellular ligand binding that can distinguish between different levels of expression of a non-target antigen, thereby allowing the second receptor to inhibit activation of an immune cell that comprises the second receptor in the presence of a non-target cell that expresses a ligand for the second receptor, but allows activation of the immune cell in the presence of cancer cells that express low levels or no ligand for the second receptor.

[0188] Inhibitor Ligand In some embodiments, the non-target antigen is not expressed by the target cells and is expressed by the non-target cells. In some embodiments, the non-target antigen is expressed by healthy cells, i.e., cells that are not cancer cells. In some embodiments, the target cells are a plurality of cancer cells that have lost expression of the non-target antigen through loss of heterozygosity (LOH). In some embodiments, the non-target cells are a plurality of healthy cells (i.e., non-cancer cells) that express both the target and non-target antigens.

[0189] Any cell surface molecule expressed by non-target cells that is not expressed by target cells can be a suitable non-target antigen for the second receptor extracellular ligand binding domain. For example, cell adhesion molecules, intercellular signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane, receptors for neurotransmitters, or voltage-gated ion channels can be used as non-target antigens. In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in complex with major histocompatibility complex class I (MHC-I).

[0190] In some embodiments, the non-target antigen is lost in the cancer cells due to loss of heterozygosity. Exemplary non-target antigens lost in the 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 that includes polymorphic residues of ICAM1, COMT, or CXCL16 in complex with major histocompatibility complex class I (MHC-I).

[0191] Non-target major histocompatibility complex class I MHC-I (or pMHC-I) antigens, including any of HLA-A, HLA-B, HLA-C, or HLA-E, are contemplated within the scope of this 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.

[0192] 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 HLA-A allele 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).

[0193] 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.

[0194] In some embodiments, the non-target antigen comprises HLA-C. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0195] In some embodiments, the non-target antigen comprises ICAM1 or an antigenic peptide thereof in complex with MHC-I. Human ICAM1 is frequently lost via LOH in cancer cells.

[0196] 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 is [ka] (SEQ ID NO: 27)

[0197] In some embodiments, ICAM1 comprises a sequence that shares 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% identity with SEQ ID NO: 27. Polymorphic residues of ICAM1 are marked as bold and underlined in SEQ ID NO: 27. For example, rs5498 is a polymorphism at position 469 of SEQ ID NO: 27 and can be K or E.

[0198] In some embodiments, the non-target antigen comprises a polymorphism of ICAM1. For example, the non-target antigen comprises a peptide derived from ICAM1 that comprises 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 that comprises 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.

[0199] 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 that has 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 that has 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.

[0200] In some embodiments, the non-target antigen comprises COMT or an antigenic peptide thereof in complex with MHC-I. Human COMT is frequently lost via LOH in cancer cells.

[0201] Wild-type human COMT is described in NCBI Accession 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: [ka]

[0202] In some embodiments, COMT comprises a sequence that shares 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% identity with SEQ ID NO: 28. Polymorphic residues of COMT are marked as 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 V or M.

[0203] In some embodiments, the non-target antigen comprises a polymorphism of COMT. For example, the non-target antigen comprises a peptide derived from COMT that comprises a polymorphic residue of COMT. The polymorphic residue of COMT1 comprises amino acid residue 158 of SEQ ID NO:28. In some embodiments, the non-target antigen comprises a peptide of COMT that comprises amino acid 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.

[0204] 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 that has 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 that has 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.

[0205] In some embodiments, the non-target antigen comprises CXC motif chemokine ligand 16 (CXCL16) or an antigenic peptide thereof in complex with MHC-I. Human CXCL16 precursor is described in NCBI Accession No. NP_001094282.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, CXCL16 is [ka] The amino acid sequence of

[0206] In some embodiments, the non-target antigen comprises a polymorphism of CXCL16. For example, the non-target antigen comprises a peptide derived from CXCL16 that comprises a polymorphic residue of CXCL16. The polymorphic residue of CXCL16 comprises positions 142 and 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises amino acid 142 or 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises an A at amino acid 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises a V at amino acid 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises an I at amino acid 142 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises a T at amino acid 142 of SEQ ID NO:29.

[0207] In some embodiments, the non-target antigen comprises a polymorphism of CXCL16. In some embodiments, the non-target antigen comprises a peptide of CXCL16 comprising an A at amino acid 200 of SEQ ID NO:29, and the second receptor comprises a ligand binding domain that has 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 comprising a V at amino acid 200 of SEQ ID NO:29, and the second receptor comprises a ligand binding domain that has 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 that comprises an I at amino acid 142 of SEQ ID NO:29, and the second receptor comprises a ligand binding domain that has a higher affinity for a CXCL16 ligand that comprises an I at position 142 of SEQ ID NO:29 than for a CXCL16 ligand that has a T at position 142 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises a T at amino acid 142 of SEQ ID NO:29, and the second receptor comprises a ligand binding domain that has a higher affinity for a CXCL16 ligand that comprises a T at position 142 of SEQ ID NO:29 than for a CXCL16 ligand that has an I at position 142 of SEQ ID NO:29.

[0208] 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. For use in the embodiments described herein, various single variable domains that bind to or recognize specific HLA alleles are listed in Table 5. (Complementarity determining regions are underlined): [Table 5-1] [Table 5-2]

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

Table 5-26

Table 5-27

Table 5-28

Table 5-29

Table 5-30

Table 5-31

Table 5-32

Table 5-33

Table 5-34

Table 5-35

Table 5-36

Table 5-37

[0209] 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*3, HLA-A*11, HLA-B*07, or HLA-C*07 and comprises a sequence selected from the group of sequences set forth 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*3, HLA-A*11, HLA-B*07, or HLA-C*07 and comprises a sequence selected from the group of sequences set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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.

[0210] Exemplary heavy and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3, respectively) for the HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, and HLA-C*07 ligand binding domains are shown in Table 6 below. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9]

[0211] 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 the CDR sequences set forth in Table 6 or Table 7.

[0212] 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 set forth in Table 6.

[0213] 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 set forth in Table 6.

[0214] 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, hi 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.

[0215] 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 HLA-A*01 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 compared to the HLA-A*01 CDRs of Table 6.

[0216] 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 HLA-A*02 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 compared to the HLA-A*02 CDRs of Table 6.

[0217] In some embodiments, the extracellular ligand-binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 103-108 or 109-114, or a CDR sequence having up to one, two, or three substitutions, deletions, or insertions compared to the CDRs of SEQ ID NOs: 103-108 or 109-114.

[0218] 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 HLA-A*03 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 compared to the HLA-A*03 CDRs of Table 6.

[0219] 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 HLA-A*11 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 7, or CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the HLA-A*11 CDRs of Table 7.

[0220] 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 HLA-B*07 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 compared to the HLA-B*07 CDRs of Table 6.

[0221] 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 HLA-C*07 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 compared to the HLA-C*07 CDRs of Table 6.

[0222] In any further embodiment of the ligand binding domain, each CDR sequence may have one, two, three or more substitutions, insertions or deletions. The CDR sequence may tolerate substitutions, deletions or insertions. Using sequence alignment tools, routine experimentation and known assays, one skilled in the art can generate and test variant sequences with one, two, three or more substitutions, insertions or deletions in the CDR sequence without undue experimentation.

[0223] 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 to HLA-A*02 independently of the 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 any one of SEQ ID NOs: 30-41. In some embodiments, the HLA-A*02 ligand binding domain comprises a sequence at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 30-41.

[0224] 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, 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*02 and the extracellular ligand binding domain of the second receptor comprises the sequence of SEQ ID NO: 30.

[0225] 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 sequence DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTSGGGTKLEIK (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 a sequence of QVQLQQSGPELVKPGASVRISCKASGYTFTSYHIHWVKQRPGQGLEWIGWIYPGNVNTEYNEKFKGKATLTADKSSSTAYMHLSSLTSEDSAVYFCAREEITYAMDYWGQGTSVTVSS (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, the VH and VL are separated by a linker, e.g., GGGGSGGGGSGGGGSGG (SEQ ID NO: 152). In some embodiments, the VH and VL are ordered from N to C-terminus, VH, linker, and VL. In some embodiments, the VH and VL are ordered from N to C-terminus, VL, linker, and VH.

