Compositions and methods for treating mesothelin-positive cancer

Abstract

The present disclosure provides immune cells comprising a first activating receptor specific for mesothelin and a second inhibitory receptor specific for a ligand that is lost in mesothelin-positive cancer cells, and methods of making and using said immune cells for the treatment of cancer. Provided herein are compositions and methods related to the treatment of MSLN(+) cancer. Advantageously, the compositions and methods disclosed herein can take advantage of loss of heterozygosity (LOH) to address MSLN(+) cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 304,409, filed January 28, 2022. The contents of the foregoing application are expressly incorporated herein by reference in their entirety, including any drawings.

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

[0003] background 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 the target cells. Identifying suitable target molecules remains challenging because many targets are expressed in normal tissues. This expression can cause 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.

[0004] Mesothelin (MSLN) was proposed as a cancer target in 1992 (Chang et al. Cancer Res 52:181-86), but no viable treatments utilizing MSLN exist. 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 present only in the mesothelium, and this tissue itself may not be essential. Several investigational therapeutics targeting MSLN, such as immunotoxin conjugates, antibody-drug conjugates, bispecific antibodies, CAR-T, and hybrid TCR-scFv, are being tested. All effective systemically administered therapeutic agents have been toxic. Thus, there is a need in the art for compositions and methods related to the treatment of MSLN(+) cancer. [Prior art documents] [Non-patent literature]

[0005] [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]

[0006] overview Provided herein are compositions and methods related to the treatment of MSLN(+) cancer. Advantageously, the compositions and methods disclosed herein can address MSLN(+) cancer by exploiting loss of heterozygosity (LOH). In some cases, the compositions and methods disclosed herein can avoid systemic toxicity to normal tissues by pairing MSLN-targeted activator receptors with blocker receptors. Without being bound by theory, differences in blocker antigen expression in tumor tissue versus normal tissue caused by LOH at the locus encoding the blocker antigen can confer high selectivity for tumor killing.

[0007] The present disclosure provides an immune cell comprising: (a) a first receptor comprising an extracellular ligand-binding domain specific for mesothelin (MSLN); and (b) an HLA-A * and a second receptor comprising an extracellular ligand-binding domain specific for HLA-A 03, wherein the first receptor is an activator receptor that responds to MSLN and the second receptor is an HLA-A 03-specific activator receptor. * 03 responsive inhibitory receptors, providing immune cells.

[0008] In some embodiments of the immune cells of the present disclosure, HLA-A * 03 is lost in MSLN+ cancer cells by loss of heterozygosity.

[0009] In some embodiments, the extracellular ligand-binding domain of the second receptor comprises complementarity determining region (CDR) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or a CDR sequence having up to 1, 2, or 3 substitutions, deletions, or insertions compared to a CDR of Table 6 or Table 7. In some embodiments, the extracellular ligand-binding domain of the second receptor is selected from the group consisting of: (i) SEQ ID NOs: 638, 645, 650, 657, 676, and 693; (ii) SEQ ID NOs: 638, 645, 650, 658, 677, and 694; (iii) SEQ ID NOs: 638, 645, 650, 659, 678, and 695; (iv) SEQ ID NOs: 638, 645, 650, 660, 678, and 696; (v) SEQ ID NOs: 638, 645, 650, 661, 679, and 697; (vi) SEQ ID NO: 6 39, 646, 651, 657, 676, and 698, (vii) SEQ ID NOs: 638, 645, 650, 657, 676, and 699, (viii) SEQ ID NOs: 639, 646, 651, 657, 676, and 700, (ix) SEQ ID NOs: 638, 645, 650, 662, 680, and 701, (x) SEQ ID NOs: 639, 646, 651, 657, 676, and 702, (xi) SEQ ID NOs: 638, 645, 650, 661, 679, and 703, (xii) SEQ ID NOs: 640, 647, 65 (xiii) SEQ ID NOs: 641, 648, 653, 663, 681 and 705, (xiv) SEQ ID NOs: 638, 645, 650, 664, 682 and 706, or (xv) the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 1260 to 1265; or (i) SEQ ID NOs: 638, 645, 650, 657, 676 and 693, (ii) SEQ ID NOs: 638, 645 , 650, 658, 677 and 694, (iii) SEQ ID NOs: 638, 645, 650, 659, 678 and 695, (iv) SEQ ID NOs: 638, 645, 650, 660, 678 and 696, (v) SEQ ID NOs: 638, 645, 650, 661, 679 and 697, (vi) SEQ ID NOs: 639, 646, 651, 657, 676 and 698, (vii) SEQ ID NOs: 638, 645, 650, 657, 676 and 699, (viii) SEQ ID NOs: 639, 646, 651, 657,676 and 700, (ix) SEQ ID NOs: 638, 645, 650, 662, 680 and 701, (x) SEQ ID NOs: 639, 646, 651, 657, 676 and 702, (xi) SEQ ID NOs: 638, 645, 650, 661, 679 and 703, (xii) SEQ ID NOs: 640, 647, 652, 657, 676 and 704, (xiii) SEQ ID NOs: 641, 648, 653, 663, 681 and 705, (xiv) SEQ ID NOs: 638, 645, 650, 664, 682 and 706, or (xv) CDR sequences having up to one, two or three substitutions, deletions or insertions compared to the CDRs of SEQ ID NOs: 1260-1265. In some embodiments, the extracellular ligand-binding domain of the second receptor is selected from the group consisting of: (i) SEQ ID NOs: 638, 645, 650, 657, 676, and 693; (ii) SEQ ID NOs: 638, 645, 650, 658, 677, and 694; (iii) SEQ ID NOs: 638, 645, 650, 659, 678, and 695; (iv) SEQ ID NOs: 638, 645, 650, 660, 678, and 696; (v) SEQ ID NOs: 638, 645, 650, 661, 679, and 697; (vi) SEQ ID NOs: 639, 646, 651, 657, 676, and 698; (vii) SEQ ID NOs: 638, 645, 650, 657, 676, and 699; (viii) SEQ ID NO: 639; (ix) SEQ ID NOs: 638, 645, 650, 662, 680 and 701; (x) SEQ ID NOs: 639, 646, 651, 657, 676 and 702; (xi) SEQ ID NOs: 638, 645, 650, 661, 679 and 703; (xii) SEQ ID NOs: 640, 647, 652, 657, 676 and 704; (xiii) SEQ ID NOs: 641, 648, 653, 663, 681 and 705; or (xiv) SEQ ID NOs: 638, 645, 650, 664, 682 and 706. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises complementarity-determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 1260-1265. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises a polypeptide sequence disclosed in Table 5,or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises any one of SEQ ID NOs: 615-628 or SEQ ID NO: 1259, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises any one of SEQ ID NOs: 615-628. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises SEQ ID NO: 1259.

[0010] 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 one, two, or three 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 set forth in Table 3 and a light chain variable (VL) portion comprising a sequence set forth 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 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 the scFv sequence of SEQ ID NO: 171, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0011] 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 the sequence of SEQ ID NO: 7, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 11, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. 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 the 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 the 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.

[0012] 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 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 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 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 of these, or a combination thereof. In some embodiments, the LILRB1 hinge domain, the LILRB1 intracellular domain, and the LILRB1 transmembrane domain comprise SEQ ID NO: 71, or a sequence at least 90%, at least 95%, at least 97%, or at least 99% identical to, or identical to, SEQ ID NO: 71. In some embodiments, the second receptor comprises the sequence of SEQ ID NO: 1268, or a sequence at least 90%, at least 95%, at least 97%, or at least 99% identical thereto.

[0013] 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.

[0014] 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 cancer.

[0015] In some embodiments of the immune cells of the present disclosure, the MSLN+ cancer cells are HLA-A * MSLN+ / HLA-A not expressing 03 * 03-Cancer cells. In some embodiments, MSLN+ / HLA-A * 03-Cancer cells have HLA-A * MSLN+ / HLA-A due to loss of heterozygosity at HLA-A, resulting in loss of HLA-A3 * In some embodiments, the first receptor and the second receptor are both MSLN+ / HLA-A cells with loss of heterozygosity. * 03—Specifically activates immune cells in the presence of cancer cells. In some embodiments, the first receptor and the second receptor both bind to HLA-A by loss of heterozygosity. * The presence of MSLN+ cells that have not lost O3 does not specifically activate immune cells.

[0016] In some embodiments of the immune cells of the present disclosure, the immune cells are T cells. In some embodiments, the T cells are CD8+CD4- T cells or CD8-CD4+ T cells.

[0017] 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 cell further comprises an interfering RNA, wherein 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 is capable of inducing 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' to 3' end, and (b) a second sequence having a sequence complementary to the first sequence from its 5' to 3' end, wherein the first sequence and the second sequence form the 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.

[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 comprise one or more modifications to a sequence encoding B2M, wherein the one or more modifications reduce expression and / or eliminate function of B2M. In some embodiments, the one or more modifications comprise one or more inactivating mutations in an endogenous gene encoding B2M. In some embodiments, the one or more inactivating mutations comprise a deletion, insertion, substitution, or frameshift mutation. In some embodiments, the one or more inactivating mutations are introduced by a nucleic acid-guided endonuclease in complex with at least one guide. In some embodiments, the at least one guide is a guide nucleic acid (gNA) that specifically targets a sequence in the endogenous gene encoding B2M. In some embodiments, the gNA comprises a sequence selected from the group of sequences set forth in Table 12, or a sequence having up to 1, 2, 3, or 4 substitutions, insertions, or deletions thereto.

[0019] In some embodiments of the immune cells of the present disclosure, the expression and / or function of MHC class I genes is reduced or eliminated. In some embodiments, the MHC class I genes are HLA-A * In some embodiments, the immune cells are HLA-A * In some embodiments, the interfering RNA comprises a polynucleotide that is complementary to the sequence of an HLA-A mRNA. * 03 mRNA. In some embodiments, the interfering RNA can induce RNA interference (RNAi)-mediated degradation of (a) HLA-A *03 is a short hairpin RNA (shRNA) comprising: (a) a first sequence having a sequence complementary to a sequence of mRNA from the 5' end to the 3' end; and (b) a second sequence having a sequence complementary to the first sequence from the 5' end to the 3' end, wherein the first sequence and the second sequence form an shRNA. In some embodiments, the shRNA comprises the sequence shown in 14. In some embodiments, the immune cells are HLA-A * and one or more modifications to the sequence of the endogenous gene encoding HLA-A 03, wherein the one or more modifications are * Reduce expression of HLA-A3 and / or * In some embodiments, the one or more modifications remove the function of HLA-A 03. * In some embodiments, the one or more inactivating mutations are in the endogenous gene encoding HLA-A 03. * The nucleotide sequence of the endogenous gene encoding 03 is introduced by a nucleic acid-guided endonuclease in a complex with at least one guide nucleic acid (gNA) that specifically targets the sequence of the endogenous gene encoding 03. In some embodiments, the gNA comprises a sequence shown in Table 11.

[0020] In some embodiments of the immune cells of the present disclosure, the first receptor comprises the sequence of SEQ ID NO: 164, and the second receptor comprises the sequence of SEQ ID NO: 1259, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the immune 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 a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351).

[0021] In some embodiments of the immune cells of the present disclosure, the immune cells are autologous.

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

[0023] The present disclosure provides pharmaceutical compositions comprising a therapeutically effective amount of the immune cells of the present disclosure. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0024] 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.

[0025] The present disclosure provides a polynucleotide or polynucleotide system, comprising: (a) a first receptor comprising an extracellular ligand-binding domain specific for mesothelin (MSLN); and (b) an HLA-A* and one or more polynucleotides comprising a polynucleotide sequence encoding a second receptor comprising an extracellular ligand-binding domain specific for HLA-A 03, wherein the first receptor is an activator receptor responsive to MSLN on MSLN+ cancer cells, and the second receptor is an activator receptor responsive to HLA-A 03. * The present invention provides a polynucleotide or polynucleotide system that is an inhibitory receptor that responds to .O3.

[0026] 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 the 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 the polypeptide of SEQ ID NO: 1268, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

[0027] The present disclosure provides vectors comprising one or more polynucleotides of the present disclosure.

[0028] The present disclosure relates to HLA-A * A method for killing MSLN+ cancer cells having loss of heterozygosity at the O3 locus is provided, comprising administering to a subject an effective amount of the immune cells or pharmaceutical composition of the present disclosure.

[0029] The present disclosure relates to HLA-A *

[0014] Provided are methods for treating MSLN+ cancer in a subject having an MSLN+ tumor with loss of heterozygosity at the O3 locus, comprising administering to the subject an effective amount of an immune cell or pharmaceutical composition of the present disclosure.

[0030] The present disclosure provides a method of treating cancer in a subject, comprising: (a) determining the HLA-A genotype or expression of normal cells and a plurality of cancer cells of the subject; (b) optionally determining the expression of MSLN in the plurality of cancer cells of the subject; and (c) determining whether the normal cells are HLA-A * 03, and multiple cancer cells express HLA-A * and if the plurality of cancer cells do not express MSLN (MSLN+) and the plurality of cancer cells are MSLN-positive, administering to the subject an effective amount of the immune cells or pharmaceutical composition of the present disclosure. In some embodiments of the methods of the present disclosure, the subject expresses MSLN (MSLN+) and HLA-A. * Heterozygous HLA-A with malignant tumors that have lost 03 expression * In some embodiments, the subject expresses MSLN and is an HLA-A 03 patient. * Heterozygous HLA-A with recurrent unresectable or metastatic solid tumors that have lost HLA-A expression *In some embodiments, the cancer comprises mesothelioma, 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 cholangiocarcinoma. 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.

[0031] In some embodiments of the disclosed methods, the cancer cells are HLA-A * MSLN+ / HLA-A not expressing 03 * In some embodiments, the MSLN+ / HLA-A cancer cells * 03-Cancer cells have HLA-A * MSLN+ / HLA-A due to loss of heterozygosity at HLA-A, resulting in loss of HLA-A3 * In some embodiments, the first receptor and the second receptor are both MSLN+ / HLA-A. * 03—Specifically activates immune cells in the presence of cancer cells. In some embodiments, the first receptor and the second receptor both bind to HLA-A * It does not specifically activate immune cells in the presence of MSLN+ cells that have not lost O3.

[0032] 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.

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

[0034] In some embodiments of the disclosed methods, administration of the immune cells or pharmaceutical composition reduces the number of tumors in the subject.

[0035] 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 a subject. In some embodiments, at least about 60% of the killed cells are cancer cells, about 65% of the killed cells are cancer cells, about 70% of the killed cells are cancer cells, about 75% of the killed cells are cancer cells, about 80% of the killed cells are cancer cells, about 85% of the killed cells are cancer cells, about 90% of the killed cells are cancer cells, about 95% of the killed cells are cancer cells, or about 100% of the killed cells are cancer cells. In some embodiments, administration of 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 subject's cancer cells.

[0036] In some embodiments of the disclosed methods, administration of the immune cells or pharmaceutical compositions results in fewer side effects in the subject than administration of otherwise equivalent immune cells that comprise the first activator receptor but do not comprise the second inhibitory receptor.

[0037] The present disclosure provides a method of producing 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 present disclosure.