[0226] In some embodiments, the HLA-A*02 scFv comprises a complementarity determining region (CDR) of any one of SEQ ID NOs: 42 to 53. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOs: 42 to 53. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOs: 42 to 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 to 53, and the light chain of the antigen-binding domain comprises a light chain CDR of any one of SEQ ID NOs: 42 to 53. In some embodiments, the HLA-A*02 antigen-binding domain comprises a heavy chain and a light chain, the heavy chain comprises a CDR selected from SEQ ID NOs: 45 to 47 and 51 to 53, and the light chain comprises a CDR selected from SEQ ID NOs: 42 to 44 and 48 to 50.

[0227] In some embodiments, the HLA-A*02 antigen-binding domain comprises a heavy chain and a light chain, wherein 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.

[0228] 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 the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 30-41.

[0229] 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 group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-A*01 scFv comprises an HLA-A*1 CDR sequence set forth in Table 6.

[0230] 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 group of sequences set forth in Table 5, or a sequence 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 set forth in Table 6.

[0231] 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 group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-A*11 scFv comprises an HLA-A*11 CDR sequence set forth in Table 7.

[0232] 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 group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-B*07 scFv comprises an HLA-B*07 CDR sequence set forth in Table 6.

[0233] 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 group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-C*07 scFv comprises an HLA-C*07 CDR sequence set forth in Table 6.

[0234] In some embodiments, the non-target antigen comprises HLA-A*11. A variety of single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*11 are suitable for use in the embodiments. Such scFvs include, but are not limited to, the following murine and humanized scFv antibodies that bind to HLA-A*11 in a peptide-independent manner as shown in Table 5 above:

[0235] Exemplary heavy and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR-L1, CDR-L2 and CDR-L3, respectively) for the HLA-A*11 ligand binding domain are shown below in Table 7. Any of the VH CDRs in Table 7 can be combined with the VL CDRs disclosed in Table 7. [Table 7]

[0236] 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 the 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 any one of SEQ ID NOs: 114-122. In some embodiments, the HLA-A*11 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 114-122.

[0237] 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 at least 95% identical to any one of SEQ ID NOs: 114-122. In some embodiments, the scFv comprises a sequence 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 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.

[0238] Exemplary heavy and light chain sequences for an HLA-A*11 antigen-binding domain are provided below in Table 8. In some embodiments, the HLA-A*11 antigen-binding domain comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 132-140, and the light chain comprises a sequence at least 95% identical to the light chain portion of SEQ ID NO: 141.

[0239] 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 the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 114-122. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0240] Differentially expressed repressor ligands The present disclosure provides suppressor ligands (non-target antigens) that are differentially expressed between cancer cells and normal cells.

[0241] The activation of inhibitory receptors is mediated by the presence of non-target antigens on the surface of cells. Cells expressing non-target antigens activate inhibitory receptors based on the level of expression of non-target antigens. In some embodiments, non-target antigens are expressed by both target cells and non-target cells. However, in these embodiments, non-target antigens are expressed by non-target cells at a higher level than target cells. Higher levels of non-target antigens expressed by non-target cells activate inhibitory receptors, thereby preventing immune cell activation. In contrast, lower levels of non-target antigens expressed by targets are not sufficient to activate inhibitory receptors, resulting in immune cell activation.

[0242] In an alternative embodiment, the non-target antigen is expressed by the non-target cell but not by the target cell, In the absence of expression of the non-target antigen, the target cell activates the target receptor, thereby activating the immune cell.

[0243] Differential expression can be determined by any technique known in the art that is used to measure expression. They include, among others, techniques for measuring the mRNA and / or protein levels of target genes in cells. Methods for measuring protein levels in a sample include analytical methods such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and liquid chromatography mass spectrometry (LC-MS). Methods for measuring mRNA levels include real-time quantitative reverse transcription PCR (qRT-PCR), and high-throughput sequencing. Differences in expression can be observed, for example, between normal cells and diseased cells, such as cancer cells.

[0244] The activation of inhibitory receptor by non-target antigen can occur according to various ways known in the art.The activation of inhibitory receptor by non-target antigen can be determined by methods known in the art.For example, the level of downstream intracellular signaling in the cell expressing inhibitory receptor can be measured by using reporter gene.

[0245] Without wishing to be bound by theory, whether the expression of non-target antigens inhibits the activation of immune cells by activating inhibitory receptors may occur depending on the ratio of non-target antigens to inhibitory factor receptors. The expression levels of non-target antigens and inhibitory receptors, and their ratios, can be determined by methods known in the art, including, inter alia, 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. Low or no expression of non-target antigens on target or non-target cells can indicate, for example, that inhibitory receptors are not activated in the immune cells of the present disclosure.

[0246] Alternatively, or in addition, without wishing to be bound by theory, inhibition of immune cell activation by a non-target antigen via activation of an inhibitory receptor may depend on the affinity of the non-target antigen for the inhibitory receptor. Methods for measuring affinity are known in the art and include, inter alia, enzyme-linked immunosorbent assay or radioimmunoassay methods.

[0247] Alternatively, or in addition, without wishing to be bound by theory, inhibition of immune cell activation by non-target antigens via activation of inhibitory receptors may occur in response to crosstalk between inhibitory and activator receptors, resulting in downregulation of activator receptor activity. For example, activation of an inhibitory receptor by a non-target antigen may result in reduced expression of the activator receptor on the surface of the immune cell.

[0248] In some embodiments, the non-target antigen is expressed in the target cells at a lower level than in normal cells. In some embodiments, the non-target antigen is expressed by healthy cells, i.e., cells that are not cancer cells. In some embodiments, the non-target antigen expression level is at least about 10 fold lower, at least about 30 fold lower, at least about 50 fold lower, at least about 70 fold lower, at least about 90 fold lower, at least about 100 fold lower, at least about 110 fold lower, at least about 150 fold lower, at least about 200 fold lower, at least about 250 fold lower, at least about 300 fold lower, at least about 350 fold lower, at least about 400 fold lower, at least about 450 fold lower, at least about 500 fold lower, at least about 600 fold lower, at least about 700 fold lower, at least about 800 fold lower, at least about 900 fold lower, or at least about 1000 fold lower in the target cells than in the non-target cells. In some embodiments, the non-target antigen expression level is about 10-fold, about 30-fold, about 50-fold, about 70-fold, about 90-fold, about 100-fold, or about 110-fold lower in the plurality of cancer cells than in the plurality of healthy cells. In some embodiments, the non-target antigen expression level is at least about 5-fold lower in the plurality of cancer cells than in the plurality of healthy cells. In some embodiments, the non-target antigen expression level is at least about 5-fold lower in the target cells than in the non-target cells. In some embodiments, the target cells are a plurality of cancer cells that have low or no expression of the non-target antigen.

[0249] Any cell surface molecule expressed by non-target cells that is not expressed (or expressed at low levels) by target cells can be a suitable non-target antigen for the second receptor extracellular ligand-binding domain. For example, cell adhesion molecules, intercellular signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane proteins, receptors for neurotransmitters, or voltage-gated ion channels can be used as non-target antigens.

[0250] In some embodiments, the non-target antigen is selected from the group consisting of leucine-rich repeat neuronal 4 (LRRN4) and uroplakin B3 (UPKB3), or a peptide antigen of either thereof, in a complex with major histocompatibility complex class I (MHC-I). In some embodiments, the non-target antigen is LRRN4 or a peptide antigen thereof in a complex with MHC-I. In some embodiments, the non-target antigen is UPKB3 or a peptide antigen thereof in a complex with MHC-I.

[0251] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in complex with major histocompatibility complex class I (MHC-I).

[0252] Non-target MHC-I (pMHC) antigens comprising any of HLA-A, HLA-B, or HLA-C are contemplated within the scope of this 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.

[0253] Non-target antigens include proteins that are poorly or not expressed at all in cancer cells, e.g., lung cancer cells, but are expressed in normal tissues, such as normal lung tissue.

[0254] In some embodiments, the non-target antigen comprises LRRN4 or an antigenic peptide thereof in complex with MHC-I. Human LRRN4 is described in NCBI Accession No. NP_689824.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, LRRN4 is [ka] The amino acid sequence of

[0255] In some embodiments, LRRN4 comprises a sequence that shares 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% identity with SEQ ID NO: 75. In some embodiments, LRRN4 comprises a sequence identical to SEQ ID NO: 75.