[0038] The present disclosure provides kits comprising the immune cells or pharmaceutical compositions of the present disclosure. In some embodiments, the kits further comprise instructions for use. [Brief explanation of the drawings]

[0039] [Figure 1] Figure 1 is a table showing the expression of candidate blocking factor genes from the TCGA database. (*) indicates genes previously identified as being expressed in the mesothelium.

[0040] [Figure 2] FIG. 2 is a plot showing mesothelin (MSLN) expression in normal tissues.

[0041] [Figure 3] Figure 3 is a plot showing the expression of MSLN across TCGA cancers (using tumor and normal samples). Abbreviations: BLCA (bladder cancer), BRCA (breast cancer), CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal carcinoma), 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 (skin cutaneous melanoma), THCA (thyroid carcinoma), THYM (thymoma), STAD (gastric adenocarcinoma), UCEC (uterine endometrial carcinoma).

[0042] [Figure 4] FIG. 4 is a plot showing MSLN expression in CCLE cell lines.

[0043] [Figure 5] FIG. 5 is a plot showing LRRN4 expression in normal tissues.

[0044] [Figure 6] FIG. 6 is a plot showing LRRN4 expression across TCGA cancers (using tumor and normal samples).

[0045] [Figure 7]FIG. 7 is a plot showing LRRN4 expression across TCGA tumors.

[0046] [Figure 8] FIG. 8 is a plot showing the expression of LRRN4 in CCLE cell lines.

[0047] [Figure 9] Figure 9 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.

[0048] [Figure 10A] FIG. 10A shows 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.

[0049] [Figure 10B] Figure 10B shows that pMHC HLA-A*02 scFv LIR-1-based inhibitory receptor can inhibit the activation of Jurkat cells by MSLN scFv CAR using the leukemia cell line K562 as target cells.

[0050] [Figure 11] Figure 11 is a diagram (left) and chart (right) showing that pMHC HLA-A*02 scFv LIR-1-based inhibitory receptors can inhibit Jurkat cell activation, as measured by fold induction of IFNγ, by MSLN scFv CARs using pMHC HLA-A*02 scFv LIR-1-based inhibitory receptors and HLA-A*02+ HeLa and SiHa cells as target cells.

[0051] [Figure 12]Figure 12 shows that pMHC HLA-A*02 scFv LIR-1-based inhibitory receptors inhibit killing by MSLN CAR activators using HLA-A*02+SiHa cells but not HLA-A*02-SiHa cells.

[0052] [Figure 13] FIG. 13 is a diagram of the bioinformatics pipeline used to identify potential inhibitory receptor targets lost in cancer cells due to loss of heterozygosity.

[0053] [Figure 14] Figure 14 is a of the bioinformatics pipeline used to identify potential inhibitory receptor targets that are not expressed in cancer cells.

[0054] [Figure 15A] Figure 15A is a pair of plots showing that HLA-A*02 blocking agent inhibits MSLN CAR activator directed against the high-density antigen, MSLN. Jurkat cells transfected with MSLN LBD1-CAR or MSLN LBD1-CAR and A2-LIR-1 cocultured with K562 cells expressing either MSLN or MSLN and HLA-A*02 show blocking of high-density antigen activation by the A2-LIR-1 blocking agent only in the presence of HLA-A*02.

[0055] [Figure 15B] Figure 15B is a pair of plots showing that HLA-A*02 blocking agent inhibits MSLN CAR activators directed against MSLN, a high-density antigen. Killing of endogenous MSLN+HeLa cells by MSLN LBD1-CAR T cells is blocked by A2-LIR-1 blocking agent in the presence of HLA-A*02.

[0056] [Figure 15C]Figure 15C is a pair of plots showing that HLA-A*02 blocker inhibits MSLN CAR activator directed against MSLN, a high-density antigen. Killing of endogenous MSLN+HeLa cells by MSLN LBD2-CAR T cells is shown. The effect of the 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.

[0057] [Figure 16] Figure 16 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 an MSLN third-generation CAR 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 third-generation CAR (top right, boxed) provides a window superior to the humanized M5 and humanized SS1 CARs.

[0058] [Figure 17] Figure 17 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 third generation CARs in the presence of MSLN+HLA-A*02+Capan-2 cells.

[0059] [Figure 18] Figure 18 is 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.

[0060] [Figure 19]Figure 19 is 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.

[0061] [Figure 20A] Figure 20A 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 Figure 20B. Humanized PA2.1 and humanized BB7.2, which have shorter LIR-1 hinges, block similarly to the original longer hinge.

[0062] [Figure 20B] Figure 20B is a plot and table showing the EC50 shift (+ / -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).

[0063] [Figure 21A] Figure 21A 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, TM: transmembrane domain, ICD: intracellular domain, s: short, tr: truncated. The LIR-1 constructs are described in more detail in Figure 21B. The murine PA2.1, which has a slightly longer hinge, functions similarly to the original LIR-1 hinge in the T2-Jurkat assay.

[0064] [Figure 21B]Figure 21B is 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 (SEQ ID NOs: 357-361) with lengths shown in the table on the left.

[0065] [Figure 22A-B] Figures 22A and 22B show 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 an MSLN(+) mesothelial lining, creating a high risk of on-target, off-tumor toxicity for MSLN-targeted drugs. By selecting patients whose tumors are heterozygous for HLA-A*02, which has lost this allele via loss of heterozygosity, 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 an MSLN-targeted Tmod construct (Tmod refers to paired activating and inhibitory receptors). The two receptors are coexpressed within a single construct, and the encoded fusion protein is cleaved intracellularly to generate the activator and blocker.

[0066] [Figure 23A-B]Figures 23A, 23B, 23C, and 23D show the isolation and characterization of selective MSLN binders. The on-target probe was labeled soluble MSLN (Acro Bio), and the off-target probe used for counterselection was a mixture of soluble CEA and EGFR proteins. Figure 23A shows enrichment of the IgG library. Figure 23B shows enrichment of the scFv library. Figure 23C shows 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 shown. 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 in which MSLN protein was attached to the surface (see Hamburger et al., 2020). Sixty-two CAR constructs (Gen3) with different scFvs were transiently transfected into Jurkat cells to express CARs, and functional responses to surface-bound recombinant human sMSLN (Acro Bio) were evaluated 6 hours later. Most produced some degree of response. [Figure 23C]Figures 23A, 23B, 23C, and 23D show the isolation and characterization of selective MSLN binders. The on-target probe was labeled soluble MSLN (Acro Bio), and the off-target probe used for counterselection was a mixture of soluble CEA and EGFR proteins. Figure 23A shows enrichment of the IgG library. Figure 23B shows enrichment of the scFv library. Figure 23C shows 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 shown. 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 in which MSLN protein was attached to the surface (see Hamburger et al., 2020). Sixty-two CAR constructs (Gen3) with different scFvs were transiently transfected into Jurkat cells to express CARs, and functional responses to surface-bound recombinant human sMSLN (Acro Bio) were evaluated 6 hours later. Most produced some degree of response. [Figure 23D]Figures 23A, 23B, 23C, and 23D show the isolation and characterization of selective MSLN binders. The on-target probe was labeled soluble MSLN (Acro Bio), and the off-target probe used for counterselection was a mixture of soluble CEA and EGFR proteins. Figure 23A shows enrichment of the IgG library. Figure 23B shows enrichment of the scFv library. Figure 23C shows 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 shown. 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 in which MSLN protein was attached to the surface (see Hamburger et al., 2020). Sixty-two CAR constructs (Gen3) with different scFvs were transiently transfected into Jurkat cells to express CARs, and functional responses to surface-bound recombinant human sMSLN (Acro Bio) were evaluated 6 hours later. Most produced some degree of response.

[0067] [Figure 24A]Figure 24A shows the sensitivity of MSLN CARs versus benchmark CARs M5, SS1, and m912. All constructs were Gen3 except for SS1 (Gen2). To assess sensitivity in a 6-hour coculture assay, the dose response (RLU) of Jurkat cells was measured. (1) HEK293 cells were transfected with titrated MSLN-encoding mRNA, (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, the EC50 was reported as less than 3,000 MSLN molecules / cell. The maximum signal (Emax) for each construct is also shown. Experiments were repeated 1 to 4 times.

[0068] [Figure 24B] Figure 24B shows CAR3 selectivity. Example of MSLN CAR3 selectivity benchmarked against M5 CAR. CAR activation was measured in Jurkat cell functional assays with MSLN(+) or MSLN(-) cell lines. For the MSLN(+) cell line, a variant MSLN(-) version was generated by MSLN knockout for comparison. For more detailed off-target characterization, see Figure 32B.

[0069] [Figure 25A] Figure 25A shows MSLN expression 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 with CAR3 or the M5 benchmark CAR.

[0070] [Figure 25B]Figure 25B shows plotted levels of MSLN and A*02 mRNA (CCLE) and protein (QIFIKIT) showing correlation. Conversion between protein and mRNA levels was calculated using a standard curve (see Methods for Example 8 below).

[0071] [Figure 26A] Figure 26A shows an overview of the cell lines used in this study. Quantification of MSLN and A*02 surface densities 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 measurements of its surface A*02 protein level using a standard curve. HLA-A*02-transduced cell lines better mimic the A*02:MSLN ratio in 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.

[0072] [Figure 26B] Figure 26B shows the quantification of MSLN molecules / cells using QIFIKIT. Approximately 100,000 cells were stained with anti-human MSLN mouse antibody clone 618923 (R&D Systems). After washing the cells, both the cells and QIFI beads were stained with anti-mouse IgG F(ab')2 secondary antibody (Invitrogen A21237). The number of MSLN molecules on the surface was quantified using a QIFI antigen standard curve.

[0073] [Figure 27A]Figure 27A shows the characterization of MSLN CAR Tmod constructs in a 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 controls (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, and CAR M5 and SS1 are Gen2.

[0074] [Figure 27B] Figure 27B shows a two-dimensional titration of MSLN and A*02 mRNA in MSLN(-) HeLa target cells to establish the EC50 for the MSLN CAR3 Tmod construct in Jurkat cells. MSLN(-) HeLa target cells were transfected with serial dilutions of MSLN mRNA and constant 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.

[0075] [Figure 27C] Figure 27C shows 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.

[0076] [Figure 28A]Figure 28A shows plotted levels of MSLN and A*02 mRNA and protein. Construct EC50 and IC50 in relation 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 the standard curves shown in Figure 25B; Methods). The HeLa and MS751 A*02 transgenic cell line variants shown in the graph better mimic the activator-to-blocker target ratio in normal tissues.

[0077] [Figure 28B] Figure 28B shows MSLN CAR and CAR3 Tmod cytotoxicity in primary T cells. Primary T cells transduced with CAR or Tmod were cocultured with either tumor or normal target cells at an effector:target (E:T) ratio of 1:1, as indicated, for 48 hours. The A*02:MSLN (B:A) target antigen ratio ranged from 2 to 27:1. M5 was a Gen2 CAR, and all others were Gen3. Tumor = MSLN(+)A*02(-) target cells; normal = MSLN(+)A*02(+) target cells.

[0078] [Figure 29A]Figure 29A shows a comparison of the lead CAR3 receptor paired with the A*02 blocker against a benchmark CAR in a cytotoxicity assay. The SS1 CAR is a Gen2 construct, while the others are Gen3. Primary T cells transduced with various CARs + / - 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(+)] 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 the A*02 blocker.

[0079] [Figure 29B] Figure 29B shows secreted IFN-g after 48 hours of co-culture with tumor or normal target cells. Significance was calculated using two-way ANOVA with multiple comparisons test (technical replicates n=3).

[0080] [Figure 29C] FIG. 29C shows T cell activation assessed by forward scatter shift measured 48 hours after co-culture of transduced T cells with tumor or "normal" target cells.

[0081] [Figure 30A] Figure 30A shows that 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, killed targets tend to remain as clusters on the surface. White arrows point to some examples of killed RFP(+) tumor cells. Examples show E:T ratios of 0.6:1 and normal:tumor ratios of 1:1.

[0082] [Figure 30B]Figure 30B shows cytotoxicity in mixed normal and tumor cocultures at E:T = 1:1 and normal:tumor = 9:1 (see Figure 31B for other ratios). Primary T cells transduced with CAR3 or CAR3 Tmod constructs were cocultured 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.

[0083] [Figure 31A] Figure 31A shows 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.

[0084] [Figure 31B] Figure 31B shows the cytotoxicity of CAR3 and CAR3 Tmod in mixed normal and tumor co-cultures ranging from 9:1 to 1:9 normal:tumor; E:T = 0.6:1.

[0085] [Figure 32A] Figure 32A shows that MSLN CAR3 Tmod constructs mediate selective, durable, and reversible cytotoxicity. CAR3- or CAR3 Tmod-transduced primary T cells were co-cultured with either tumor or normal target cells at E:T = 1.2:1 for 48 hours. T cells were then harvested, depleted of dead or non-adherent target cells, and replated on fresh tumor or normal target cells for an additional 48 hours. RACA, repeated antigen challenge assay. R1, round 1; R2, round 2.

[0086] [Figure 32B]Figure 32B shows that MSLN CAR3 Tmod constructs selectivity in Jurkat cell functional assays on subsets of MSLN(+) and MSLN(-) control target cells showed no off-target activity (see Methods for Example 8 below). Bar heights correspond to the average from technical replicates.

[0087] [Figure 33A] Figure 33A shows a schematic of the 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 at an E:T ratio of 1.2:1 for 48 hours. T cells were then harvested, any dead or lifted target cells were removed, and the cells were re-seeded onto fresh tumor or normal targets for an additional 48 hours.

[0088] [Figure 33B] Figure 33B shows that soluble circulating MSLN (sMSLN) does not affect CAR-T activity. Acute cytotoxicity of tumor or normal target cells by the M5 benchmark CAR or CAR3 was not affected by the presence of 500 ng / mL sMSLN (Acro Bio).

[0089] [Figure 33C] Figure 33C shows staining of transiently transfected CAR(+) Jurkat cells with labeled sMSLN monomer or tetramer analyzed by flow cytometry, demonstrating that sMSLN is structurally intact and capable of binding to the receptor.

[0090] [Figure 34A-B]Figures 34A, 34B, and 34C show that Tmod constructs mediate selective killing of tumor cells in a xenograft model. Figure 34A shows a schematic of a bilateral flank tumor and normal MS751 xenograft model. Figure 34B shows bioluminescence values ​​on the right side of the color scale in flux units of photons / second / 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] Figures 34A, 34B, and 34C show that Tmod constructs mediate selective killing of tumor cells in a xenograft model. Figure 34A shows a schematic of a bilateral flank tumor and normal MS751 xenograft model. Figure 34B shows bioluminescence values ​​on the right side of the color scale in flux units of photons / second / 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).

[0091] [Figure 35A] Figures 35A, 35B, and 35C show that MSLN CAR Tmod selectively kills tumors in xenograft models. Figure 35A shows primary T cells transduced with MSLN CAR or CAR3 Tmod were co-cultured in vitro with either HLA-A KO tumor or A*02-transgenic normal MS751 target cells at an E:T ratio of 1.4:1 for 48 hours. M5 is a Gen2 CAR, and 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]Figures 35A, 35B, and 35C show that MSLN CAR Tmod selectively kills tumors in xenograft models. Figure 35A shows primary T cells transduced with MSLN CAR or CAR3 Tmod were co-cultured in vitro with either HLA-A KO tumor or A*02-transgenic normal MS751 target cells at an E:T ratio of 1.4:1 for 48 hours. M5 is a Gen2 CAR, and 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] Figures 35A, 35B, and 35C show that MSLN CAR Tmod selectively kills tumors in xenograft models. Figure 35A shows primary T cells transduced with MSLN CAR or CAR3 Tmod were co-cultured in vitro with either HLA-A KO tumor or A*02-transgenic normal MS751 target cells at an E:T ratio of 1.4:1 for 48 hours. M5 is a Gen2 CAR, and 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.

[0092] [Figure 36] Figure 36 shows 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 assay shown with E:T=0.5:1.

[0093] [Figure 37A-C]Figures 37A, 37B, and 37C show that the MSLN Tmod system can be extended to autologous T cells. Figure 37A shows that cis-binding of autologous A*02 in the A*02(+) donor eliminates binding to the A*02 tetramer, and CRISPR-mediated knockout (KO) of B2M restores blocker availability, as demonstrated by binding to the A*02 tetramer at levels similar to those observed in the A*02(-) donor. 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 A*02(+) donor, blockade is achieved only via B2M CRISPR KO. E:T = 1.2:1. Figure 37C shows representative images at 48 hours. Figure 37D shows that CAR3 Tmod, like SS1 Tmod, has reduced binding to A*02 tetramers in A*02(+) T cells. B2M knockdown (KD) with shRNA restores blocker availability. Figure 37E shows that B2M shRNA also restores blockade of cytotoxicity against MSLN(+)A*02(+) "normal" HeLa cells. Figure 37F shows that CAR3 paired with a humanized A*02 blocker retains the ability to block "normal" cell killing in A*02(+) donor T cells, even in the absence of B2M KO or KD. [Figure 37D-E]Figures 37A, 37B, and 37C show that the MSLN Tmod system can be extended to autologous T cells. Figure 37A shows that cis-binding of autologous A*02 in the A*02(+) donor eliminates binding to the A*02 tetramer, and CRISPR-mediated knockout (KO) of B2M restores blocker availability, as demonstrated by binding to the A*02 tetramer at levels similar to those observed in the A*02(-) donor. 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 A*02(+) donor, blockade is achieved only via B2M CRISPR KO. E:T = 1.2:1. Figure 37C shows representative images at 48 hours. Figure 37D shows that CAR3 Tmod, like SS1 Tmod, has reduced binding to A*02 tetramers in A*02(+) T cells. B2M knockdown (KD) with shRNA restores blocker availability. Figure 37E shows that B2M shRNA also restores blockade of cytotoxicity against MSLN(+)A*02(+) "normal" HeLa cells. Figure 37F shows that CAR3 paired with a humanized A*02 blocker retains the ability to block "normal" cell killing in A*02(+) donor T cells, even in the absence of B2M KO or KD. [Figure 37F]Figures 37A, 37B, and 37C show that the MSLN Tmod system can be extended to autologous T cells. Figure 37A shows that cis-binding of autologous A*02 in the A*02(+) donor eliminates binding to the A*02 tetramer, and CRISPR-mediated knockout (KO) of B2M restores blocker availability, as demonstrated by binding to the A*02 tetramer at levels similar to those observed in the A*02(-) donor. 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 A*02(+) donor, blockade is achieved only via B2M CRISPR KO. E:T = 1.2:1. Figure 37C shows representative images at 48 hours. Figure 37D shows that CAR3 Tmod, like SS1 Tmod, has reduced binding to A*02 tetramers in A*02(+) T cells. B2M knockdown (KD) with shRNA restores blocker availability. Figure 37E shows that B2M shRNA also restores blockade of cytotoxicity against MSLN(+)A*02(+) "normal" HeLa cells. Figure 37F shows that CAR3 paired with a humanized A*02 blocker retains the ability to block "normal" cell killing in A*02(+) donor T cells, even in the absence of B2M KO or KD.

[0094] [Figure 38A] Figure 38A shows the enrichment of anti-HLA-A*11 binders from an scFv library through multiple rounds of cell sorting. The on-target probe was a labeled HLA-A*11 tetramer, and the off-target protein was a mixture of unrelated MHC tetramers.

[0095] [Figure 38B]Figure 38B shows 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 hours of co-culture. PE, phycoerythrin.

[0096] [Figure 39A] Figure 39A 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.

[0097] [Figure 39B-C] Figures 39B and 39C show cytotoxicity assays of primary T cells transduced with MSLN CAR3 + A*11:01 blocking agent. Primary T cells transduced with CAR3 and A*11:01-directed blocking agent efficiently blocked HeLa target cells with A*11:01 and killed wild-type HeLa cells as effectively as CAR-only cells. Transduced primary T cells were co-cultured with HeLa cells at an E:T ratio of 0.8:1, with or without HLA-A*11:01. Note that both tumor and normal target cells used here expressed GFP. Figure 39C shows representative co-culture images at 48 hours for Figure 39B.

[0098] [Figure 40] Figure 40 shows that MSLN CAR3 Tmod killing of tumor target cells and blocking of "normal" target cells is unaffected by the presence of PD-L1 induced by overnight treatment of HeLa target cells with 50 ng / mL IFN-γ. Similarly, blocking with an anti-PD-1 antibody has no effect on CAR3 Tmod.

[0099] [Figure 41]Figure 41 shows the characterization of MSLN CAR A*03 Tmod constructs in Jurkat cell functional assays. mBA GAP-A3 = construct with mouse blocker and activator (no shRNA).

[0100] [Figure 42] Figure 42 shows functional characterization of MSLN CAR A*03 Tmod constructs in primary T cells from five different donors against MS751 target cells. mBAsh(GAP-A3) = construct with murine blocker and activator (plus β2M shRNA).

[0101] [Figure 43] Figure 43 shows functional characterization of MSLN CAR A*03 Tmod constructs in primary T cells from five different donors against HeLa target cells. mBAsh(GAP-A3) = construct with mouse blocker and activator (plus β2M shRNA).

[0102] [Figure 44A-B] Figures 44A and 44B show that Tmod constructs mediate selective killing of tumor cells in xenograft models. Figure 44A shows a schematic of bilateral flank tumor and normal MS751 xenograft models. Figure 44B shows xenograft size assessed by caliper measurement. Day 0 = xenograft injection. The horizontal dashed line indicated the time of T cell injection. mBAsh(A3) = construct with murine blocking and activating factors (plus β2M shRNA). DETAILED DESCRIPTION OF THE INVENTION

[0103] Detailed Description Provided herein are compositions and methods for treating cancer using immune cells containing two receptor systems that respond to differences in gene expression of a ligand between cancer and normal (i.e., healthy or wild-type) cells. These expression differences may be due to loss of heterozygosity in cancer cells. Alternatively, the expression differences may be because a gene is not expressed in cancer cells or is expressed at a lower level in cancer cells than in normal cells. The two-receptor system is expressed in immune cells, such as immune cells used in adoptive cell therapy, and directs the activity of these immune cells toward cancer cells that exhibit loss of heterozygosity or expression differences. In this two-receptor system, the first receptor (an activator receptor, sometimes referred to herein as the A module) activates or promotes the activation of the immune cell, while the second receptor (an 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. Upon ligand binding, signals from the two receptors 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 immune cell activation by target cancer cells that express the first activator ligand but not the second inhibitory ligand.

[0104] Loss of heterozygosity (LOH) due to large chromosomal deletions is a cause of genetic variation in tumors. LOH is a common event in tumorigenesis, affecting nearly every locus in the genome; approximately 20% of genes exhibit LOH in an average tumor. LOH provides a means to clearly distinguish tumors from normal tissue, as all malignant cells can be found in tumors lacking specific germline alleles. 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 uses one 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 HLA-A*02 or HLA-A*03 proteins. The dual receptor system described herein (sometimes referred to herein as "Tmod") has other advantageous properties as a cell therapy, including, but not limited to, reversible activation / blocking of immune cells and selectivity in mixtures of tumor and "normal" cells.

[0105] 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 cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, renal cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or cholangiocarcinoma. In some embodiments, the cancer has recurred in the subject. In some embodiments, the cancer is refractory to one or more previously administered anticancer 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 adipose tissue, fallopian tube tissue, lung tissue, and salivary gland tissue, among others (Figure 2). Due to its expression in certain tumors, MSLN is an attractive tumor-specific antigen that can mediate 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 the MSLN activator receptor with an inhibitory receptor, the methods provided herein increase the specificity of adoptive cell therapy and reduce adverse effects associated with these therapies, such as dose-limiting toxicity.

[0106] 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, thereby increasing the safety window of the activator by blocking its cytolytic activity against normal MSLN-positive tissue. However, because tumor cells do not express a ligand for the inhibitory, or blocking, receptor, the activator receptor still induces targeted killing of tumor cells by immune cells comprising a two-receptor system. A target for the second inhibitory receptor is expressed by MSLN-positive tissues, such as lung tissue, mesothelial tissue, and adipose tissue, 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 allelic 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 HLAs. * 01. HLA-A * 02. HLA-A * 03. HLA-C *These are encoded by variant genes such as .07, which can be lost from MSLN-positive cancer cells through loss of heterozygosity. Alternatively, additional exemplary targets of inhibitory receptors include, but are not limited to, intercellular adhesion molecule 1 (ICAM1), catechol-O-methyltransferase (COMT), and C-X-C motif chemokine ligand 16 (CXCL16). Each of these has a common nonsynonymous variant form with amino acid changes in its extracellular domain accessible to antibodies, which can be used as an inhibitory receptor or blocker receptor target for cellular integrators designed to safely treat patients with MSLN-positive cancers using engineered T cells activated by MSLN or activator receptors such as MSLN pMHC-responsive activator receptors.

[0107] The disclosed compositions and methods can reduce or eliminate dose-limiting toxicities (DLTs) caused by MSLN expression on normal tissues. The present disclosure provides methods for 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, called a non-target antigen or alternatively a blocker antigen). Using the compositions and methods described herein, MSLN-expressing tumor cells are attacked by adoptive cells, such as immune cells, that express both receptors because these tumor cells express only MSLN, the activator ligand. In contrast, normal cells that express MSLN and the non-target antigen are protected from the adoptive immune cells. The inhibitory receptor's response to the non-target antigen on normal cells prevents immune cell activation by the MSLN-targeted activator receptor. This dual-targeting approach creates a therapeutic window that allows for the safe and effective administration of MSLN-directed cell therapy in MSLN-positive cancer patients.

[0108] 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.

[0109] 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.

[0110] The methods and compositions described in U.S. Patent Application Publication No. 2022 / 0370497 are incorporated herein by reference in their entirety.

[0111] definition Before setting forth the present disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms used herein.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of a particular embodiment, preferred embodiments of the compositions, methods, and materials are described herein. For purposes of this disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0113] 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% of 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 ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0114] 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.

[0115] 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 domestic animals or pets (such as cats or dogs). Non-human primates and preferably human patients are included.

[0116] As used herein, "treatment" or "treating" includes any beneficial or desired effect, and may include even minimal amelioration of symptoms. "Treatment" does not necessarily indicate a complete eradication or cure of a disease or condition, or its associated symptoms.

[0117] As used herein, "prevent" and similar words such as "prevented," "preventing," and the like refer to an approach for preventing, inhibiting, or reducing the likelihood of disease symptoms. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the occurrence or recurrence of disease symptoms. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease before onset or recurrence.

[0118] As used herein, the term "amount" refers to an "effective amount" or "effective dose" of a virus to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0119] A "therapeutically effective amount" of a virus or cells can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cells to elicit 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).

[0120] An "increased" or "augmented" amount of 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 or more fold (e.g., 500, 1000 fold) (including all integers and decimal points greater than 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) above the level of activity in untreated cells.

[0121] A "reduced" or "diminished" amount of 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 decimal points greater than 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the level of activity in untreated cells.

[0122] "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 different or has no measurable difference from the reference response.

[0123] 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 involve determining the nucleotide sequence of a polynucleotide and / or 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 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 an 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). Desired sequence identity ranges are 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%.

[0124] As used herein, a "polynucleotide system" refers to one or more polynucleotides that can be designed to work together for a particular use or to produce a desired transformed cell.

[0125] The term "exogenous" is used herein to refer to any molecule, including nucleic acids, proteins or peptides, small molecules, etc., that originate outside an organism. In contrast, the term "endogenous" refers to any molecule that originates within an organism (i.e., is naturally produced by the organism).

[0126] The term "MOI" is used herein to refer to the multiplicity of infection, which is the ratio of agent (e.g., virus particles) to infected target (e.g., cell).

[0127] In this description, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within 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%.

[0128] 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 transferred T cell of adoptive cell therapy.The target cell of the present disclosure expresses the target antigen as described herein and does not express non-target antigen.

[0129] 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 the target antigen and the non-target antigen. The non-target cells in a subject may express the non-target antigen regardless of whether these cells also express the target antigen.

[0130] As used herein, a "non-target allelic variant" refers to an allele of a gene whose product is expressed by non-target cells but not by target cells. For example, a non-target allelic variant is an allele of a gene that is expressed by normal, non-cancer cells of a subject but not by cancer cells of the subject. Expression of a non-target allelic variant can be lost in cancer cells by any mechanism, including, but not limited to, loss of heterozygosity, mutation, or epigenetic modification of the gene encoding the non-target allelic variant.

[0131] 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 designated 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. Compared to 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, HLA-A * The ligand-binding domain specific for HLA-A * 01 or HLA-A * It does not bind, or only weakly binds, other HLA-A alleles, such as 03. One skilled in the art will appreciate that a ligand-binding domain can be said to be specific for a particular target, but may still have a low level of binding to one or more additional targets that does not affect its function in the receptor system described herein.

[0132] 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 the target antigen is not limited to the target cell. The target antigen can be expressed by both cancer cells and normal, non-cancerous cells in a subject.

[0133] As used herein, a "non-target antigen" (or "blocker antigen") refers to an antigen that is expressed by normal, non-cancerous cells but not 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.

[0134] Polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population. Polymorphisms 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 variations that occur when a single nucleotide, adenine (A), thymine (T), cytosine (C), or guanine (G), is changed.

[0135] 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.

[0136] The 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. A high Kd indicates a high concentration of ligand is required to occupy the receptor, indicating a low affinity of the receptor for the ligand. Conversely, a low Kd indicates a high affinity of the ligand for the receptor.

[0137] As used herein, a receptor "responsive to" or "responsive to" refers to a receptor comprising an intracellular domain that, upon binding of a ligand (i.e., an antigen), generates a signal corresponding to the known function of the intracellular domain. An activator receptor bound to a target antigen can generate a signal that causes activation of immune cells that express the activator receptor. An inhibitory receptor bound to a non-target antigen can generate an inhibitory signal that prevents or reduces activation of immune cells that express 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.