[0256] In some embodiments, the non-target antigen comprises UPK3B or an antigenic peptide thereof in complex with MHC-I. All isoforms of UPK3B are contemplated within the scope of this disclosure. Human UPK3B isoform a precursor is described in NCBI Accession No. NP_085047.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the UPK3B isoform a precursor is [ka] The amino acid sequence of

[0257] The human UPK3B isoform b precursor is described in NCBI accession number NP_872625.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the UPK3B isoform b precursor is [ka] The amino acid sequence of

[0258] Human UPK3B isoform c precursor is described in NCBI accession number NP_872624.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the UPK3B isoform c precursor is [ka] The amino acid sequence of

[0259] The human UPK3B isoform d precursor is described in NCBI accession number NP_001334613.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the UPK3B isoform c precursor is [ka] The amino acid sequence of

[0260] In some embodiments, UPKB3 comprises a sequence or subsequence that shares 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% identity with any one of SEQ ID NOs: 76-79. In some embodiments, UPKB3 comprises a sequence or subsequence identical to SEQ ID NOs: 76-79.

[0261] Inhibitory Chimeric Antigen Receptor 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 to and recognizes a non-target antigen or a peptide derivative thereof in the MHC-I complex.

[0262] As used herein, the term "inhibitory receptor" refers to a ligand-binding domain fused to an intracellular signaling domain that can transduce an inhibitory signal that inhibits or suppresses the immune activity of an immune cell. Inhibitory receptors have immune cell inhibitory potential and are distinct from and distinguishable from CARs, which are receptors that have immune cell activation potential. For example, CARs contain an intracellular stimulatory domain and / or a costimulatory domain, making them activating receptors. Inhibitory receptors are inhibitory receptors that contain an intracellular inhibitory domain.

[0263] As used herein, an "inhibitory signal" refers to a signal transduction or change in protein expression in an immune cell that results in the suppression of an immune response (e.g., reduced cytokine production or reduced immune cell activation). The inhibition or suppression of immune cells can be selective and / or reversible, or it may not be selective and / or reversible.

[0264] The inhibitory receptor of the present disclosure may comprise an extracellular ligand-binding domain. Any type of ligand-binding domain that can regulate the activity of the receptor in a ligand-dependent manner is contemplated 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 to or contacts the inhibitory receptor, the inhibitory receptor is responsive and activates an inhibitory signal in immune cells that express the inhibitory receptor upon binding of the non-target antigen by the inhibitory receptor's extracellular ligand-binding domain.

[0265] Inhibitory receptors of the present disclosure can include an extracellular ligand-binding domain. Any type of ligand-binding domain capable of regulating the activity of the receptor in a ligand-dependent manner is contemplated within the scope of the present disclosure.

[0266] In some embodiments, the ligand binding domain is an antigen binding domain. Exemplary antigen binding domains include, inter alia, scFv, SdAb, Vβ-only domains, and TCR antigen binding domains derived from TCR α and β chain variable domains.

[0267] Any type of antigen binding domain is contemplated within the scope of this disclosure.

[0268] In some embodiments, the extracellular ligand binding domain of the second receptor is an scFv.

[0269] In some embodiments, the extracellular ligand-binding domain of the second receptor binds to 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 antigenic peptides 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 an scFv.

[0270] In some embodiments, the extracellular ligand-binding domain of the second receptor is fused to the extracellular domain of the inhibitory receptor.

[0271] 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, e.g., IgG1. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0272] 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 transmembrane domains of the same or different surface membrane proteins and to minimize interactions with other members of the receptor complex.

[0273] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region may be isolated or derived from (i.e., including at least the transmembrane region thereof) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or an immunoglobulin such as IgG4. Alternatively, the transmembrane domain may be synthetic, in which case it comprises mainly hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular domain of the inhibitory receptor. A glycine-serine doublet provides a particularly suitable linker.

[0274] The present disclosure provides an inhibitory receptor comprising an intracellular domain. The intracellular domain of the inhibitory receptor of the present disclosure is responsible for suppressing activation of an immune cell comprising the inhibitory receptor that would otherwise be activated in response to an activation signal by a first receptor. In some embodiments, the inhibitory intracellular domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor, such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immune inhibitory receptors expressed on the surface of T cells and play an important role in dampening or terminating T cell responses.

[0275] In some embodiments, the inhibitory intracellular domain is isolated from the 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.

[0276] In some embodiments, the inhibitory intracellular domain is isolated from a phosphoprotein membrane anchor having glycosphingolipid microdomain 1 (PAG1). In some embodiments, the inhibitory intracellular domain is isolated from leukocyte immunoglobulin-like receptor B1 (LILRB1).

[0277] 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.

[0278] 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.

[0279] 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 called LIR-1 and LILRB1), programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin-like receptor K1 (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N- and C-terminal SH2 domains), or ZAP70 KI_K369A (kinase-inactive ZAP70).

[0280] In some embodiments, the inhibitory domain is isolated or derived from a human protein.

[0281] 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.

[0282] 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 the transmembrane domain.

[0283] In some embodiments, the second receptor is a TCR comprising an inhibitory domain (an inhibitory TCR). 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 a TCR of TCR alpha, TCR beta, CD3 delta, CD3 gamma, or CD3 epsilon intracellular domain, or a portion thereof. In some embodiments, the inhibitory intracellular domain is fused to a TCR of TCR alpha, TCR beta, CD3 delta, CD3 gamma, or CD3 epsilon transmembrane domain. In some embodiments, the second receptor is a TCR that includes an inhibitory domain (an inhibitory TCR). In some embodiments, the inhibitory domain is isolated or derived from LILRB1.

[0284] LILRB1 inhibitory receptor The present disclosure provides a second inhibitory receptor comprising a LILRB1 inhibitory domain and, optionally, a LILRB1 transmembrane domain and / or hinge domain, or a functional variant thereof. By including the LILRB1 transmembrane domain and / or the LILRB1 hinge domain in an inhibitory receptor, the inhibitory signal generated by the inhibitory receptor may be increased compared to a reference inhibitory receptor having a different transmembrane domain or a different hinge domain. The second inhibitory receptor comprising the LILRB1 inhibitory domain may be a CAR or TCR as described herein. Any suitable ligand binding domain as described herein may be fused to the LILRB1-based second inhibitory receptor.

[0285] 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 are members of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1 protein belongs to the subfamily B class of LIR receptors. These receptors contain 2-4 extracellular immunoglobulin domains, a transmembrane domain, and 2-4 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 stimulation of the immune response. LILRB1 is believed to play a role in regulating inflammatory responses and cytotoxicity, and limiting autoreactivity. There are multiple transcriptional variants that encode different isoforms of LILRB1, all of which are contemplated to be within the scope of the present disclosure.

[0286] 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, 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 or identical to the sequence or subsequence of SEQ ID NO:54. In some embodiments, 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 or identical to the sequence or subsequence of SEQ ID NO:54. In some embodiments, one or more domains of LILRB1 consist of ...

[0287] In some embodiments of a receptor having one or more domains isolated or derived from LILRB1, 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 subsequence of SEQ ID NO:55.

[0288] In some embodiments of a receptor having one or more domains of LILRB1, one or more domains of LILRB1 are encoded by a polynucleotide sequence that is identical to the sequence or subsequence of SEQ ID NO:55.

[0289] In various embodiments, an inhibitory receptor is provided comprising a polypeptide, the polypeptide comprising one or more of 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 inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and SIYATL (SEQ ID NO:59).

[0290] As used herein, "immunoreceptor tyrosine-based inhibitory motif" or "ITIM" refers to a conserved sequence of amino acids with a consensus sequence, such as S / I / V / LxYxxI / V / L (SEQ ID NO: 547), found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-bearing inhibitory receptors interact with their ligands, the ITIM motif becomes phosphorylated, allowing the inhibitory receptors to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or an inositol-phosphatase called SHIP.

[0291] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), at least two ITIMs, at least three ITIMs, at least four ITIMs, at least five ITIMs, or at least six ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0292] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one ITIM selected from the group of ITIMs consisting of NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0293] In further specific embodiments, the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and SIYATL (SEQ ID NO:59).