[0138] As used herein, "activation" of an immune cell or an "activated" immune cell refers to an immune cell that is capable of performing one or more functions characteristic of an immune response. These functions include proliferation, cytokine release, and cytotoxicity, i.e., killing of target cells. Activated immune cells express markers that will be apparent to those skilled in the art. For example, activated T cells can express one or more of CD69, CD71, CD25, and HLA-DR. Immune cells that express an activator receptor (e.g., MSLN CAR) can be activated by an activator receptor when they become responsive to receptor binding to a target antigen (e.g., MSLN) expressed by the target cell. A "target antigen" can also be referred to as an "activator antigen," and can be isolated or expressed by the target cell. Activation of an immune cell that expresses an inhibitory receptor can occur when the inhibitory receptor binds to a non-target antigen (e.g., HLA-A CAR) even when the activator receptor is bound to a target activator ligand. * 02) can be prevented when the target cell becomes responsive to the antigen. "Non-target antigens" can also be called "inhibitory ligands" or "blocking factors" and can be isolated or expressed by the target cell.

[0139] Receptor expression on immune cells can be verified by assays that report the presence of activator and inhibitory receptors 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 the 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 the immune cells, pharmaceutical compositions, and kits described herein. It should be understood that for the immune cells, pharmaceutical compositions, and kits described herein, the appropriate percentage of immune cells expressing both activator and inhibitory receptors will be determined specifically for the methods described herein. For example, a suitable percentage of immune cells expressing both activator receptors and inhibitory receptors can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. 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 receptors and inhibitory receptors. For example, a suitable ratio of activator receptors to inhibitory receptors on 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.

[0140] Responsive receptors expressed by immune cells described herein can be verified by assays measuring the generation of signals predicted 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 signaling marker for T cell activation. Jurkat cells can be transduced or transfected with activating and / or inhibitory receptors described herein. Activating 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. When co-expressed with an activator receptor in Jurkat cells, an inhibitory receptor responds to ligand binding and prevents normally responding immune cells from expressing luciferase in response to the activator receptor. For example, the responsiveness of an inhibitory receptor can be determined and quantified in Jurkat cells expressing both an activator and an inhibitor by observing: 1) the Jurkat cells express luciferase in the presence of an activator receptor ligand and in the absence of an inhibitory receptor ligand; and 2) luciferase expression in Jurkat cells is reduced or eliminated in the presence of both an activator receptor ligand and an inhibitory receptor ligand. This approach can be used to determine the sensitivity, potency, and selectivity of activator receptors and specific pairs of activator and inhibitory receptors. 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-cultures 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 increasing amounts of the activator or inhibitory ligand. Increased amounts of the activator or inhibitory ligand can be achieved in the target cells, for example, by titrating mRNA encoding the activator or inhibitory ligand 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 target cells expressing various amounts of target and non-target ligands include an EC50 of 10 transcripts per million (TPM) or less for activator receptors, e.g., an EC50 of 2-10 TPM, and an IC50 of 25 TPM or less for inhibitory receptors, e.g., an IC50 of 5-21 TPM.

[0141] Activation of immune cells described herein expressing an activator receptor or a specific pair of an activator receptor and an inhibitory receptor can be further determined by an assay measuring 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 ligand 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 measures active mitochondrial enzymes using tetrazolium substrate. 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 target cell viability. A decrease in target cell viability after incubation with immune cells expressing an activator receptor or a specific pair of an activator receptor and an inhibitory receptor is interpreted as target cell-mediated activation of the immune cells. A measure of immune cell selectivity 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 of target cells that express the activator receptor ligand but not the inhibitory receptor ligand, and 2) there is no decrease in viability of target cells that express both the activator and inhibitory receptor ligands. From these measurements, a "specific killing" value can be derived, which quantifies the percentage of immune cell activation based on the decrease in target cell viability as a percentage of the negative control (immune cells that do not express the activator receptor). Furthermore, from these measurements, a "selectivity ratio" value can be derived, which represents the ratio of the specific killing observed in target cells that express the activator receptor ligand in the absence of the inhibitory receptor ligand to the specific killing observed in target cells that express both the activator and inhibitory receptor ligands.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) specific killing of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% of target cells expressing an activator receptor ligand after 48 hours of co-incubation in the absence of an inhibitory receptor ligand, and 2) specific killing of 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% of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand.

[0142] As further examples, suitable specific kill values ​​for immune cells, pharmaceutical compositions, and kits may be the following criteria: 1) 30% to 99%, 40% to 99%, 50% to 99%, 55% to 95%, 60% to 95%, 60% to 90%, 50% to 80%, 50% to 70%, or 50% to 60% of target cells that express an activator ligand but not an inhibitory ligand are killed; and 2) 1% to 40%, 3% to 40%, 5% to 40%, 5% to 30%, 10% to 30%, 15% to 30%, or 5% to 20% of target cells that express an activator ligand and an inhibitory ligand are killed. By way of still further example, suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits may 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, the immune cells may kill 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-hour, 12-hour, 18-hour, 24-hour, 30-hour, 36-hour, 42-hour, 48-hour, 54-hour, 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.

[0143] Suitable specific killing values ​​for target cells 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%. Suitable specific killing values ​​for target cells 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%. Suitable specific killing values ​​for target cells expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, or at most about 95%. Suitable specific killing values ​​for target cells expressing both activator and inhibitory receptor ligands for immune cells, pharmaceutical compositions, and kits can be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. Suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits can be determined after 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.

[0144] Suitable specific killing values ​​for target cells 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%. Suitable specific killing values ​​for target cells 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%. Suitable specific killing values ​​for target cells expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, or at most about 95%. Suitable specific killing values ​​for target cells expressing both activator and inhibitory receptor ligands for immune cells, pharmaceutical compositions, and kits can be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. Suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits can be determined after 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.

[0145] 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.

[0146] All publications and patents mentioned herein are incorporated 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, the present application, including any definitions herein, will control. However, 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 anywhere in the world.

[0147] 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 responds 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).

[0148] In some embodiments, the first receptor is humanized. As used herein, "humanization" refers to replacing a sequence or subsequence isolated from or transgene derived from a non-human species with a homologous or functionally equivalent human sequence. For example, a humanized antibody can be created by grafting murine CDRs onto human framework sequences, followed by back-substitution of specific human framework residues for the corresponding murine residues from the original antibody.

[0149] 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 cells can be a suitable target antigen for the first receptor ligand binding domain.For example, cell adhesion molecules, intercellular signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane receptors, receptors for neurotransmitters, or voltage-dependent ion channels can be used as target antigens.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] Cancer cell-specific pMHC antigens, including any of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G, are contemplated within the scope of this disclosure. In some embodiments, the cancer cell-specific antigen comprises HLA-A. HLA-A receptors are heterodimers comprising a heavy α chain and a smaller β chain. The α chain is encoded by a variant of HLA-A, while the β chain (β2-microglobulin) is invariant. Thousands of variant HLA-A genes exist, all of which are within the scope of this disclosure. In some embodiments, the MHC-I is encoded by human leukocyte antigen A. * 02 allele (HLA-A * 02).

[0154] In some embodiments, the cancer cell-specific antigen comprises HLA-B. Hundreds of versions (alleles) of the HLA-B gene are known, each of which has a specific number (HLA-B * 27, etc.) is given.

[0155] 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.

[0156] 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.

[0157] 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 40 KDa protein typically expressed in mesothelial cells, as well as lung, fallopian tube, salivary gland, and adipose tissue ( FIG. 2 ). MSLN is expressed in multiple human tumor types, including mesothelioma, ovarian, cervical, colorectal, esophageal, head and neck, renal, uterine, gastric, pancreatic, lung, colorectal, or cholangiocarcinoma. In some embodiments, the cancer has recurred in the subject. In some embodiments, the cancer is refractory to one or more previously administered anticancer therapies. In some embodiments, the cancer is metastatic.

[0158] All isoforms of MSLN are contemplated as cancer cell-specific antigens in 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]

[0159] 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.

[0160] 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]

[0161] 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.

[0162] 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.

[0163] 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.

[0164] Any type of ligand-binding domain capable of modulating receptor activity in a ligand-dependent manner is contemplated within the scope of this disclosure. In some embodiments, the ligand-binding domain is an antigen-binding domain. Exemplary antigen-binding domains include, among others, scFvs, SdAbs, Vβ-only domains, and TCR antigen-binding domains derived from TCR α and β chain variable domains.

[0165] Any type of antigen binding domain is contemplated within the scope of this disclosure.

[0166] 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 murine, 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 sdAb.

[0167] 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.

[0168] The term "antibody fragment" or "antibody binding domain" refers to at least a portion of an antibody or 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 its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), 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.

[0169] 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 contiguously linked 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.

[0170] "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.

[0171] 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 may comprise a VL-linker-VH or a VH-linker-VL.

[0172] 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 connected 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. An exemplary signal sequence includes MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 362), which is encoded by ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGT (SEQ ID NO: 153).

[0173] 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.

[0174] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, e.g., an antibody expressed in 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 the 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 technology that is available and well known in the art.

[0175] The terms "Vβ domain," "Vβ-only domain," "β-chain variable domain," or "single variable domain TCR (svd-TCR)" refer 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. Vβ-only domains engage antigen using complementarity-determining regions (CDRs). Each Vβ-only domain contains three complementarity-determining regions (CDR1, CDR2, and CDR3). Additional elements may be combined, provided that the Vβ domain is configured to bind to an epitope in the absence of a second TCR variable domain.

[0176] 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).

[0177] In some embodiments, the extracellular ligand-binding domain of the first receptor is a TCR It contains an α chain variable domain and a TCR β chain variable domain.

[0178] 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

[0179] 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 a CAR. Exemplary scFv domains specific for MSLN are shown in Table 1 above. In Table 1, the underlines indicate the CDR sequences.

[0180] 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 the 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 the sequence of SEQ ID NO: 3-6, 80, or 154-215 set forth in Table 1.

[0181] 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.

[0182] 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]

[0183] In Table 2, the light chain (LC) CDRs paired with the indicated heavy chain (HC) CDRs are shown in the left column.

[0184] 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 of Table 2, etc.), or a sequence having up to one, two, or three 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, 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), or a sequence having up to one, two, or three 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, or line #3 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).

[0185] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises an HC CDR1, an HC CDR2, an HC CDR3, an LC CDR1, an LC CDR2, and an 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 an HC CDR1, an HC CDR2, and an 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 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), or a 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 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., an HC CDR1, an HC CDR2, and an HC CDR3 in line #1, line #2, or line #3 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., an LC CDR1, an LC CDR2, and an LC CDR3 in 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, using routine testing to confirm the desired expression and binding activity. However, preferred pairings between heavy and light chains in some embodiments are shown in the right-hand column of Table 2.

[0186] 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), and an HC CDR3 comprising the sequence of CAREDVVKGAFDIW (SEQ ID NO: 533), 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), and an HC CDR3 comprising the sequence of CAREDVVKGAFDIW (SEQ ID NO: 533). In some embodiments, the extracellular ligand-binding domain of the first receptor comprises 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 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). In some embodiments, the extracellular ligand-binding domain of the first receptor comprises an HC CDR1 comprising the sequence SGDYYWS (SEQ ID NO: 438), an HC CDR2 comprising the sequence YIYYSGSTYYNPSLKS (SEQ ID NO: 454), an HC CDR3 comprising the sequence CAREDVVKGAFDIW (SEQ ID NO: 533), an LC CDR1 comprising the sequence RASQSISSYLN (SEQ ID NO: 535), an LC CDR2 comprising the sequence AASSLQS (SEQ ID NO: 539), and an LC CDR3 comprising the sequence QQSYSTPLT (SEQ ID NO: 542), or CDR sequences having up to 1, 2, or 3 amino acid substitutions, insertions, or deletions compared thereto.In some embodiments, the extracellular ligand-binding domain of the first receptor comprises an HC CDR1 comprising the sequence SGDYYWS (SEQ ID NO: 438), an HC CDR2 comprising the sequence YIYYSGSTYYNPSLKS (SEQ ID NO: 454), an HC CDR3 comprising the sequence CAREDVVKGAFDIW (SEQ ID NO: 533), an LC CDR1 comprising the sequence RASQSISSYLN (SEQ ID NO: 535), an LC CDR2 comprising the sequence AASSLQS (SEQ ID NO: 539), and an LC CDR3 comprising the sequence QQSYSTPLT (SEQ ID NO: 542).

[0187] 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).

[0188] 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]

[0189] 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 sequence 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 sequence set forth in Table 4.

[0190] 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., an HC CDR1, HC CDR2, and HC CDR3 sequence set forth in line #1, line #2, or line #3 of Table 2), and (ii) comprises 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 set forth in Table 4.

[0191] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises (i) a VH sequence listed 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 listed in Table 3, and (ii) a VL sequence listed in Table 4 or 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 sequence listed 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 preferred pairings between Tables 3 and 4 are shown in the "LC" column of Table 3, which corresponds to the # column of Table 4.

[0192] 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.

[0193] 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.

[0194] 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 substituted with another amino acid. The substitution may be "conservative," meaning that the substitution is within the same family of amino acids. Naturally occurring amino acids can be divided into four families, and conservative substitutions are made within these families: (1) amino acids with basic side chains: lysine, arginine, and histidine; (2) amino acids with acidic side chains: aspartic acid and glutamic acid; (3) amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, and tyrosine; and (4) amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine. Altering the amino acid sequence of an antibody CDR by adding, deleting, or substituting amino acids can produce various effects, such as increased binding affinity for a target antigen.

[0195] Chimeric antigen receptor (CAR) 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.

[0196] 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 specific immune effector cells. CARs can be used to confer the specificity of a monoclonal antibody to T cells, thereby enabling the generation of large numbers of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, a CAR directs the specificity of a cell to, for example, a tumor-associated antigen. Exemplary CARs include an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen-binding region. In some embodiments, a CAR further comprises a hinge domain. In certain aspects, a 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 may 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.

[0197] In some embodiments, the extracellular ligand-binding domain of the first receptor is fused to the extracellular domain of the CAR.

[0198] 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 in vivo expansion of CAR-T cells. Exemplary hinges can be isolated from or derived from IgD and CD8 domains, e.g., IgG1. In some embodiments, the hinge is isolated from or derived from CD8α or CD28.

[0199] In some embodiments, the hinge is isolated from 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 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 7). In some embodiments, the CD8α hinge comprises SEQ ID NO: 7. In some embodiments, the CD8α hinge consists essentially of SEQ ID NO: 7. In some embodiments, the CD8α hinge is [ka] In some embodiments, the CD8 alpha 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 is identical to the sequence of SEQ ID NO: 8. In some embodiments, the CD8 alpha hinge is encoded by SEQ ID NO: 8.

[0200] 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 comprises [ka] 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 SEQ ID NO: 10.

[0201] The CAR of the present disclosure can be designed to include a transmembrane domain 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 can 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 proteins and minimize interaction with other members of the receptor complex.

[0202] The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region can be isolated from or derived from (i.e., comprising at least the transmembrane region(s) 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 can be synthetic, in which case it comprises primarily 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 cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0203] 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 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 comprises [ka] 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 is identical to the sequence of SEQ ID NO: 12.

[0204] 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] 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 is identical to the sequence of SEQ ID NO: 14.

[0205] The cytoplasmic domain, or otherwise, intracellular signaling domain, of a 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 the 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.

[0206] 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 cooperatively to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences that have the same functional capability.

[0207] 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 the T cell receptor (TCR) signaling necessary to activate the effector function of the CAR T cell is present. In some embodiments, the at least one cytoplasmic activation domain is a CD247 molecule (CD3ζ) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX activation protein of 12 kDa (DAP12) activation domain.

[0208] In some embodiments, the CD3ζ activation domain is [ka] The present invention includes 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

[0209] 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 comprises [ka] 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 is identical to the sequence of SEQ ID NO: 16.

[0210] It is known that signals generated through the TCR alone 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).

[0211] 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 a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. 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 CD3ζ 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 TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d.

[0212] 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, the 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] 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 is identical to the sequence of SEQ ID NO: 18.

[0213] 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. A costimulatory domain refers to a 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 costimulatory domains, which are 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 intracellular domain of the CAR of the present disclosure is selected from the group consisting of: cytochrome P450 superfamily member 4 (OX40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory domain (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 costimulatory domain. In some embodiments, the costimulatory domain is isolated from or derived from CD28.

[0214] 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 from 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 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 that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC (SEQ ID NO: 20). In some embodiments, the CD28 costimulatory domain is encoded by SEQ ID NO: 20.

[0215] In some embodiments, the costimulatory domain is isolated from or derived from 4-1BB. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to 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 AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGGCCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 284).

[0216] In some embodiments, the intracellular domain of the CAR comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ 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.

[0217] The cytoplasmic domains within the cytoplasmic signaling portion of a CAR of the present disclosure can be linked to each other in a random or specified order. If desired, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides an example of a suitable linker. An exemplary linker comprises the sequence GGGGSGGGGSGGGGSGG (SEQ ID NO: 152).

[0218] The cytoplasmic domains within the cytoplasmic signaling portion of a CAR of the present disclosure can be linked to each other in random or specified order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides an example of a suitable linker. Exemplary full-length activator receptors of the present disclosure are listed in Table 20. In some embodiments, the first activator receptor comprises the sequence of SEQ ID NOs: 286-347 listed 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 the sequence of SEQ ID NOs: 286-347 listed in Table 20. In some embodiments, the first activator receptor comprises the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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.

[0219] The cytoplasmic domains within the cytoplasmic signaling portion of a CAR of the present disclosure can 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, can form the linkage. A glycine-serine doublet provides an example of a suitable linker.

[0220] 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).

[0221] As used herein, "TCR," sometimes referred to as "TCR complex" or "TCR / CD3 complex," refers to a protein complex comprising one or more of the TCR alpha chain, TCR beta chain, and invariant CD3 chains (zeta, gamma, delta, and epsilon), sometimes referred to as subunits. The TCR alpha and beta chains may be disulfide-linked to function as a heterodimer that binds to peptide-MHC complexes. Engagement of the TCR alpha / beta heterodimer induces a conformational change in the TCR complex within the associated invariant CD3 subunit, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal. In an exemplary TCR complex, 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 and two CD3 zetas form homodimers.

[0222] Any suitable ligand binding domain may be fused to the extracellular domain, hinge domain or transmembrane domain of a TCR as described herein. For example, the ligand binding domain may be an antibody or 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).

[0223] 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 portion may be fused to both TCR alpha and TCR beta.

[0224] 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 chains, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta, or fragments or derivatives thereof, can be fused to one or more domains capable of providing a stimulatory signal of the present disclosure, thereby enhancing TCR function and activity.

[0225] 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.

[0226] In some embodiments, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex is bound to a target. Transmembrane domains of particular use in the present disclosure may include, for example, at least the transmembrane region(s) 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.

[0227] In some embodiments, the transmembrane domain can be connected 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 from or derived from CD8α or CD28.

[0228] In some embodiments, the extracellular ligand-binding domain is linked to one or more transmembrane domains of a 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.

[0229] A transmembrane domain may 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).

[0230] In some embodiments, transmembrane domains may 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, e.g., to minimize interactions with other members of the receptor complex.

[0231] 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 limitation, a native or artificial transmembrane domain may often comprise a hydrophobic a-helix of about 20 amino acids, 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.

[0232] 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).

[0233] 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 ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO: 24).

[0234] TCRs of the present disclosure can comprise one or more intracellular domains. Exemplary TCRs comprising an intracellular domain 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 comprises one or more domains capable of providing a stimulatory signal to the transmembrane domain. In some embodiments, the intracellular domain comprises 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 comprises first, second, and third intracellular domains 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 the 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.

[0235] 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 a 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.

[0236] 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.

[0237] 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, and intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences, and any recombinant sequence having the same functional capability.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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 ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 26).

[0242] 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 a CD3 delta, CD3 gamma, or CD3 epsilon intracellular domain, 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 a CD3 delta, CD3 gamma, or CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.

[0243] 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 may 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, a ligand that specifically binds to CD83, 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.

[0244] 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 containing only the CD3 zeta signaling domain. Similarly, including a costimulatory domain, e.g., a CD28 and 4-1BB domain, in a TCR can increase T cell effector function and specifically enable 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.

[0245] 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 cancer cells, e.g., an allelic variant of a gene. The non-target allelic variant may be lost in cancer cells through any mechanism, including, but not limited to, epigenetic changes affecting the expression of the non-target allelic variant, mutations in the gene encoding the non-target allelic variant, disruption of cell signaling regulating the expression of the non-target allelic variant, chromosomal loss, partial or complete deletion of a genomic locus, gene silencing due to nucleic acid or heterochromatin modifications, or loss of expression due to other mechanisms. In variations of the compositions and methods disclosed herein, treated cells or treated subjects may exhibit 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 lacking expression of the non-target antigen due to any cause, including, but not limited to, loss of heterozygosity.

[0246] The non-target antigen may be a protein in a complex with major histocompatibility complex class I (MHC-I) or an antigenic peptide thereof, and the non-target antigen may include polymorphism. Because the non-target antigen is polymorphic, loss of a single copy of the gene encoding the non-target antigen, which may occur due to 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, if the HLA locus contains HLA-A * 02 and HLA-A * A subject with the HLA-A*01 allele may have a cancer in which only the HLA-A*02 allele is lost. * 03 and HLA-A * Subjects with the 01 allele are HLA-A * In such subjects, the HLA-A gene may be present in a cancer where only the 03 allele is lost. * The O1 protein is still present, but the inhibitory receptor is * 02. HLA-A * They are designed to be specific for HLA-A or other non-target antigens, so they are not recognized by the inhibitory receptors of immune cells that encounter cancer cells. * 02. HLA-A * HLA-A3 or other non-target antigens may be present and inhibit the activation of engineered immune cells. * HLA-A*02, HLA-A*03, or allelic variants are lost. Inhibitory receptors are HLA-A*02, which are not present in cancer cells. * 02. HLA-A * Immune cells engineered to express inhibitory receptors do not receive inhibitory signals from inhibitory receptors because they respond only to HLA-A or other non-target antigens. By this mechanism, immune cells can be selectively activated and express MSLN but not HLA-A through loss of heterozygosity. *This selectively kills cancer cells that have lost HLA-A (or another non-target antigen). Herein, HLA-A is used as an example. Similar polymorphic variations occur in other MHC gene clusters and 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), C-X-C 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). The non-target antigen may include nonsynonymous extracellular domain polymorphisms (e.g., in the extracellular domains of ICAM1, COMT, and CXCL16) and immune cells containing them. In some embodiments, the second receptor is an inhibitory chimeric antigen receptor. Alternatively, non-target antigens may include proteins whose expression is lost in tumors but which are present in significant MSLN-expressing normal tissues (e.g., LRRN4, UPK3B).

[0247] 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 herein by reference.

[0248] In some embodiments, the second receptor is humanized.

[0249] The present disclosure provides a second receptor that is an inhibitory receptor comprising an extracellular ligand binding domain capable of distinguishing between single amino acid variant alleles of a non-target antigen. This ability to distinguish between allelic variants of the non-target antigen allows the second receptor to suppress the activation of immune cells containing the second receptor in the presence of non-target cells that express the allele recognized by the ligand-binding domain. However, immune cell activation is not suppressed in the presence of target cells that have lost the allele, such as cancer cells that have lost one allele of the gene due to loss of heterozygosity.

[0250] The present disclosure provides a second receptor that is an inhibitory receptor, comprising extracellular ligand binding that can distinguish between different levels of expression of a non-target antigen, thereby allowing the second receptor to suppress activation of an immune cell that contains the second receptor in the presence of non-target cells that express 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.

[0251] Repressor Ligand In some embodiments, the non-target antigen is not expressed by target cells but is expressed by 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.

[0252] 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 extracellular ligand-binding domain of the second receptor. For example, cell adhesion molecules, intercellular signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane receptors, 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).

[0253] 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 comprising polymorphic residues of ICAM1, COMT, or CXCL16 in complex with major histocompatibility complex class I (MHC-I).

[0254] 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 the present disclosure. In some embodiments, the non-target antigen comprises a major histocompatibility complex (MHC) protein. In some embodiments, the MHC is MHC class I. In some embodiments, the MHC class I protein comprises a human leukocyte antigen (HLA) protein. In some embodiments, the non-target antigen comprises an allele of an HLA class I protein selected from the group consisting of HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the HLA-A allele is an allele of an HLA class I protein selected from the group consisting of HLA-A, HLA-B, HLA-C, or HLA-E. * 01. HLA-A * 02. HLA-A * 03, or HLA-A *In some embodiments, the HLA-B allele comprises HLA-B * In some embodiments, the HLA-C allele comprises HLA-C * Including 07.

[0255] In some embodiments, the non-target antigen comprises an HLA-A. In some embodiments, the non-target antigen comprises an allele of HLA-A. In some embodiments, the allele of HLA-A is HLA-A * 01. HLA-A * 02. HLA-A * 03, or HLA-A * In some embodiments, the non-target antigen is HLA-A * 69. In some embodiments, the non-target antigen is human leukocyte antigen A * 02 allele (HLA-A * In some embodiments, the non-target antigen is human leukocyte antigen A. * 03 allele (HLA-A * In some embodiments, the non-target antigen is human leukocyte antigen A. * 11 alleles (HLA-A * 11).

[0256] In some embodiments, the non-target antigen comprises an allele of HLA-B. In some embodiments, the allele of HLA-B * Including 07.

[0257] In some embodiments, the non-target antigen comprises HLA-C. In some embodiments, the HLA-C allele comprises HLA-C * Including 07.

[0258] 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 loss of heterozygosity in cancer cells.

[0259] Wild-type human ICAM1 is described in NCBI accession number NP_000192.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, ICAM1 is [ka] It contains the amino acid sequence of

[0260] 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.

[0261] 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.

[0262] 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 with 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 with 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.

[0263] 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.

[0264] Wild-type human COMT is described in NCBI accession number 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]

[0265] 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.

[0266] 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.

[0267] 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 with 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 with 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.

[0268] In some embodiments, the non-target antigen comprises a C-X-C motif chemokine ligand 16 (CXCL16) or an antigenic peptide thereof in complex with MHC-I. Human CXCL16 precursor is described in NCBI accession number NP_001094282.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, CXCL16 is [ka] It contains the amino acid sequence of

[0269] 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.

[0270] In some embodiments, the non-target antigen comprises a CXCL16 polymorphism. 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 with 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 with higher affinity for a CXCL16 ligand having a V at position 200 of SEQ ID NO:29 than for a CXCL16 ligand having an A at position 200 of SEQ ID NO:29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 comprising an I at amino acid 142 of SEQ ID NO: 29, and the second receptor comprises a ligand binding domain with higher affinity for a CXCL16 ligand having an I at position 142 of SEQ ID NO: 29 than for a CXCL16 ligand having a T at position 142 of SEQ ID NO: 29. In some embodiments, the non-target antigen comprises a peptide of CXCL16 comprising a T at amino acid 142 of SEQ ID NO: 29, and the second receptor comprises a ligand binding domain with higher affinity for a CXCL16 ligand having a T at position 142 of SEQ ID NO: 29 than for a CXCL16 ligand having an I at position 142 of SEQ ID NO: 29.

[0271] In some embodiments, the non-target antigen is 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

[0272] In some embodiments, the ligand-binding domain of the second inhibitory receptor comprises an scFv. In some embodiments, the scFv binds to HLA-A * 01. HLA-A * 02. HLA-A * 03. HLA-A * 11. HLA-B * 07, or HLA-C * In some embodiments, the scFv binds to HLA-A 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. * 01. HLA-A * 02. HLA-A * 03. HLA-A * 11. HLA-B * 07, or HLA-C * 07 and comprises a sequence selected from the group of sequences set forth in Table 5. In some embodiments, the non-target antigen is HLA-A * 01, and the non-target extracellular ligand-binding domain of the second receptor is an HLA-A receptor listed in Table 5. *In some embodiments, the non-target antigen comprises an HLA-A scFv sequence, 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. * 02, and the non-target extracellular ligand-binding domain of the second receptor is an HLA-A receptor listed in Table 5. * In some embodiments, the non-target antigen comprises an HLA-A scFv sequence, 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. * 03, and the non-target extracellular ligand-binding domain of the second receptor is an HLA-A receptor listed in Table 5. * In some embodiments, the non-target antigen comprises an HLA-A scFv sequence, 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. * 11, and the non-target extracellular ligand-binding domain of the second receptor is an HLA-A * In some embodiments, the non-target antigen comprises an HLA-B scFv sequence, 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. * 07, and the non-target extracellular ligand-binding domain of the second receptor is an HLA-B * In some embodiments, the non-target antigen comprises an HLA-C scFv sequence, 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. * 07, and the non-target extracellular ligand-binding domain of the second receptor is an HLA-C * 07 scFv sequence, 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.

[0273] HLA-A * 01. HLA-A * 02. HLA-A * 03. HLA-A * 11. HLA-B * 07, and HLA-C * Exemplary heavy and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3, respectively) for the 07 ligand binding domain 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] [Table 6-10] [Table 6-11]

[0274] In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the ligand-binding domain of the second inhibitory receptor comprises a CDR sequence set forth in Table 6 or Table 7. * 01. HLA-A * 02. HLA-A * 03, or HLA-A * Contains 11 ligand-binding domains.

[0275] 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 CDR sequence set forth in Table 6. * 07 containing the ligand-binding domain.

[0276] 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 comprising the CDR sequences set forth in Table 6. * 07 containing the ligand-binding domain.

[0277] 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. * 01. HLA-A * 02. HLA-A * 03. HLA-A * 11. HLA-B * 07, or HLA-C * Binds specifically to 07.

[0278] In some embodiments, the extracellular ligand-binding domain of the second receptor is HLA-A * In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A 01 as disclosed in Table 6. *01 Complementarity determining region (CDR) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, or HLA-A in Table 6 * 01 CDRs.

[0279] In some embodiments, the extracellular ligand-binding domain of the second receptor is HLA-A * In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A 02 as disclosed in Table 6. * 02 Complementarity determining region (CDR) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, or HLA-A in Table 6 * 02 CDRs.

[0280] 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 CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs of SEQ ID NOs: 103-108 or 109-114.

[0281] In some embodiments, the extracellular ligand-binding domain of the second receptor is HLA-A * In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A 03 as disclosed in Table 6. * 03 Complementarity determining region (CDR) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, or HLA-A in Table 6 * 03 CDRs.

[0282] In some embodiments, the extracellular ligand-binding domain of the second receptor is HLA-A * In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A 11 as disclosed in Table 7. * 11. Complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, or HLA-A in Table 7 * 11 CDRs.

[0283] In some embodiments, the extracellular ligand-binding domain of the second receptor is HLA-B * In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-B 07 as disclosed in Table 6. * 07 Complementarity determining region (CDR) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, or HLA-B in Table 6 * 07 CDRs.