[0294] In some embodiments, the intracellular domain comprises both the ITIMs NLYAAV (SEQ ID NO:56) and 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.

[0295] In some embodiments, the intracellular domain comprises both the ITIMs VTYAEV (SEQ ID NO:57) and 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.

[0296] In some embodiments, the intracellular domain comprises both the ITIMs VTYAQL (SEQ ID NO:58) and 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.

[0297] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), and 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.

[0298] In some embodiments, the intracellular domain comprises the ITIMs VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and 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.

[0299] In some embodiments, the intracellular domain comprises the ITIMs 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 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.

[0300] 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).

[0301] The LILRB1 intracellular domain or functional variant thereof of the present disclosure can 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 functional variant thereof has 2, 3, 4, 5, or 6 ITIMs.

[0302] In certain embodiments, the intracellular domain comprises two, three, four, five, or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO:56), VTYAEV (SEQ ID NO:57), VTYAQL (SEQ ID NO:58), and SIYATL (SEQ ID NO:59).

[0303] In certain embodiments, the intracellular domain comprises at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0304] In certain embodiments, the intracellular domain comprises three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0305] In certain embodiments, the intracellular domain comprises four immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0306] In certain embodiments, the intracellular domain comprises five immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0307] In certain embodiments, the intracellular domain comprises six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0308] In certain embodiments, the intracellular domain comprises at least seven immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0309] The LILRB1 protein has four immunoglobulin (Ig)-like domains, designated D1, D2, D3, and D4. In some embodiments, the LILRB1 hinge domain comprises a LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1 D3D4 domain comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or identical to, SEQ ID NO:66. In some embodiments, the LILRB1 D3D4 domain comprises or essentially consists of SEQ ID NO:66.

[0310] 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 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.

[0311] 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.

[0312] 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.

[0313] 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 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 at least 95% identical to SEQ ID NO:74. In some embodiments, the LILRB1 transmembrane domain comprises a sequence identical to SEQ ID NO:74. In embodiments, the LILRB1 transmembrane domain consists essentially of a sequence identical to SEQ ID NO:74.

[0314] In some embodiments, the transmembrane domain can be attached to the extracellular region of a second inhibitory receptor, e.g., an antigen-binding domain or a ligand-binding domain, via a hinge, e.g., a hinge derived from a human protein. For example, in some embodiments, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, a CD8a hinge, or a LILRB1 hinge.

[0315] 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.

[0316] Inhibitory receptors containing combinations of LILRB1 domains 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.

[0317] In certain 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, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 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.

[0318] In further 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).

[0319] 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, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 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.

[0320] 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), each ITIM independently selected from LYAAV (SEQ ID NO: 56), VTYAE (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0321] 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.

[0322] In some embodiments, the polypeptide comprises a sequence 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.

[0323] In some embodiments, the polypeptide comprises a sequence 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. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0324] Exemplary inhibitory receptors of the present disclosure comprise an scFv specific for either an HLA-A, HLA-B or HLA-C non-target antigen, the sequence of which is set forth in Table 5, fused N-terminally to the LILRB1 hinge, transmembrane and intracellular domains. 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, a second inhibitory receptor comprises an scFv sequence of Table 5 fused N-terminally to SEQ ID NO: 71.

[0325] As a further example, the non-target antigen comprises HLA-A*02 and the second inhibitory receptor comprises DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTSGGTKLEIKGGGGSGGGGSGGGGSGGQVQLQQSGPELVKPGASVRISCKASGYTFTSYHIHWVKQRPGQGLEWIGWIYPGNVNTEYNEKFKGKATLTADKSSSTAYMHLSSLTSEDSAVYFCAREEITYAMDYWGQGTSVTVSSYGSQSSKPYLLTHPSDPLE In some embodiments, the non-target antigen comprises HLA-A*02 and the second inhibitory receptor comprises the 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.

[0326] Polynucleotides and Vectors The present disclosure provides polynucleotides encoding the sequences of the first and second receptors of the present disclosure.The present disclosure provides immune cells comprising the polynucleotides and vectors described herein.

[0327] In some embodiments, the sequence of the first and / or second receptor is operably linked to a promoter, hi 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.

[0328] The disclosure provides vectors comprising the polynucleotides described herein.

[0329] 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 and / or second vector comprises an shRNA, e.g., a B2M shRNA.

[0330] In some embodiments, both receptors are encoded by a single vector. In some embodiments, the vector comprises an shRNA, e.g., a B2M shRNA.

[0331] In some embodiments, the first and second receptors are encoded by a single vector. Methods of encoding multiple polypeptides using a single vector are known to those of skill in the art and may include, among others, encoding multiple polypeptides under the control of different promoters or, when using a single promoter to control the transcription of multiple polypeptides, the use of 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 (SEQ ID 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 comprise an N-terminal GSG linker. For example, the T2A self-cleaving peptide may also comprise the sequence GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 351), which may be encoded by the sequence GGATCCGGAGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCC (SEQ ID NO: 768).

[0332] In some embodiments, the vector is an expression vector, ie, for expression of the first and / or second receptor in a suitable cell.

[0333] Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, because they allow long-term stable integration of transgenes and their proliferation in daughter cells.Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of low immunogenicity.

[0334] Expression of natural or synthetic nucleic acid encoding a receptor is typically achieved by operably linking the nucleic acid encoding the receptor or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0335] The polynucleotide encoding the receptor can be cloned into several 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.

[0336] Furthermore, the expression vector can be provided to cells, such as immune cells, in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0337] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of interest either 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, lentiviral vectors are used.

[0338] Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 base pairs (bp) upstream of the start site, although many promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. 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, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0339] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation 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. Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0340] To assess receptor expression, the expression vector introduced into the cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the cell population to be transfected or infected via the viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA or used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0341] Reporter genes are used to identify potentially transfected or transduced cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. At a suitable time after the DNA is introduced into the recipient cells, the expression of the reporter gene is assayed. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0342] Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0343] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. 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.

[0344] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0345] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0346] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to an inhibitor of the present disclosure, various assays can be performed to confirm the presence of the recombinant DNA sequence within the host cell. Such assays include "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents falling within the scope of the present disclosure.

[0347] immune cells The present disclosure provides immune cells comprising the receptors, vectors and polynucleotides described herein.

[0348] In some embodiments, the immune cells comprise: (a) a first receptor comprising a first extracellular ligand-binding domain specific for a target antigen selected from (i) a cancer cell-specific antigen in complex with major histocompatibility complex class I (MHC-I), or a peptide antigen thereof, or (ii) MSLN in complex with major histocompatibility complex class I (MHC-I), or a peptide antigen thereof; and (b) a second receptor comprising a second extracellular ligand-binding domain 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 antigenic peptides 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 a TCR.

[0349] The present disclosure provides an immune cell comprising a first receptor comprising a sequence of SEQ ID NO: 303 and a 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 SEQ ID NO: 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 comprising a sequence of SEQ ID NO: 303, a second receptor comprising a sequence of SEQ ID NO: 348, and a sequence encoding an 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 the sequences encoding the first and second receptors are separated by a sequence encoding an autocleaving polypeptide. In some embodiments, the autocleaving polypeptide comprises a T2A autocleaving polypeptide comprising a sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351).

[0350] As used herein, the term "immune cell" refers to a cell involved 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.

[0351] As used herein, "T cells" refers to a type of lymphocyte derived from myeloid precursors that develop in the thymus. There are several different types of T cells that develop upon migration to the thymus, including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells, and stem memory T cells. Different types of T cells can be distinguished by those skilled in the art based on their expression of markers. Methods for distinguishing types of T cells will be readily apparent to those skilled in the art.

[0352] In some embodiments, the first receptor and the second receptor together specifically activate an immune cell in the presence of a target cell.

[0353] In some embodiments, the immune cells are CD4+, CD8+, gamma delta T cells, invariant T cells, iNK cells, NK cells, macrophages, or combinations thereof. In some embodiments, the immune cells are gamma delta (γδ) T cells. In some embodiments, the immune cells are invariant T cells. In some embodiments, the immune cells are invariant natural killer T cells (iNKT cells). In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are B cells. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are CD8-. In some embodiments, the immune cells are CD8+. In some embodiments, the immune cells are CD4+. In some embodiments, the immune cells are CD4-. In some embodiments, the immune cells are CD8- / CD4+. In some embodiments, the immune cells are CD8+CD4- T cells.