[0284] In some embodiments, the extracellular ligand-binding domain of the second receptor is HLA-C * In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-C 07 as disclosed in Table 6. * 07 Complementarity determining region (CDR) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, or HLA-C in Table 6 * 07 CDRs.

[0285] In further embodiments of any of the ligand-binding domains, each CDR sequence may have one, two, three, or more substitutions, insertions, or deletions. CDR sequences 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 sequences without undue experimentation.

[0286] In some embodiments, HLA-A * The non-target antigen, including 02, and the ligand-binding domain of the second receptor are HLA-A * In some embodiments, the ligand binding domain comprises an HLA-A 02 ligand binding domain. * In pMHC complexes containing 02, HLA-A * 02. In some embodiments, * In some embodiments, the HLA-A 02 ligand binding domain comprises an scFv domain. * The HLA-A 02 ligand-binding domain comprises any one of SEQ ID NOs: 30 to 41. * The O2 ligand binding domain comprises a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 30-41.

[0287] In some embodiments, the non-target antigen is 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 is HLA-A * 02, and the extracellular ligand-binding domain of the second receptor comprises the sequence of SEQ ID NO:30.

[0288] In some embodiments, the non-target antigen is HLA-A * 02, and the extracellular ligand-binding domain of the second receptor comprises a VL comprising the sequence of 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 the sequence 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.

[0289] In some embodiments, HLA-A * 02 scFv comprises the 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 the heavy chain CDR of any one of SEQ ID NOs: 42 to 53, and the light chain of the antigen-binding domain comprises the light chain CDR of any one of SEQ ID NOs: 42 to 53. In some embodiments, the heavy chain of the antigen-binding domain comprises the heavy chain CDR of any one of SEQ ID NOs: 42 to 53. In some embodiments, the heavy chain of the antigen-binding domain comprises the light chain CDR of any one of SEQ ID NOs: 42 to 53.* The O2 antigen-binding domain comprises a heavy chain and a light chain, the heavy chain comprising a CDR selected from SEQ ID NOs: 45 to 47 and 51 to 53, and the light chain comprising a CDR selected from SEQ ID NOs: 42 to 44 and 48 to 50.

[0290] In some embodiments, HLA-A * The O2 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 to 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 to 41.

[0291] 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.

[0292] In some embodiments, the non-target antigen is HLA-A * 01, and the extracellular ligand-binding domain of the second receptor comprises HLA-A * In some embodiments, the HLA-A ligand-binding domain * In some embodiments, the HLA-A 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. * 01 scFv binds to HLA-A listed in Table 6 * 01 includes CDR sequences.

[0293] In some embodiments, the non-target antigen is HLA-A * 03, and the extracellular ligand-binding domain of the second receptor is HLA-A * In some embodiments, the HLA-A 03 ligand binding domain is *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. * 03 scFv binds to HLA-A listed in Table 6 * 03 Contains CDR sequences.

[0294] In some embodiments, the non-target antigen is HLA-A * 03, and the ligand-binding domain of the second receptor is HLA-A * In some embodiments, the ligand binding domain comprises an HLA-A 03 ligand binding domain. * In pMHC complexes containing 03, HLA-A * 03. In some embodiments, * In some embodiments, the HLA-A ligand binding domain comprises an scFv domain. * The HLA-A 03 ligand-binding domain comprises any one of SEQ ID NOs: 615-628 or SEQ ID NO: 1259. In some embodiments, the HLA-A 03 ligand-binding domain comprises any one of SEQ ID NOs: 615-628 or SEQ ID NO: 1259. * The 03 ligand binding domain comprises a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 615-628 or SEQ ID NO: 1259.

[0295] In some embodiments, the non-target antigen is HLA-A * 03, and the extracellular ligand-binding domain of the second receptor comprises a sequence of SEQ ID NOs: 615-628, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen is HLA-A * 03, and the extracellular ligand-binding domain of the second receptor comprises the sequence of SEQ ID NOs: 615-628.

[0296] In some embodiments, the non-target antigen is HLA-A* 03, and the extracellular ligand-binding domain of the second receptor comprises the sequence of SEQ ID NO: 1259, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen is HLA-A * 03, and the extracellular ligand-binding domain of the second receptor comprises the sequence of SEQ ID NO: 1259.

[0297] In some embodiments, the non-target antigen is HLA-A * 03, and the extracellular ligand-binding domain of the second receptor comprises a VL comprising the sequence DIVMTQSHKFMSTSVGDRVSITCKASQDVSTTVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPPTFGGGTKLEIK (SEQ ID NO: 1266), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises a VH comprising the sequence of EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSTNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTTLITPDYWGQGTTLTVSS (SEQ ID NO: 1267), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, 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.

[0298] In some embodiments, HLA-A *The O3 extracellular ligand-binding domain comprises the complementarity determining regions (CDRs) of any one of SEQ ID NOs: 638-641, 645-648, 650-653, 657-664, 676-682, 693-706, or 1260-1265. In some embodiments, the extracellular ligand-binding domain comprises a sequence at least 95% identical to any one of SEQ ID NOs: 638-641, 645-648, 650-653, 657-664, 676-682, 693-706, or 1260-1265. In some embodiments, the extracellular ligand-binding domain comprises a sequence identical to any one of SEQ ID NOs: 638-641, 645-648, 650-653, 657-664, 676-682, 693-706, or 1260-1265. In some embodiments, the heavy chain of the antigen-binding domain comprises the heavy chain CDR of any one of SEQ ID NOs: 638 to 641, 645 to 648, 650 to 653, 657 to 664, 676 to 682, 693 to 706, or 1260 to 1265, and the light chain of the antigen-binding domain comprises the light chain CDR of any one of SEQ ID NOs: 638 to 641, 645 to 648, 650 to 653, 657 to 664, 676 to 682, 693 to 706, or 1260 to 1265. In some embodiments, the heavy chain of the antigen-binding domain comprises the heavy chain CDR of any one of SEQ ID NOs: 638 to 641, 645 to 648, 650 to 653, 657 to 664, 676 to 682, 693 to 706, or 1260 to 1265. *The 03 antigen-binding domain comprises a heavy chain and a light chain, the heavy chain comprising CDRs selected from SEQ ID NOs: 657 to 664, 676 to 682, 693 to 706, and 1263 to 1265, and the light chain comprising CDRs selected from SEQ ID NOs: 638 to 641, 645 to 648, 650 to 653, and 1260 to 1262. In some embodiments, the extracellular ligand-binding domain of the second receptor is selected from the group consisting of (i) SEQ ID NOs: 638, 645, 650, 657, 676, and 693, (ii) SEQ ID NOs: 638, 645, 650, 658, 677, and 694, (iii) SEQ ID NOs: 638, 645, 650, 659, 678, and 695, (iv) SEQ ID NOs: 638, 645, 650, 660, 678, and 696, (v) SEQ ID NOs: 638, 645, 650, 661, 679, and 697, (vi) SEQ ID NOs: 639, 646, 651, 657, 676, and 698, (vii) SEQ ID NOs: 638, 645, 650, 657, 676, and 699, and (viii) SEQ ID NOs: 639, 646, 651, 6 (ix) SEQ ID NOs: 638, 645, 650, 662, 680, and 701; (x) SEQ ID NOs: 639, 646, 651, 657, 676, and 702; (xi) SEQ ID NOs: 638, 645, 650, 661, 679, and 703; (xii) SEQ ID NOs: 640, 647, 652, 657, 676, and 704; (xiii) SEQ ID NOs: 641, 648, 653, 663, 681, and 705; (xiv) SEQ ID NOs: 638, 645, 650, 664, 682, and 706; or (xv) SEQ ID NOs: 1260 to 1265.

[0299] In some embodiments, the HLA-A*03 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: 615-628 or 1259, and the light chain comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOs: 615-628 or 1259. In some embodiments, the heavy chain comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 615-628 or 1259, and the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 615-629 or 1259.

[0300] In some embodiments, the non-target antigen is HLA-A * 11, and the extracellular ligand-binding domain of the second receptor is HLA-A * In some embodiments, the HLA-A * The 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 binds to HLA-A listed in Table 7 * Contains 11 CDR sequences.

[0301] In some embodiments, the non-target antigen is HLA-B * 07, and the extracellular ligand-binding domain of the second receptor is HLA-B * In some embodiments, the HLA-B 07 ligand binding domain * The 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 binds to HLA-B antigens listed in Table 6. * 07 Contains CDR sequences.

[0302] In some embodiments, the non-target antigen is HLA-C * 07, and the extracellular ligand-binding domain of the second receptor is HLA-C * In some embodiments, the HLA-C 07 ligand binding domain *The 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 binds to HLA-C as listed in Table 6 * 07 Contains CDR sequences.

[0303] In some embodiments, the non-target antigen is HLA-A * Contains 11. HLA-A * 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, for example, those that bind to and recognize HLA-A 11 in a peptide-independent manner as shown in Table 5 above. * Examples of antibodies that bind to IgG11 include, but are not limited to, the following murine and humanized scFv antibodies:

[0304] HLA-A * Exemplary heavy and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3, respectively) for the 11 ligand-binding domains 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 7-1] [Table 7-2]

[0305] In some embodiments, the non-target antigen is HLA-A * 11, and the ligand-binding domain of the second receptor is HLA-A * In some embodiments, the ligand binding domain comprises an HLA-A 11 ligand binding domain. * In pMHC complexes containing 11, HLA-A *11. In some embodiments, * 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 to 122. * The 11 ligand-binding domains comprise a sequence at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 114-122.

[0306] In some embodiments, HLA-A * 11 scFv comprises the complementarity determining regions (CDRs) of any one of SEQ ID NOs: 114 to 122. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOs: 114 to 122. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOs: 114 to 122. In some embodiments, the heavy chain of the antigen-binding domain comprises the heavy chain CDRs of any one of SEQ ID NOs: 132 to 140, and the light chain of the antigen-binding domain comprises the light chain CDRs of SEQ ID NO: 141 ... * The 11 antigen-binding domains comprise a heavy chain and a light chain, the heavy chain comprising one, two, or three CDRs selected from SEQ ID NOs: 92 to 110, and the light chain comprising one, two, or three CDRs selected from SEQ ID NOs: 111 to 113.

[0307] HLA-A * Exemplary heavy and light chain sequences for the 11 antigen-binding domains are provided below in Table 8. In some embodiments, HLA-A * The 11 antigen-binding domains comprise 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 to 140, and the light chain comprises a sequence at least 95% identical to the light chain portion of SEQ ID NO: 141.

[0308] 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]

[0309] Differentially expressed repressor ligands The present disclosure provides suppressor ligands (non-target antigens) that are differentially expressed between cancer cells and normal cells.

[0310] 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 the non-target antigens. In some embodiments, the non-target antigens are expressed by both target cells and non-target cells. However, in these embodiments, the non-target antigens are expressed at higher levels by non-target cells than by 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.

[0311] In an alternative embodiment, the non-target antigen is expressed by the non-target cell but not by the target cell, and in the absence of expression of the non-target antigen, the target cell activates the target receptor, thereby activating the immune cell.

[0312] Differential expression can be determined by any technique known in the art that is used to measure expression. These include, among others, techniques for measuring mRNA and / or protein levels of target genes in cells. Methods for measuring protein levels in a sample include immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and liquid chromatography. Analytical methods include 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.

[0313] The activation of inhibitory receptor by non-target antigen can occur according to various methods 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 signal transduction in the cell that expresses inhibitory receptor can be measured by using reporter gene.

[0314] Without wishing to be bound by theory, whether the expression of a non-target antigen inhibits immune cell activation through the activation of an inhibitory receptor may depend on the ratio of the non-target antigen to the inhibitory receptor. The expression levels of non-target antigens and inhibitory receptors, as well as their ratio, 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 selective targeting of immune cells expressing inhibitory receptors. Low or no expression of non-target antigens on target cells or non-target cells may indicate, for example, that inhibitory receptors are not activated in the immune cells of the present disclosure.

[0315] 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.

[0316] 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 due to crosstalk between inhibitory receptors and activator receptors, resulting in downregulation of the activity of activator receptors. For example, activation of inhibitory receptors by non-target antigens may result in reduced expression of activator receptors on the surface of immune cells.

[0317] In some embodiments, the non-target antigen is expressed at a lower level in target cells 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 in target cells 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 target cells than in 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 target cells than in the non-target cells. In some embodiments, the target cells are a plurality of cancer cells that express low or no non-target antigens.

[0318] 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 extracellular ligand-binding domain of a second receptor. 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.

[0319] 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.

[0320] 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).

[0321] Non-target MHC-I (pMHC) antigens including any of HLA-A, HLA-B, or HLA-C are contemplated within the scope of the present disclosure. In some embodiments, the non-target antigen includes HLA-A. In some embodiments, the non-target antigen includes HLA-B. In some embodiments, the non-target antigen includes HLA-C.

[0322] Non-target antigens include proteins that are poorly expressed or not expressed at all in cancer cells, e.g., lung cancer cells, but are expressed in normal tissues, such as normal lung tissue.

[0323] In some embodiments, the non-target antigen comprises LRRN4 or an antigenic peptide thereof in a complex with MHC-I. Human LRRN4 is described in NCBI accession number NP_689824.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, LRRN4 is [ka] [ka] It contains the amino acid sequence of

[0324] 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.

[0325] In some embodiments, the non-target antigen comprises UPK3B or an antigenic peptide thereof in a 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 number 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] It contains the amino acid sequence of

[0326] 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] It contains the amino acid sequence of

[0327] 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] [ka] It contains the amino acid sequence of

[0328] 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] It contains the amino acid sequence of

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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., decreased cytokine production or decreased immune cell activation). Immune cell inhibition or suppression can be selective and / or reversible, or it may not be selective and / or reversible.

[0333] The inhibitory receptors of the present disclosure may comprise 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. Inhibitory receptors may bind to non-target antigens (e.g., HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F ... * 02). For example, non-target antigens (e.g., HLA-A * 02) binds to or contacts an inhibitory receptor, the inhibitory receptor becomes responsive and activates an inhibitory signal in immune cells that express the inhibitory receptor upon binding of a non-target antigen by the inhibitory receptor's extracellular ligand-binding domain.

[0334] Inhibitory receptors of the present disclosure can include 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.

[0335] In some embodiments, the ligand-binding domain is an antigen-binding domain. Exemplary antigen-binding domains include, inter alia, scFvs, SdAbs, Vβ-only domains, and TCR antigen-binding domains derived from TCR α and β chain variable domains.

[0336] Any type of antigen binding domain is contemplated within the scope of this disclosure.

[0337] In some embodiments, the extracellular ligand-binding domain of the second receptor is an scFv.

[0338] In some embodiments, the extracellular ligand-binding domain of the second receptor is a major histocompatibility complex class I (MHC-I) or HLA-A * It binds to and recognizes polymorphic variants of intercellular adhesion molecule 1 (ICAM1), catechol-O-methyltransferase (COMT), C-X-C motif chemokine ligand 16 (CXCL16), leucine-rich repeat neuronal 4 (LRRN4), and uroplakin 3B UPK3B, or antigenic peptides thereof, in complex with 02. In some embodiments, the extracellular ligand-binding domain of the second receptor is an scFv.