[0354] In some embodiments, the immune cells are non-naturally occurring, hi some embodiments, the immune cells are isolated.

[0355] It will be readily apparent to one of skill in the art how to transduce a population of immune cells, such as T cells, using the vectors of the present disclosure. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi) following the manufacturer's instructions. T cells can be cultured at a density of 1x10^6 cells / mL in X-Vivo 15 medium supplemented with 5% human A / B serum and 1% Pen / strep in the presence of CD3 / 28 Dynabeads (1:1 cell to bead ratio) and 300 units / mL IL-2 (Miltenyi). After 2 days, T cells can be transduced with a viral vector, such as a lentiviral vector, using methods known in the art. In some embodiments, the viral vector is transduced at a multiplicity of infection (MOI) of 5. The cells can then be cultured in IL-2 or other cytokines, such as a combination of IL-7 / 15 / 21, for an additional 5 days before enrichment. Methods for isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells, will be readily apparent to those skilled in the art. This method outlines potential approaches, but it should be noted that these methodologies are rapidly evolving. For example, successful viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of expansion to generate a cell population that is more than 99% CD3+ highly transduced.

[0356] Methods for activating and culturing populations of T cells containing a TCR, CAR, inhibitory receptor, receptor or vectors encoding them will be readily apparent to one of skill in the art.

[0357] Whether before or after genetic modification of the T cells to express a TCR, the T cells may be engineered to express a TCR using any of the methods described in, e.g., 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, The cells can be activated and expanded using methods generally described in U.S. Patent Application Publication Nos. 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.

[0358] In some embodiments, the T cells of the present disclosure are expanded and activated in vitro. In general, the T cells of the present disclosure are expanded in vitro by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, a population of T cells can be stimulated as described herein, for example, by contact with an anti-CD3 antibody. For costimulation of an accessory molecule on the surface of the T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate proliferation of either CD4+ T cells or CD8+ T cells. Exemplary anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and can be used as well as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0359] In some embodiments, the primary stimulatory signal and the costimulatory signal for T cells may be provided by different protocols. For example, the agents providing each signal may be in solution or may be bound to a surface. When bound to a surface, the agents may be bound to the same surface (i.e., in a "cis" formation) or to separate surfaces (i.e., in a "trans" formation). Alternatively, one agent may be bound to a surface and the other agent may be in solution. In some embodiments, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents may be in solution. In another embodiment, the agents may be in soluble form and then crosslinked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that binds to the agent. In this regard, see, for example, U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810 for artificial antigen presenting cells (aAPCs) contemplated for use in activating and expanding T cells in the present disclosure.

[0360] 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". As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents are co-immobilized on the same bead with comparable molecular weights. In one embodiment, a 1:1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell proliferation is used. 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 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 disclosure, the ratio of anti-CD28 antibodies to anti-CD3 antibodies bound to the beads is greater than 2:1.

[0361] To stimulate T cells or other target cells, particle to cell ratios of 1:500 to 500:1 and any integer value therebetween may be used. As one of ordinary skill in the art can readily appreciate, the particle to cell ratio may depend on the particle size relative to the target cells. For example, small sized beads may only bind to a few cells, while larger beads may bind to many cells. In certain embodiments, the cell to particle ratio ranges from 1:100 to 100:1 and any integer value therebetween, and in further embodiments, the ratio includes 1:9 to 9:1 and any integer value therebetween, and may also be used to stimulate T cells. In some embodiments, a 1:1 cell to bead ratio is used. One of ordinary skill in the art will appreciate that various other ratios may be suitable for use in the present disclosure. In particular, the ratio will vary depending on the particle size, as well as the size and type of cells.

[0362] In further embodiments of the present disclosure, cells such as T cells are combined with agent-coated beads, followed by separation of the beads and cells, and then culturing the cells. In alternative embodiments, the agent-coated beads and cells are not separated prior to culturing, but are cultured together. In further embodiments, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0363] As an example, cell surface proteins can be ligated by allowing anti-CD3 and anti-CD28 to bind and contact the T cells with paramagnetic beads. In one embodiment, cells (e.g., CD4+ T cells) and beads (e.g., DYNABEADS CD3 / CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer. Again, one of skill in the art can readily appreciate that any cell concentration can be used. In certain embodiments, it is desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, more than 100 million cells / ml is used. In further embodiments, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet other embodiments, concentrations of cells from 75, 80, 85, 90, 95, or 100 million cells / ml are used. In further embodiments, concentrations of 125 or 150 million cells / ml may be used. In some embodiments, concentrations of 1×10 6 Cells cultured at a density of 10 cells / mL are used.

[0364] In some embodiments, the mixture may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value of time units therebetween. In another embodiment, the beads and T cells are cultured together for 2 to 3 days. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine 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 the growth of cells known to those of skill in the art. Other additives for the growth of cells include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in amounts sufficient for T cell growth and expansion. In some embodiments, the media comprises X-VIVO-15 medium supplemented with 5% human A / B serum, 1% penicillin / streptomycin (pen / strep), and 300 units / ml of IL-2 (Miltenyi).

[0365] The T cells are maintained under the necessary conditions to support proliferation, for example, at an appropriate temperature (eg, 37° C.) and atmosphere (eg, air+5% CO 2 ).

[0366] In some embodiments, the T cells comprising the TCR, CAR, and inhibitory receptors of the present disclosure are autologous. Prior to expansion and genetic modification, a source of T cells is obtained from the subject. Immune cells such as T cells can be obtained from a number of 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, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation.

[0367] In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, may lack magnesium, or may lack many, but not all, divalent cations. As one of skill in the art would readily appreciate, the washing steps may be accomplished by methods known to those of skill 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) following the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca2+-free PBS, Mg2+-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0368] In some embodiments, immune cells such as T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugation. Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD4 conjugated beads for a period of time sufficient for positive selection of the desired T cells.

[0369] Enrichment of immune cell populations, such as T cell populations, by negative selection can be achieved by a combination of antibodies directed to surface markers specific to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0370] For isolation of a desired population of immune cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and beads.

[0371] In some embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at either 2-10° C. or at room temperature.

[0372] PBMCs from which immune cells such as stimulatory T cells or T cells are isolated can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogenous product by removing granulocytes, and to some extent monocytes, in the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and the cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled instantaneous freezing at -20°C or in liquid nitrogen, may also be used.

[0373] The present disclosure provides immune cells expressing an activator and / or blocker receptor as described herein, wherein the immune cells have reduced expression and / or function of major histocompatibility (MHC) class I complex.

[0374] In some embodiments, the immune cells are autologous. For example, the immune cells are isolated or derived from the same subject who receives the cells as part of the treatment regimen. The modification of autoimmune cells to reduce MHC class I expression and / or function by blocker receptors can be advantageously specific to MHC class I antigens. Without wishing to be bound by theory, the modification of autoimmune cells to reduce MHC class I expression and / or function reduces the binding of blocker receptors by MHC class I expressed by the immune cells, either in cis or trans.

[0375] In some embodiments, the immune cells are all allogeneic. Allogeneic immune cells may be derived from a donor other than the subject to whom the immune cells are administered. Because allogeneic cells may be prepared and stored for use in subjects of various genotypes, allogeneic immune cells are commonly referred to as "off the shelf" or "universal" in cell therapy.

[0376] Any suitable method of reducing expression and / or function of an MHC class I complex is contemplated within the scope of the present disclosure, including, inter alia, expression of an interfering RNA that knocks down one or more RNAs encoding an MHC class I component, or modification of a gene encoding an MHC class I component.

[0377] Major histocompatibility complex (MHC) is a genetic locus on the vertebrate genome that encodes a set of polypeptides required for the adaptive immune system. Among these are MHC class I polypeptides, including 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 are present in complex with antigens on the surface of cells. As referred to herein, MHC class I genes or polypeptides may refer to any polypeptide found in the MHC or the corresponding gene encoding the polypeptide. In some embodiments, the immune cells of the present disclosure are inactivated by inhibitory ligands, including MHC class I polypeptides, for example, HLA-A, HLA-B, and HLA-C, and their alleles. An 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 an HLA-A*02 protein. Thus, to prevent the autocrine signaling / binding described herein, it is desirable to remove or reduce expression of polypeptides encoded by HLA-A, HLA-B, and HLA-C, and their alleles, in immune cells.

[0378] Immune cells with reduced expression of MHC class I polypeptides In some embodiments, the immune cells described herein are modified to inactivate or reduce or eliminate 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 the 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 contemplated within the scope of the present disclosure. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A*02. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A*02:01. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A*02:01:01. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A*02:01:01:01.

[0379] In some embodiments, the engineered immune cells described herein are modified to reduce or eliminate expression of the B2M gene product. The beta-2 microglobulin (B2M) gene encodes a protein that associates with the major histocompatibility complex (MHC) class I, i.e., the MHC-I complex. The MHC-I complex is required for the presentation of antigens on the cell surface. The MHC-I complex is disrupted and non-functional when B2M is deleted (Wang D et al. Stem Cells Transl Med. 4:1234-1245 (2015)). Furthermore, the B2M gene can be disrupted with high efficiency using gene editing techniques known in the art (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.

[0380] The present disclosure provides a gene editing system for editing endogenous target genes in immune cells. The present disclosure provides an interfering RNA specific to the sequence of the target gene. Gene editing systems such as CRISPR / Cas system, TALEN and zinc finger 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 broken ends or improper end joining can be used to introduce deletions. In some embodiments, the target gene comprises a gene encoding a subunit of the MHC-I complex.

[0381] Target gene sequences include, but are not limited to, promoters, enhancers, introns, exons, intron / exon junctions, transcripts (pre-mRNA, mRNA, and splice variants), and / or 3' and 5' untranslated regions (UTRs). Any genetic element or combination of genetic elements can be targeted for the purposes of gene editing in immune cells as described herein. Modifications to target genes can be achieved by editing the target gene, resulting in alteration or disruption of expression or function of the target gene or gene product, using any method known in the art.

[0382] In some embodiments, modifying a gene encoding an MHC class I polypeptide comprises deleting all or a portion 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, an insertion, a substitution, or a frameshift mutation. In some embodiments, modifying the gene comprises using a nucleic acid-guided endonuclease.

[0383] Gene sequences for the target genes described herein are known in the art. The sequences can be found in public databases such as NCBI GenBank or the NCBI nucleotide database. The sequences can be found using gene identifiers, for example, the HLA-A gene has NCBI gene ID: 3105, the HLA-B gene has NCBI gene ID: 3106, the HLA-C gene has NCBI gene ID: 3107, and the B2M gene has NCBI gene ID: 567 and 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 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 as described herein. [Table 10]

[0384] One of skill in the art will understand 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 in the present disclosure.

[0385] In some embodiments, the target gene is edited in the immune cells described herein using a nucleic acid-guided endonuclease. Exemplary nucleic acid-guided endonucleases include class II endonucleases such as CRISPR / Cas9.

[0386] As used herein, "CRISPR" or "CRISPR gene editing" refers to a set of clustered regularly interspaced short palindromic repeats or a system that includes a set of such repeats. "Cas" as used herein refers to a CRISPR-associated protein. "CRISPR / Cas" system refers to a system derived from CRISPR and Cas that can be used to silence, knock out, or mutate target genes. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR / Cas systems have been modified for use in gene editing. This is accomplished by introducing into a eukaryotic cell one or more specifically designed guide nucleic acids (gNAs), typically guide RNAs (gRNAs), and an appropriate Cas endonuclease that forms a ribonucleoprotein complex with the gNA. The gNA guides the gNA-endonuclease protein complex to the target genomic location, and the endonuclease introduces a strand break at the target genomic location. This strand break can be repaired by cellular mechanisms such as non-homologous end joining (leading to deletions) or homologous repair (which can generate insertions), thereby introducing the genetic modification into the host cell genome.

[0387] CRISPR / Cas systems are classified into classes and types. Class 2 systems represent a single interfering protein that is currently classified into three different types (type II, type V, and type VI). Any class 2 CRISPR / Cas system suitable for gene editing, such as type II, type V, or type VI systems, is contemplated 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.

[0388] CRISPR sequence, sometimes called CRISPR locus, comprises alternating repeats and spacers. In naturally occurring CRISPR, the spacer usually comprises a sequence foreign to bacteria, such as a plasmid or phage sequence. As described herein, the spacer sequence may also be referred to as "targeting sequence". In the CRISPR / Cas system for genetic manipulation, the spacer is derived from the target gene sequence (gNA).

[0389] Exemplary class 2 type II CRISPR system relies on 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 a system for gene editing. Pennisi (2013) Science 341:833-836. In some embodiments, the Cas protein used to modify immune cells is Cas9.

[0390] Thus, CRISPR / Cas system can be used to edit target genes, such as the genes targeted for editing in immune cells described herein, by adding or deleting base pairs, or by introducing early stops, thereby reducing the expression of the target. Alternatively, CRISPR / Cas system can be used like RNA interference to reversibly turn off target genes. In mammalian cells, for example, RNA can guide Cas protein to target gene promoters and sterically block RNA polymerase.

[0391] The Cas protein may be derived from any bacterial or archaeal Cas protein. Any suitable CRISPR / Cas system is contemplated within the scope of the present disclosure. In other embodiments, the Cas protein is selected from the group consisting 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, The Cas protein may comprise one or more of 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, Cas5, Cas7, Cas8, Cas10, or a combination or complex thereof. In some embodiments, the Cas protein is a Cas9 protein.

[0392] The artificial CRISPR / Cas system that inhibits target genes can be generated using techniques known in the art, such as those described in US Publication No. 2014 / 0068797 and Cong (2013) Science 339:819-823. For example, those 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, but other artificial CRISPR / Cas systems that inhibit target genes known in the art can also be generated. Methods for designing suitable gNAs for specific Cas proteins will be known to those skilled in the art.

[0393] The present disclosure provides gene-targeting guide nucleic acids (gNAs) that can guide the activity of an associated polypeptide (e.g., a nucleic acid-guided endonuclease) to a specific target gene sequence within a target nucleic acid genome. The genome-targeting nucleic acid can be an RNA. The genome-targeting RNA is referred to herein as a "guide RNA" or "gRNA." The guide RNA can include at least a targeting sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In some type II systems, the gRNA also includes a second RNA, called a tracrRNA sequence, also referred to herein as a "scaffold" sequence. In type II guide RNAs (gRNAs), the CRISPR repeat sequence and the scaffold sequence hybridize to each other to form a duplex. In type V guide RNAs (gRNAs), the crRNA forms a duplex. In both systems, the duplex can bind to a site-directed polypeptide such that the guide RNA and the site-directed polypeptide form a complex. The gene-targeting nucleic acid can provide target specificity to the complex by association with the site-directed polypeptide. Thus, the gene targeting nucleic acid can direct the activity of a site-specific polypeptide.

[0394] 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.

[0395] Exemplary guide RNAs include a targeting sequence of about 15-20 bases. As will be appreciated by those skilled in the art, each gRNA can be designed to include a targeting sequence complementary to its genomic target sequence. For example, each of the targeting sequences, e.g., the RNA versions of the DNA sequences shown in Tables 11 and 14, minus the three 3' nucleotides representing their PAM sites, can be placed into a single RNA chimera or crRNA.

[0396] The gene targeting nucleic acid may be a bimolecular guide RNA.The gene targeting nucleic acid may be a single-molecule guide RNA.The gene targeting nucleic acid may be any known configuration of guide RNA known in the art, including, for example, paired gRNA, or multiple gRNAs used in a single step.The coding sequence and splice junction are clear from the genome sequence, but other features required for gene expression may be specific and unclear.

[0397] The bimolecular guide RNA can comprise two strands of RNA. The first strand comprises a 5' to 3' sequence, an optional spacer extension sequence, a targeting sequence, and a minimal CRISPR repeat sequence. The second strand can comprise a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3'tracrRNA sequence, and an optional tracrRNA extension sequence.

[0398] The single molecule guide RNA (sgRNA) in the type II system can include, from 5' to 3', an optional spacer extension sequence, a targeting sequence, a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension can include elements that contribute additional functionality (e.g., stability) to the guide RNA. The single molecule guide linker can link the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can include one or more hairpins.