[0339] In some embodiments, the extracellular ligand-binding domain of the second receptor is fused to the extracellular domain of the inhibitory receptor.

[0340] In some embodiments, an inhibitory receptor of the present disclosure comprises an extracellular hinge region. Exemplary hinges can be isolated from or derived from IgD and CD8 domains, e.g., IgG1. In some embodiments, the hinge is isolated from or derived from CD8α or CD28.

[0341] 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 domain to transmembrane domains of the same or different surface membrane proteins and to minimize interactions with other members of the receptor complex.

[0342] The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region can be isolated from or derived from (i.e., comprising at least the transmembrane region(s) 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 can be synthetic, in which case it comprises primarily 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.

[0343] The present disclosure provides inhibitory receptors comprising an intracellular domain. The intracellular domain of the inhibitory receptor of the present disclosure is responsible for suppressing activation of immune cells comprising the inhibitory receptor that would otherwise be activated in response to an activating signal from 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 from or derived from immune checkpoint inhibitors such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immunoinhibitory receptors expressed on the surface of T cells and play important roles in dampening or terminating T cell responses.

[0344] 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.

[0345] 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).

[0346] In some embodiments, the inhibitory domain is isolated from or derived from a human protein, for example, a human TRAIL receptor, CTLA-4, PD-1, PAG1, or LILRB1 protein.

[0347] 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.

[0348] In some embodiments, the inhibitory domain is isolated from or derived from killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin-like receptor B1 (LIR1, also known as LIR-1 and LILRB1), programmed 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).

[0349] In some embodiments, the inhibitory domain is isolated from or derived from a human protein.

[0350] 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.

[0351] In some embodiments, the second inhibitory receptor comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, or portions thereof, isolated or derived from the same protein, e.g., an ITIM-containing protein. In some embodiments, the second inhibitory receptor comprises an intracellular domain and / or a transmembrane domain and a hinge region isolated or derived from the same protein, e.g., an ITIM-containing protein.

[0352] In some embodiments, the second receptor is a TCR comprising an inhibitory domain (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 the intracellular domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma, or CD3 epsilon, or a portion thereof. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma, or CD3 epsilon. In some embodiments, the second receptor is a TCR comprising an inhibitory domain (inhibitory TCR). In some embodiments, the inhibitory domain is isolated from or derived from LILRB1.

[0353] 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 LILRB1 hinge domain in an inhibitory receptor, the inhibitory signal generated by the inhibitory receptor can 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 can be fused to the LILRB1-based second inhibitory receptor.

[0354] 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 two to four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces negative signals that inhibit stimulation of the immune response. LILRB1 is thought to play a role in regulating inflammatory responses and cytotoxicity and limiting autoreactivity. Multiple transcriptional variants encoding different isoforms of LILRB1 exist, all of which are contemplated within the scope of the present disclosure.

[0355] In some embodiments of the inhibitory receptors described herein, the inhibitory receptor comprises one or more domains isolated from or derived from LILRB1. In some embodiments of receptors having one or more domains isolated from 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%, or 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%, or 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 an amino acid sequence identical to the sequence or subsequence of SEQ ID NO:54.

[0356] In some embodiments of a receptor having one or more domains isolated from or derived from LILRB1, one or more domains of LILRB1 are at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or encoded by a polynucleotide sequence identical to, the sequence or subsequence of SEQ ID NO:55.

[0357] In some embodiments of a receptor having one or more domains of LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is identical to the sequence or subsequence of SEQ ID NO:55.

[0358] In various embodiments, an inhibitory receptor comprising a polypeptide is provided, wherein the polypeptide comprises 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 inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 56), VTYAEV (SEQ ID NO: 57), VTYAQL (SEQ ID NO: 58), and SIYATL (SEQ ID NO: 59).

[0359] 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 receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or an inositol-phosphatase called SHIP.

[0360] 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.

[0361] 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).

[0362] 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).

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] In some embodiments, the intracellular domain comprises the ITIM 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.

[0368] 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.

[0369] 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).

[0370] 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.

[0371] 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).

[0372] 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).

[0373] 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).

[0374] 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).

[0375] 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).

[0376] 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).

[0377] 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).

[0378] 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%, or at least 99% identical to, or identical to, SEQ ID NO:66. In some embodiments, the LILRB1 D3D4 domain comprises or consists essentially of SEQ ID NO:66.

[0379] 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%, or 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.

[0380] 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.

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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 from or derived from LILR1B.

[0385] 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 functional equivalents 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.

[0386] 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 identical to SEQ ID NO: 68.

[0387] 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 inhibition 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).

[0388] 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 identical to SEQ ID NO: 69.

[0389] 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).

[0390] In some embodiments, the inhibitory receptor comprises a sequence 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.

[0391] 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.

[0392] 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]

[0393] 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 to the N-terminus with a LILRB1 hinge, transmembrane, and intracellular domain. In some embodiments, the LILRB1 hinge comprises the sequence of SEQ ID NO: 73, the LILRB1 transmembrane domain comprises the sequence of SEQ ID NO: 74, and the LILRB1 intracellular domain comprises the sequence of SEQ ID NO: 70. For example, a second inhibitory receptor comprises an scFv sequence from Table 5 fused to the N-terminus of SEQ ID NO: 71.

[0394] As a further example, the non-target antigen may be HLA-A * 02, and the second inhibitory receptor is [ka] In some embodiments, the non-target antigen comprises a sequence of HLA-A, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. * 02, and the second inhibitory receptor comprises the sequence of SEQ ID NO: 348.

[0395] In yet another example, the non-target antigen is HLA-A * 03, and the second inhibitory receptor is [ka] or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-A*03 and the second inhibitory receptor comprises the sequence of SEQ ID NO: 1268.

[0396] The corresponding nucleotide sequences include: [ka] [ka]

[0397] As a further example, the non-target antigen may be HLA-A * 11, and the second inhibitory receptor is [ka] In some embodiments, the non-target antigen comprises a sequence of HLA-A * 11, and the second inhibitory receptor comprises the sequence of SEQ ID NO: 1269.

[0398] The corresponding nucleotide sequences include: [ka] [ka]

[0399] As a further example, the non-target antigen may be HLA-B * 07, and the second inhibitory receptor is [ka] In some embodiments, the non-target antigen comprises a sequence of HLA-B or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. * 07, and the second inhibitory receptor comprises the sequence of SEQ ID NO: 1270.

[0400] The corresponding nucleotide sequences include: [ka]

[0401] Polynucleotides and Vectors The present disclosure provides polynucleotides encoding the sequence(s) of the first and second receptors of the present disclosure.The present disclosure provides immune cells comprising the polynucleotides and vectors described herein.

[0402] In some embodiments, the sequences of the first and / or second receptors are operably linked to promoters, ie, 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.

[0403] The present disclosure provides vectors comprising the polynucleotides described herein.

[0404] 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.

[0405] In some embodiments, both receptors are encoded by a single vector. In some embodiments, the vector comprises an shRNA, e.g., a B2M shRNA.

[0406] In some embodiments, the first and second receptors are encoded by a single vector. Methods for encoding multiple polypeptides using a single vector are known to those of skill in the art and include, among other things, encoding multiple polypeptides under the control of different promoters, or, when using a single promoter to control transcription of multiple polypeptides, the use of an internal ribosome entry site (IRES) and / or a sequence encoding a self-cleaving peptide. Exemplary self-cleaving peptides include the 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 can also include an N-terminal GSG linker. For example, the T2A self-cleaving peptide can also comprise the sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351), which can be encoded by the sequence GGATCCGGAGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCC (SEQ ID NO: 768).

[0407] In some embodiments, the vector is an expression vector, ie, for expression of the first and / or second receptor in a suitable cell.

[0408] Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow the long-term and stable integration of transgenes and their propagation in daughter cells.Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of low immunogenicity.

[0409] Expression of natural or synthetic nucleic acids encoding receptors 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.

[0410] 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.

[0411] Furthermore, expression vectors can be provided to cells, such as immune cells, in the form of viral vectors. Viral vector technology is well known in the art and is described, 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, suitable vectors contain 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).

[0412] 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 target cells 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, a lentiviral vector is used.

[0413] Additional promoter elements, such as 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 as much as 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription.

[0414] 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-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, the 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, and U6 promoter, as well as human gene promoters such as, but not limited to, the 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.

[0415] To assess receptor expression, the expression vector introduced into cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells to be transfected or infected via the viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA or used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0416] Reporter genes are used to identify potentially transfected or transduced cells and 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 encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (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 commercially available. Generally, the construct with the minimal 5'-flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0417] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0418] 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.

[0419] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and particularly 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, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0420] 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).

[0421] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to inhibitors of the present disclosure, various assays can be performed to confirm the presence of recombinant DNA sequences within the host cells. 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 within the scope of the present disclosure.

[0422] immune cells The present disclosure provides immune cells comprising the receptors, vectors, and polynucleotides described herein.

[0423] In some embodiments, the immune cells are comprised of: (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 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. * and a second receptor comprising a second extracellular ligand binding specific for a non-target antigen selected from intercellular adhesion molecule 1 (ICAM1), catechol-O-methyltransferase (COMT), C-X-C motif chemokine ligand 16 (CXCL16), leucine-rich repeat neuronal 4 (LRRN4), and uroplakin 3B UPK3B, or antigenic peptides thereof, in a complex with 02. In some embodiments, the first receptor is a CAR or TCR. In some embodiments, the second receptor is an inhibitory receptor, such as an inhibitory chimeric antigen receptor or TCR.

[0424] The present disclosure provides immune cells comprising a first receptor comprising the sequence of SEQ ID NO: 303 and a second receptor comprising 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. In some embodiments, the immune cells comprise 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 cells comprise a first receptor comprising the sequence of SEQ ID NO: 303, a second receptor comprising the sequence of SEQ ID NO: 348, and a sequence encoding an shRNA comprising the 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 a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence of GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 351).

[0425] The present disclosure also provides an immune cell comprising a first receptor comprising the sequence of SEQ ID NO: 303, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a second receptor comprising the sequence of SEQ ID NO: 1268, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the immune cell comprises an shRNA encoded by a sequence comprising SEQ ID NO: 349 or 350, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the immune cell comprises a first receptor comprising the sequence of SEQ ID NO: 303, a second receptor comprising the sequence of SEQ ID NO: 1268, and a sequence encoding an shRNA comprising the 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 a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 351).

[0426] 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.

[0427] 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 the expression of their markers. Methods for distinguishing between types of T cells are readily apparent to those skilled in the art.

[0428] In some embodiments, the first receptor and the second receptor together specifically activate an immune cell in the presence of a target cell.

[0429] In some embodiments, the immune cells are CD4+, CD8+, gamma delta T cells, invariant T cells, iNK cells, NK cells, macrophages, or a combination 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.

[0430] In some embodiments, the immune cells are non-naturally occurring. In some embodiments, the immune cells are isolated.

[0431] Methods for transducing populations of immune cells, such as T cells, using the vectors of the present disclosure will be readily apparent to those skilled in the art. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi) according to the manufacturer's instructions. T cells can be isolated at 1 x 10 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 of IL-2 (Miltenyi). 6 T cells can be cultured at a density of 10 ...

[0432] Methods for activating and culturing populations of T cells containing TCRs, CARs, inhibitory receptors, receptors or vectors encoding them will be readily apparent to one of skill in the art.

[0433] Whether before or after genetic modification of the T cells to express a TCR, the T cells may be engineered to express TCRs as described in, for example, 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, Activation and expansion can be achieved using methods generally described in U.S. Patent 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.

[0434] In some embodiments, T cells of the present disclosure are expanded and activated in vitro. Generally, T cells of the present disclosure are expanded in vitro by contact with a surface having bound 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. Costimulation of an accessory molecule on the surface of the T cells uses a ligand that binds to the accessory molecule. 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. Examples of anti-CD28 antibodies include 9.3, B-T3, and 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).

[0435] In some embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be bound to a surface and the other agent can 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 can be in solution. In another embodiment, the agents can be in soluble form and then cross-linked 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.

[0436] In some embodiments, the two agents are immobilized on beads either on the same bead, i.e., "cis," or on separate beads, i.e., "trans." For example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, with both agents co-immobilized on the same bead with comparable molecular weights. In one embodiment, a 1:1 ratio of each antibody bound to beads for CD4+ T cell expansion and T cell growth is used. In some embodiments, the ratio of CD3:CD28 antibody bound to beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one aspect of the present disclosure, more anti-CD28 antibody than anti-CD3 antibody is bound to the particles, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the present disclosure, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to beads is greater than 2:1.

[0437] To stimulate T cells or other target cells, particle-to-cell ratios ranging from 1:500 to 500:1 and any integer value therebetween can be used. As one of ordinary skill in the art will readily appreciate, the particle-to-cell ratio can depend on the particle size relative to the target cells. For example, small beads can bind only a few cells, while larger beads can bind many cells. In certain embodiments, the cell-to-particle ratio ranges from 1:100 to 100:1 and any integer value therebetween. In further embodiments, the ratio includes 1:9 to 9:1 and any integer value therebetween, and can 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 particle size and the size and type of cells.

[0438] In further embodiments of the present disclosure, cells, such as T cells, are bound to 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 but are cultured together prior to culturing. In further embodiments, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0439] As an example, cell surface proteins can be ligated by contacting T cells with anti-CD3 and anti-CD28 conjugated 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 solution. 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 cell concentration) to ensure maximum contact between the cells and particles. For example, in one embodiment, a concentration of approximately 2 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In further embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml are 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 x 10 6 Cells cultured at a density of 10 cells / mL are used.

[0440] In some embodiments, the mixture may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value in between. In another embodiment, the beads and T cells are cultured together for 2 to 3 days. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimal 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 additive for cell growth known to those of skill in the art. Other additives for cell growth 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, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, and are supplemented with amino acids, sodium pyruvate, and vitamins, and are serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokine(s) in an amount sufficient for T cell growth and expansion. In some embodiments, the media comprises X-VIVO-15 media supplemented with 5% human A / B serum, 1% penicillin / streptomycin (pen / strep), and 300 units / ml of IL-2 (Miltenyi).

[0441] The T cells are maintained under conditions necessary to support growth, eg, an appropriate temperature (eg, 37° C.) and atmosphere (eg, air plus 5% CO 2 ).

[0442] In some embodiments, T cells comprising the TCR, CAR, and inhibitory receptor 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 the subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation.

[0443] 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, 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 ordinary skill in the art would readily understand, washing steps can be accomplished by methods known to those of ordinary skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to 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 salt solutions with or without buffer. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0444] In some embodiments, immune cells such as T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, e.g., 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 sufficient for positive selection of the desired T cells.

[0445] Enrichment of immune cell populations, such as T cell populations, by negative selection can be achieved by combining antibodies directed against surface markers unique 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 against 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.

[0446] 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.

[0447] In some embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at either 2-10° C. or room temperature.

[0448] 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 to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40, 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, 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled immediate freezing at -20°C or in liquid nitrogen, may also be used.

[0449] The present disclosure provides immune cells that express an activator receptor and / or a blocker receptor described herein, wherein the immune cells have reduced expression and / or function of major histocompatibility (MHC) class I complex.

[0450] In some embodiments, the immune cells are autologous. For example, the immune cells are isolated from or derived from the same subject who will receive the cells as part of a therapeutic regimen. Modifying autoimmune cells to reduce MHC class I expression and / or function via blocker receptors can be advantageously specific to MHC class I antigens. Without wishing to be bound by theory, modifying autoimmune cells to reduce MHC class I expression and / or function reduces blocker receptor binding by MHC class I expressed by the immune cells, either in cis or trans.

[0451] 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 can 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.

[0452] Any suitable method of reducing the expression and / or function of the 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 MHC class I components, or modification of a gene encoding an MHC class I component.

[0453] The major histocompatibility complex (MHC) is a genetic locus on the vertebrate genome that encodes a series 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 complexes with antigens on the surface of cells. As referred to herein, an MHC class I gene or polypeptide 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, such as HLA-A, HLA-B, and HLA-C, and their alleles. HLA-A alleles include, but are not limited to, HLA-A. * 02. HLA-A * 02:01, HLA-A * 02:01:01, HLA-A * 02:01:01:01, and / or HLA-A * The HLA-A, HLA-B, and HLA-C polypeptides, and their alleles, may be any gene encoding a protein identical or similar to the HLA-A, HLA-B, and HLA-C protein. Therefore, it is desirable to remove or reduce the expression of HLA-A, HLA-B, and HLA-C encoded polypeptides, and their alleles, in immune cells to prevent the autocrine signaling / binding described herein.

[0454] Immune cells with reduced MHC class I polypeptide expression In some embodiments, the immune cells described herein are modified to inactivate, or reduce or eliminate, the expression or function of endogenous genes encoding alleles of endogenous MHC class I polypeptides. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A, HLA-B, and / or HLA-C. HLA-A, HLA-B, and HLA-C are encoded by 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 gene encoding the MHC class I polypeptide is HLA-A. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A. * In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A. * In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A * In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A * The time is 02:01:01:01.

[0455] In some embodiments, the genetically 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 antigen presentation on the cell surface. When B2M is deleted, the MHC-I complex is disrupted and nonfunctional (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.

[0456] The present disclosure provides a gene editing system for editing endogenous target genes in immune cells. The present disclosure provides interfering RNA specific to the sequence of the target gene. Gene editing systems such as CRISPR / Cas systems, TALENs, and zinc fingers can be used to generate double-strand breaks, which can be used to introduce mutations through gene repair mechanisms such as homology-directed repair or non-homologous end joining (NHEJ). NHEJ after resection of the broken end, or improper end joining, can be used to introduce deletions. In some embodiments, the target gene includes a gene encoding a subunit of the MHC-I complex.

[0457] 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 described herein. Modifications to the target gene can be achieved by editing the target gene, resulting in altered or disrupted expression or function of the target gene or gene product, using any method known in the art.

[0458] 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, insertion, substitution, or frameshift mutation. In some embodiments, modifying the gene comprises using a nucleic acid-guided endonuclease.

[0459] 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 using the keyword "HLA-A * 02," "HLA-A * 02:01," "HLA-A * 02:01:01" or "HLA-A* These sequences can be found in the NCBI nucleotide database by searching "SEQ ID NO: 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]

[0460] 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.

[0461] 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.

[0462] As used herein, "CRISPR" or "CRISPR gene editing" refers to a system that includes a series of clustered regularly interspaced short palindromic repeats, or a series of such repeats. "Cas" as used herein refers to a CRISPR-associated protein. The "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. The CRISPR / Cas system has been modified for use in gene editing. This is achieved 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 gNAs. The gNAs guide 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 a deletion) or homologous repair (which can generate an insertion), thereby introducing the genetic modification into the host cell genome.

[0463] CRISPR / Cas systems are classified by class and type. 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 a Type II, Type V, or Type VI system, 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.

[0464] 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).

[0465] An exemplary class 2 type II CRISPR system relies on the protein Cas9, which is a nuclease with two active cleavage sites, one for each strand of the double helix. The combination of 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.

[0466] Therefore, the 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 premature stops, thereby reducing the expression of the target. Alternatively, the CRISPR / Cas system can be used like RNA interference to reversibly turn off target genes. In mammalian cells, for example, RNA can guide Cas proteins to the target gene promoter and sterically block RNA polymerase.

[0467] The Cas protein can be derived from any bacterial or archaeal Cas protein. Any suitable CRISPR / Cas system is contemplated within the scope of this 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, Csm5, Csm6, Csm7, Csm8, Csm9, Csm10, Csm11, Csm12, Csm13, Csm14, Csm15, Csm16, Csm17, Csm18, Csm19, Csm20, Csm21, Csm22, Csm23, Csm24, Csm25, Csm26, Csm27, Csm28, Csm29, Csm21, Csm21, Csm22, Csm23, Csm24, Csm25, Csm26, Csm27, Csm28, Csm29 ... In some embodiments, the Cas protein comprises 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.

[0468] Artificial CRISPR / Cas systems that inhibit target genes can be generated using techniques known in the art, such as those described in U.S. Publication No. 2014 / 0068797 and Cong (2013) Science 339:819-823. Other artificial CRISPR / Cas systems that inhibit target genes known in the art can also be generated, such as 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. Methods for designing suitable gNAs for specific Cas proteins are known to those skilled in the art.

[0469] The present disclosure provides gene-targeting guide nucleic acids (gRNAs) that can direct 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 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 through its association with the site-directed polypeptide. Thus, the gene-targeting nucleic acid can direct the activity of the site-specific polypeptide.

[0470] 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.

[0471] Exemplary guide RNAs include a targeting sequence of approximately 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 incorporated into a single RNA chimera or crRNA.

[0472] 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 gRNAs, or multiple gRNAs used in a single step. While coding sequences and splice junctions are evident from the genome sequence, other features required for gene expression may be idiosyncratic and unclear.

[0473] A 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 comprises a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0474] In a Type II system, a single-molecule guide RNA (sgRNA) 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.

[0475] In some embodiments, the guide RNA or single-molecule guide RNA (sgRNA) can comprise 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 sharing 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).

[0476] In some embodiments, for example, in those embodiments in which the CRISPR / Cas system is a Cas9 system, the sgRNA can comprise a 20-nucleotide targeting sequence at the 5' end of the sgRNA sequence. The sgRNA can comprise a targeting sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a targeting sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a variable-length targeting sequence having 17 to 30 nucleotides at the 5' end of the sgRNA sequence.

[0477] Suitable scaffold sequences and the placement of scaffold target sequences will depend on the choice of endonuclease and will be known to those skilled in the art.

[0478] The single molecule guide RNA (sgRNA) in the Type II system, e.g., Cas9, can comprise, from 5' to 3', a minimal CRISPR repeat sequence and a targeting sequence.

[0479] By way of example, guide RNAs or other smaller RNAs used in CRISPR / Cas9 or CRISPR / Cpf1 systems can be easily synthesized by chemical means, as exemplified below and described in the art. While chemical synthesis procedures continue to expand, as polynucleotide lengths increase significantly beyond approximately 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 encoding Cas9 or Cpf1 endonucleases, are more easily produced enzymatically. Various types of RNA modifications, such as those that improve stability, reduce the likelihood or severity of innate immune responses, and / or improve other attributes described in the art, can be introduced during or after chemical synthesis and / or enzymatic production of RNA.

[0480] The targeting sequence of gRNA hybridizes with the sequence in the target nucleic acid of interest.The targeting sequence of genome targeting nucleic acid can interact with target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing).The nucleotide sequence of targeting sequence can vary according to the sequence of the target nucleic acid of interest.

[0481] 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 perfectly match the target sequence or can have a mismatch. Each CRISPR / Cas system protein may have a specific PAM sequence recognized in a specific orientation and position in the target DNA. For example, S. pyogenes Cas9 recognizes a PAM containing the sequence 5'-NRG-3' in the target nucleic acid, 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.

[0482] The target sequence is complementary to and hybridizes with the targeting sequence of the gRNA. The target nucleic acid sequence can comprise 20 nucleotides. The target nucleic acid can comprise fewer than 20 nucleotides. The target nucleic acid can comprise more than 20 nucleotides. The target nucleic acid can comprise 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 comprise the 20 nucleotides immediately 5' to 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.

[0483] In some cases, 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 cases, 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 instances, 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 on the complementary strand of the target nucleic acid. The percent complementarity between the targeting sequence and the target nucleic acid may be at least 60% over about 20 contiguous nucleotides. The lengths of the targeting sequence and the target nucleic acid may vary by 1 to 6 nucleotides, which may be considered a bulge or multiple bulges.

[0484] Targeting sequences can be designed or selected using computer programs known to those skilled in the art. Computer programs can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic 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, and the presence of SNPs. Available computer programs can take as input an NCBI gene ID, official gene symbol, Ensembl Gene ID, genomic coordinates, or DNA sequence and create an output file containing sgRNAs targeting the appropriate genomic region specified as input. Computer programs 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 guide RNAs comprising targeting sequences. In some embodiments, the guide RNA further comprises a guide RNA scaffold sequence. In some embodiments, the targeting sequence is complementary to 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.

[0485] In some embodiments, a gNA specifically targets a sequence at the endogenous HLA-A locus. In some embodiments, a gNA that 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, a gNA that specifically targets a sequence at the HLA-A locus comprises a sequence selected from the sequences disclosed in Table 11.

[0486] In some embodiments, the gNA is HLA-A * For example, gRNAs target all HLA-A alleles. * It specifically targets and hybridizes with sequences shared by the HLA-A allele, but not with the HLA-A * 02 and HLA-A * In some embodiments, the gNA is not shared by the HLA-A 03 allele. * In some embodiments, the gNA specifically targets the sequence of the HLA-A 02:01 allele. * In some embodiments, the gNA specifically targets the sequence of the HLA-A 02:01:01 allele. * In some embodiments, the gNA specifically targets the sequence of the HLA-A 02:01:01:01 allele. * It specifically targets the sequence of the 02:01:01:01 allele.

[0487] In some embodiments, the gNA is HLA-A * It specifically targets the coding DNA sequence of 02.

[0488] In some embodiments, the gNA is greater than 1000 HLA-A * In some embodiments, the coding DNA sequence shared by more than 1000 HLA-A alleles is specifically targeted. *gNAs that specifically target coding DNA sequences in the 02 allele include sequences that share about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity with, or are identical to, a sequence selected from the sequences listed in Table 11.

[0489] 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 set forth in Tables 11-14, to arrive at the corresponding RNA sequence.

[0490] [Table 11-1] [Table 11-2]

[0491] The sequences disclosed in Table 11 include the corresponding genomic sequences, including the PAM sequence. One skilled in the art will understand that the targeting sequence of a gRNA does not include the three 3'-most nucleotides of the sequences in Table 11, which represent the corresponding PAM site for the gRNA.

[0492] 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.

[0493] 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%, or about 99% identity thereto.

[0494] 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%, or about 99% identity with a B2M sequence shown in Table 10. [Table 12-1] [Table 12-2]

[0495] In some embodiments, the immune cells described herein are edited using TALEN gene editing.

[0496] "TALEN" or "TALEN gene editing" refers to Transcription Activator-Like Effector Nuclease, an artificial nuclease used to edit target genes.

[0497] 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 portions of target genes such as TCR subunits, MHC class I complex components, or CD52. By combining engineered TALEs with DNA cleavage domains, restriction enzymes specific to any desired DNA sequence, including target gene sequences, can be produced. These can then be introduced into cells and used for genome editing.

[0498] 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.

[0499] The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains to be properly oriented and spaced apart to target sites in the 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.

[0500] TALENs specific to sequences within target genes can be constructed using any method known in the art, including various schemes that use modular components.

[0501] In some embodiments, target genes are edited in immune cells described herein using ZFN gene editing.

[0502] "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.

[0503] 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.

[0504] Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. A zinc finger can contain, for example, 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...

Claims

1. a. a first receptor comprising an extracellular ligand-binding domain specific for mesothelin (MSLN); b. HLA-A ＊ a second receptor comprising an extracellular ligand-binding domain specific for .O3; The first receptor is an activator receptor that responds to MSLN, and the second receptor is HLA-A ＊ 03-responsive inhibitory receptors, immune cells.

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

3. 2. The immune cell of claim 1, wherein 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.

4. 2. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the second receptor comprises a heavy chain variable (VH) portion comprising SEQ ID NO: 1267, or a sequence having at least 85% identity thereto; the extracellular ligand-binding domain of the second receptor comprises a light chain variable (VL) portion comprising SEQ ID NO: 1266, or a sequence having at least 85% identity thereto; and / or the extracellular ligand-binding domain of the second receptor comprises SEQ ID NO: 1268, or a sequence having at least 85% identity thereto.

5. The immune cell of claim 1 , wherein the first receptor is a chimeric antigen receptor (CAR).

6. 2. The immune cell of claim 1, wherein 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.

7. 2. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising a sequence set forth in Table 3, and a light chain variable (VL) portion comprising a sequence set forth in Table 4, or a sequence with at least 80% identity thereto.

8. 2. The immune cell of claim 1, wherein 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% identity thereto, and a light chain variable (VL) portion comprising SEQ ID NO: 279, or a sequence having 85% identity thereto.

9. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the first receptor comprises the scFv sequence of SEQ ID NO: 171, or a sequence having at least 85% identity thereto.

10. The immune cell of claim 1 , wherein the first receptor comprises a hinge domain, a transmembrane domain, and an intracellular domain.

11. The immune cell of claim 1, wherein the second receptor comprises a LILRB1 intracellular domain, a LILRB1 transmembrane domain, a LILRB1 hinge domain, a functional variant of any of these, or a combination thereof.

12. The immune cell of claim 11, wherein the LILRB1 hinge domain, LILRB1 intracellular domain, and LILRB1 transmembrane domain comprise SEQ ID NO: 71, or a sequence that is at least 90% identical to SEQ ID NO:

71.

13. The MSLN+ cancer cells are HLA-A ＊ MSLN+ / HLA-A not expressing 03 ＊ 03- The immune cell of claim 1, which is a cancer cell.

14. The immune cell of claim 1 , wherein the immune cell is a T cell.

15. The immune cell of claim 1 , wherein the expression and / or function of MHC class I genes is reduced or eliminated in the immune cell.

16. The immune cell of claim 1 , further comprising a polynucleotide comprising an interfering RNA, wherein the interfering RNA comprises a sequence complementary to a sequence of B2M mRNA.

17. A pharmaceutical composition comprising a therapeutically effective amount of the immune cells of any one of claims 1 to 16.

18. A composition for use in generating immune cells described in any one of claims 1 to 16, comprising a polynucleotide or polynucleotide system including one or more polynucleotides comprising polynucleotide sequences encoding the first receptor and the second receptor.

19. A vector comprising the polynucleotide or polynucleotide system of claim 18.

20. HLA-A ＊ 17. A composition for use in a method of killing MSLN+ cancer cells having loss of heterozygosity at the O3 locus, the composition comprising an immune cell of any one of claims 1 to 16.

21. HLA-A ＊ 17. A composition for use in a method of treating MSLN+ cancer in a subject having a MSLN+ tumor with loss of heterozygosity at the O3 locus, the composition comprising an immune cell of any one of claims 1 to 16.

22. 1. A method for producing a plurality of immune cells, comprising: a. providing a plurality of immune cells; b) transforming said plurality of immune cells with the polynucleotide or polynucleotide system of claim 18.

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