[0399] In some embodiments, the guide RNA or single molecule guide RNA (sgRNA) can include a targeting sequence and a scaffold sequence. In some embodiments, the scaffold sequence is a Cas9 gRNA sequence. In some embodiments, the scaffold sequence is encoded by a DNA sequence comprising a sequence that shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 773). In some embodiments, the scaffold sequence is encoded by a DNA sequence comprising GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 773).

[0400] In some embodiments, for example those embodiments where 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 17-30 nucleotides at the 5' end of the sgRNA sequence.

[0401] Suitable scaffold sequences, and the location of the scaffold target sequence, will depend on the choice of endonuclease and will be known to one of skill in the art.

[0402] A single molecule guide RNA (sgRNA) in a Type II system, such as Cas9, can comprise, from 5' to 3', a minimal CRISPR repeat sequence and a targeting sequence.

[0403] By way of example, guide RNAs or other smaller RNAs used in the CRISPR / Cas9 or CRISPR / Cpf1 system can be easily synthesized by chemical means, as exemplified below and described in the art. While chemical synthesis procedures are continually expanding, as polynucleotide lengths increase significantly beyond about 100 nucleotides, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult. One approach used to generate longer RNAs is to produce two or more molecules that are ligated together. Much longer RNAs, such as those that code for Cas9 or Cpf1 endonucleases, are more easily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic generation of RNA, for example, modifications that improve stability, reduce the likelihood or extent of innate immune responses, and / or improve other attributes, as described in the art.

[0404] The targeting sequence of gRNA hybridizes to a sequence in the target nucleic acid of interest. The targeting sequence of genome targeting nucleic acid can interact with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The nucleotide sequence of the targeting sequence can vary depending on the sequence of the target nucleic acid of interest.

[0405] In the Cas9 system described herein, the targeting sequence can be designed to hybridize to the target nucleic acid located 5' of the reverse complement of the PAM of the Cas9 enzyme used in the system. The targeting sequence can be a perfect match to the target sequence or can have a mismatch. Each CRISPR / Cas system protein may have a specific PAM sequence in a specific orientation and position that is recognized in the target DNA. For example, S. pyogenes Cas9 recognizes a PAM in the target nucleic acid that contains the sequence 5'-NRG-3', where R contains either A or G, and N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the targeting sequence. The selection of an appropriate PAM sequence will be apparent to one of skill in the art.

[0406] The target sequence is complementary to and hybridizes with the targeting sequence of the gRNA. The target nucleic acid sequence can include 20 nucleotides. The target nucleic acid can include less than 20 nucleotides. The target nucleic acid can include more than 20 nucleotides. The target nucleic acid can include at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, for example, in those embodiments where the CRISPR / Cas system is a Cas9 system, the target nucleic acid sequence can include 20 nucleotides immediately 5' of the first nucleotide of the reverse complement of the PAM sequence. This target nucleic acid sequence is often referred to as the PAM strand or target strand, and the complementary nucleic acid sequence is often referred to as the non-PAM strand or non-target strand. Those skilled in the art will recognize that the targeting sequence hybridizes to the non-PAM strand of the target nucleic acid. See, for example, US2019 / 0185849A1.

[0407] In some instances, 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 instances, the percent complementarity between the targeting sequence and the target nucleic acid is up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%. In some examples, the percent complementarity between the targeting sequence and the target nucleic acid is 100% over the six contiguous 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 contiguous nucleotides. The length of the targeting sequence and the target nucleic acid can vary by 1-6 nucleotides, which can be considered a bulge or multiple bulges.

[0408] The targeting sequence can be designed or selected using computer programs known to those skilled in the art. The computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic occurrence frequency (e.g., of sequences that are identical or similar but vary in one or more spots as a result of mismatches, insertions, or deletions), methylation status, presence of SNPs, etc. Available computer programs can take as input NCBI gene IDs, official gene symbols, Ensembl Gene IDs, genomic coordinates, or DNA sequences, and create an output file that includes sgRNAs that target the appropriate genomic regions specified as input. The computer program can also provide summary statistics and scores indicating on-target and off-target binding of the sgRNA for the target gene (Doench et al. Nat Biotechnol. 34:184-191 (2016)). The present disclosure provides a guide RNA that includes a targeting sequence. In some embodiments, the guide RNA further includes a guide RNA scaffold sequence. In some embodiments, the targeting sequence is complementary to a sequence of a target gene selected from the group consisting of HLA-A, HLA-B, HLA-C, B2M, or an allele thereof. In some embodiments, the target gene is an HLA-A gene. In some embodiments, the target gene is an HLA-B gene. In some embodiments, the target gene is an 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 that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity with or is identical to a sequence disclosed in Tables 11 and 14.

[0409] In some embodiments, the gNA specifically targets a sequence at the endogenous HLA-A locus. In some embodiments, the gNA specifically targets a sequence at the HLA-A locus comprises a sequence that shares 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 targets a sequence at the HLA-A locus comprises a sequence selected from the sequences disclosed in Table 11.

[0410] In some embodiments, the gNA specifically targets a sequence of the HLA-A*02 allele. For example, the gRNA specifically targets and hybridizes to a sequence shared by all HLA-A*02 alleles, but not shared by HLA-A*02 and HLA-A*03 alleles. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele.

[0411] In some embodiments, the gNA specifically targets the coding DNA sequence of HLA-A*02.

[0412] In some embodiments, the gNA specifically targets a coding DNA sequence shared by more than 1000 HLA-A*02 alleles. In some embodiments, the gNA specifically targets a coding DNA sequence in more than 1000 HLA-A*02 alleles comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity with or is identical to a sequence selected from the sequences set forth in Table 11.

[0413] The sequences in Tables 11-14 are presented as DNA sequences. One of skill in the art will understand that thymine (T) can be substituted for uracil (U) in any DNA sequence, including those listed in Tables 11-14, to arrive at the corresponding RNA sequence. [Table 11-1] [Table 11-2]

[0414] The sequences disclosed in Table 11 include the corresponding genomic sequences, including the PAM sequence. One of skill in the art will understand that the targeting sequence of a gRNA does not include the three 3' terminal nucleotides of the sequences in Table 11, which represent the corresponding PAM site for the gRNA.

[0415] The present disclosure provides a gNA comprising a targeting sequence 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 that targets the B2M gene promoter sequence.

[0416] In some embodiments, the gNA comprises a targeting sequence and a gNA scaffold sequence. In some embodiments, the targeting sequence comprises a sequence set forth in Table 12, or a sequence sharing about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity therewith.

[0417] In some embodiments, the targeting sequence is complementary to a sequence of the B2M gene. In some embodiments, the B2M gene comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity with the B2M sequence shown in Table 10. [Table 12-1] [Table 12-2]

[0418] In some embodiments, the immune cells described herein are edited using TALEN gene editing.

[0419] "TALEN" or "TALEN gene editing" refers to Transcription Activator-Like Effector Nucleases, artificial nucleases used to edit target genes.

[0420] TALENs are artificially produced by fusing a TAL effector DNA binding domain to a 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. The engineered TALE can be combined with a DNA cleavage domain to produce a restriction enzyme specific to any desired DNA sequence, including a target gene sequence. These can then be introduced into cells and used for genome editing.

[0421] To produce a TALEN, a TALE protein is fused to a nuclease (N), which is a wild-type or mutant Fold endonuclease. Several mutations to FokI have been made for use in TALENs, which improve, for example, cleavage specificity or activity.

[0422] The FokI domain functions as a dimer, requiring two constructs with their own DNA binding domains to be properly oriented and spaced to a site in the target genome. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain, and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity.

[0423] TALENs specific for sequences within a target gene can be constructed using any method known in the art, including a variety of schemes that use modular components.

[0424] In some embodiments, target genes are edited in immune cells described herein using ZFN gene editing.

[0425] "ZFN" or "zinc finger nuclease" or "ZFN gene editing" refers to zinc finger nucleases, which are artificial nucleases that can be used to edit target genes.

[0426] Similar to TALENs, ZFNs contain a Fold nuclease domain (or a derivative thereof) fused to a DNA-binding domain, which in the case of ZFNs contains one or more zinc fingers.

[0427] Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. Zinc fingers can, for example, contain Cys2His2 and can recognize approximately 3 bp sequences. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize approximately 6, 9, 12, 15, or 18 bp sequences. A variety of selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid system, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0428] Like TALENs, ZFNs must dimerize to cleave DNA. Thus, a pair of ZFNs is required to target non-palindromic DNA sites. Two individual ZFNs must bind to opposite strands of DNA with their nucleases appropriately spaced apart.

[0429] Also, like TALENs, ZFNs can create double-stranded breaks in DNA, which, if improperly repaired, can create frameshift mutations, resulting in reduced expression and amount of target genes or gene products in cells.ZFNs can also be used in conjunction with homologous recombination to make mutations in target genes.

[0430] ZFNs specific for a sequence within a target gene can be constructed using any method known in the art.

[0431] In some embodiments, the expression and function of one or more MCH-I components are reduced using RNA interference. "RNAi" or "RNA interference" refers to the process of sequence-specific post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA). Double-stranded RNAs, such as siRNA (small interfering RNA), miRNA (microRNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeat-associated siRNA), and modified forms thereof, can all mediate RNA interference. These dsRNA molecules can be commercially available or can be designed and prepared based on known sequence information. The antisense strand of these molecules can include RNA, DNA, PNA, or combinations thereof. DNA / RNA chimeric polynucleotides include, but are not limited to, double-stranded polynucleotides consisting of DNA and RNA that inhibit the expression of a target gene. The dsRNA molecule can also include one or more modified nucleotides, as described herein, which can be incorporated into either or both strands.

[0432] In RNAi gene silencing or knockdown, dsRNA is introduced into an organism, comprising a first (antisense) strand that is complementary to a portion of a target gene, and a second (sense) strand that is fully or partially complementary to the first antisense strand. After being introduced into an organism, the target gene-specific dsRNA is processed into relatively small fragments (siRNAs), which are then distributed throughout the organism, reducing the messenger RNA of the target gene and resulting in a phenotype that can be very similar to the phenotype resulting from complete or partial deletion of the target gene.

[0433] Certain dsRNA in cells can be acted upon by the Dicer enzyme, which is a ribonuclease III enzyme. Dicer can process dsRNA into shorter pieces of dsRNA, namely siRNA. RNAi also involves an endonuclease complex known as the RNA-induced silencing complex (RISC). After cleavage by Dicer, siRNA enters the RISC complex and directly cleaves the single-stranded RNA target that has a sequence complementary to the antisense strand of the siRNA duplex. The other strand of the siRNA is the passenger strand. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex. Thus, siRNA can downregulate or knock down gene expression by mediating RNA interference in a sequence-specific manner.

[0434] As used herein with respect to RNA interference, a "target gene" or a "target sequence" refers to a gene or gene sequence whose corresponding RNA is targeted for degradation via the RNAi pathway using dsRNA or siRNA as described herein. Exemplary target gene sequences are shown in Table 10. For example, to target a gene using siRNA, the siRNA comprises an antisense region that is complementary or substantially complementary to at least a portion of the target gene or sequence, and a sense strand that is complementary to the antisense strand. Once introduced into a cell, the siRNA directs the RISC complex to cleave the RNA that contains the target sequence, thereby degrading the RNA. The present disclosure provides an interfering RNA. The double-stranded RNA molecule of the present disclosure may be in the form of any type of RNA interference molecule 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 that is processed in the cell to produce an siRNA. In other embodiments, the double-stranded RNA molecule is a part of a microRNA precursor molecule.

[0435] In some embodiments, the shRNA is of a length suitable as a Dicer substrate and can be processed to produce a RISC-active siRNA molecule. See, e.g., Rossi et al., US2005 / 0244858.

[0436] A Dicer substrate double-stranded RNA (e.g., shRNA) can be of sufficient length to be processed by Dicer to produce an active siRNA and can further include one or more of the following properties: (i) a Dicer substrate shRNA can be asymmetric, e.g., with a 3' overhang on the antisense strand, (ii) a 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, e.g., incorporation of one or more DNA nucleotides, and (iii) the first and second strands of a Dicer substrate dsRNA can be 21-30 bp in length.

[0437] In some embodiments, the interfering RNA comprises a sequence complementary to a sequence of B2M mRNA. In some embodiments, the interfering RNA can induce RNAi-mediated degradation of B2M mRNA. In some embodiments, the B2M mRNA sequence comprises a coding sequence. In some embodiments, the B2M mRNA sequence comprises an untranslated region.

[0438] In some embodiments, the interfering RNA comprises a sequence complementary to the sequence of HLA-A*02 mRNA. In some embodiments, the interfering RNA can induce RNAi-mediated degradation of HLA-A*02 mRNA. In some embodiments, the HLA-A*02 mRNA sequence comprises a coding sequence. In some embodiments, the HLA-A*02 mRNA sequence comprises an untranslated region.

[0439] In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises a first sequence having a sequence complementary to B2M mRNA at its 5' to 3' end, and a second sequence having a sequence complementary to the first sequence at its 5' to 3' end, and the first sequence and the second sequence form the shRNA.

[0440] In some embodiments, the first sequence is 18, 19, 20, 21, or 22 nucleotides. In some embodiments, the first sequence is complementary to a sequence selected from the sequences shown in Tables 13 and 14. In some embodiments, the first sequence has a GC content of 25% or more and less than 60%. In some embodiments, the first sequence is complementary to a sequence selected from the sequences shown in Tables 13 and 14. In some embodiments, the first sequence does not contain four nucleotides of the same base, or a run of seven C or G nucleotide bases. In some embodiments, the first sequence is 21 nucleotides.

[0441] Exemplary target B2M sequences complementary to the first sequence are shown in Table 13.

[0442] In some cases, the first sequence may have 100% identity, i.e., complete identity, homology, complementarity, with the target nucleic acid sequence.In other cases, there may be one or more mismatches between the first sequence and the target nucleic acid sequence.For example, there may be 1, 2, 3, 4, 5, 6, or 7 mismatches between the sense region and the target nucleic acid sequence.

[0443] The sequences shown in Table 13 are presented as DNA sequences. In all sequences shown in Table 13, thymine (T) can be replaced by uracil (U) to arrive at the sequence of the target mRNA sequence. [Table 13-1] [Table 13-2] [Table 13-3]

[0444] Exemplary sequences encoding B2M shRNAs include the sequence GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 349), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. Further exemplary sequences encoding B2M shRNAs include the sequence GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 350), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0445] In some embodiments, the interfering RNA comprises a sequence complementary to the sequence of HLA-A*02 mRNA. In some embodiments, the interfering RNA can induce RNAi-mediated degradation of HLA-A*02 mRNA. In some embodiments, the HLA-A*02 mRNA sequence comprises a coding sequence. In some embodiments, the HLA-A*02 mRNA sequence comprises an untranslated region.

[0446] In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises a first sequence having a sequence complementary to HLA-A*02 mRNA at its 5' to 3' end and a second sequence having a sequence complementary to the first sequence at its 5' to 3' end, wherein the first sequence and the second sequence form the shRNA.

[0447] Exemplary target HLA sequences complementary to the first sequence are shown in Table 14. [Table 14-1] [Table 14-2]

[0448] In some embodiments, the first and second sequences are separated by a linker, sometimes referred to as a loop. In some embodiments, both the first and second sequences are encoded by one single-stranded RNA or DNA vector. In some embodiments, the loop is between the first and second sequences. In these embodiments, the first and second sequences hybridize to form a double-stranded region. The first and second sequences are linked by a linker sequence to form a "hairpin" or "stem-loop" structure. The shRNA can have a complementary first and second sequence at opposite ends of a single-stranded molecule, such that the molecule can form a double-stranded region with the complementary sequence portion, and the strands are linked at one end of the double-stranded region by a linker (i.e., loop sequence). The linker, or loop sequence, can be either a nucleotide or a non-nucleotide linker. The linker can interact with the first sequence, and optionally the second sequence, via covalent or non-covalent interactions.

[0449] Any suitable nucleotide loop sequence is contemplated within the scope of the present disclosure. ...

Claims

[Claim 1] The invention described in the specification.

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