Compositions and methods for treating cancers with hla-a*03 loss of expression

Abstract

The disclosure provides immune cells comprising a first activator receptor specific to a cancer antigen, such as a CAR, and a second inhibitory receptor specific to a ligand that has been lost in the cancer cell, in particular HLA-A*03, and methods of making and using same for the treatment of cancer. The disclosure also relates to HLA-A*03 specific antibodies and related products and uses.

Description

COMPOSITIONS AND METHODS FOR TREATING CANCERS WITH HLA-A*03 LOSSOF EXPRESSIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application 63 / 517,255 filed on August 2, 2023, the contents of which are incorporated herein by reference in its entireties.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy created on July 19, 2024, is named 061250-556001WO.xml and is 1010 KB in size.TECHNICAL FIELD

[0003] The disclosure relates to the fields of adoptive cell therapy and cancer therapeutics.BACKGROUND

[0004] Human leukocyte antigen (HLA) is a subunit of the major histocompatibility complex (MHC) class I (MHC-I), which are encoded by a set of linked, polymorphic genes. MHC is involved in the binding and presentation of antigens on the cell surface for recognition by immune cells. MHC-I molecules, including HL A- A, are highly polymorphic. For example, there are at least 6,425 HL A- A alleles known in humans, which encode at least 3,929 HLA-A proteins. Human leukocyte antigen A*03 (HLA-A*03) is a human leukocyte antigen encoded by the HLA-A locus with a serotype within the HLA-A serotype group. The disclosure provides antigen binding domains that can specifically target A*03 alleles of HLA-A.

[0005] Such antigen binding domains can be used in cell therapy methods. Cell therapy is a powerful tool for the treatment of various diseases, particularly cancers. In conventional adoptive cell therapies, immune cells are engineered to express specific receptors, for example chimeric antigen receptors (CARs) or T cell receptors (TCRs), which direct the activity of the immune cells to cellular targets via interaction of the receptor with a ligand expressed by the target cell. Identification of suitable target molecules remains challenging, as many targets are expressed in normal tissues. This expression can lead to toxicity when the transplanted cells target normal tissues expressing target molecules. There is thus a need in the art for compositions and methods useful in the treatment of disease, particularly cancers, by adoptive cell therapy.

[0006] Mesothelin (MSLN) was proposed as a cancer target in 1992 (Chang et al. Cancer Res 52: 181-86), yet there is still no viable therapy that utilizes MSLN. Not only is it expressed on mostmesotheliomas but also large 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 mesothelium, a tissue that itself may be nonessential. Several investigational therapeutics directed at MSLN have been tested; for example, immunotoxin-conjugates, antibody-drug conjugates, bispecific antibodies, CAR-Ts, and a hybrid TCR-scFv.

[0007] All active systemically administered therapeutics have been toxic. Accordingly, there exists a need in the art for compositions and methods related to treatment of MSLN(+) cancers.SUMMARY

[0008] Provided herein are compositions and methods related to treatment of MSLN(+) cancers. Advantageously, the compositions and methods disclosed herein may exploit loss of heterozygosity (LOH) to address MSLN(+) cancer. The compositions and methods disclosed herein may, in some cases, avoid systemic toxicity to normal tissues by pairing a MSLN-targeted activator receptor with a blocker receptor. Without being bound by theory, the difference in blocker antigen expression in tumor versus normal tissues caused by LOH at the locus encoding the blocker antigen may confer high selectivity for tumor killing.

[0009] The disclosure provides an immune cell comprising: a first receptor, comprising an extracellular ligand binding domain specific to a target antigen; and a second receptor, comprising a humanized extracellular ligand binding domain specific to HLA-A*03 wherein the first receptor is an activator receptor responsive to a target antigen; and wherein the second receptor is an inhibitory receptor responsive to HL A- A* 03.

[0010] In some embodiments, the HLA-A*03 is lost through loss of heterozygosity.

[0011] In some embodiments, the extracellular ligand binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed Table 1 A or Table IB and Table 2A or Table 2B; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 1 A or Table IB and Table 2A or Table 2B.

[0012] In some embodiments, the humanized 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: 2-4 or 33-38 or a sequence disclosed in Table 1 A and 2A; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of SEQ ID NOS: 2-4 or 33-38 or a sequence disclosed in Table 1 A or Table 2A.

[0013] In some embodiments, the humanized 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: 2-4 or 33-38.

[0014] In some embodiments, the humanized extracellular ligand binding domain of the second receptor comprises any one of SEQ ID NO: 83-94 or a sequence disclosed in Table 5A, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0015] In some embodiments, the humanized extracellular ligand binding domain of the second receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 51-57 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto; wherein the extracellular ligand binding domain of the second receptor comprises a variable light (VL) portion comprising SEQ ID NO: 72-78 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto; and / or wherein the humanized extracellular ligand binding domain of the second receptor comprises SEQ ID NO: 83-94, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0016] In some embodiments, the target antigen is a cancer cell-specific antigen. In some embodiments, the cancer cell-specific antigen is selected from the group consisting of EGFR, CEA, MSLN, and HER2. In some embodiments, the cancer cell-specific antigen is MSLN. In some embodiments, the cancer cell-specific antigen is EGFR. In some embodiments, the cancer cellspecific antigen is CEA. In some embodiments, the cancer cell-specific antigen is HER2.

[0017] In some embodiments, the first receptor is a chimeric antigen receptor (CAR).

[0018] 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 Table 7; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 7.

[0019] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a sequence as set forth in Table 8 and a variable light (VL) portion comprising a sequence as set forth in Table 9; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0020] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 917-978 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 979-982 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0021] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 143-208 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0022] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv sequence of SEQ ID NO: 164; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0023] 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 Table 13; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 13.

[0024] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a sequence as set forth in Table 12 and a variable light (VL) portion comprising a sequence as set forth in Table 12; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0025] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 504-510 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 511-517 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0026] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 484-493 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0027] 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 Table 12; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 12.

[0028] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a sequence as set forth in Table 12 and a variable light (VL) portion comprising a sequence as set forth in Table 12; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0029] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 468 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 470 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0030] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 454-460 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0031] 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 Table 16; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 16.

[0032] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a sequence as set forth in Table 16 and a variable light (VL) portion comprising a sequence as set forth in Table 16; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0033] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 16 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 16 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0034] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 590-595 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0035] In some embodiments, the first receptor comprises a hinge domain, a transmembrane domain and an intracellular domain.

[0036] In some embodiments, the hinge domain comprises a CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises a sequence of SEQ ID NO: 602, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0037] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises a sequence of SEQ ID NO: 606, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0038] In some embodiments, the intracellular domain comprises a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, and a CD3(^ activation domain. In some embodiments, the intracellular domain comprises a sequence of SEQ ID NO: 618, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0039] In some embodiments, the first receptor comprises a sequence of SEQ ID NO: 1000, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0040] In some embodiments, the second receptor comprises a hinge domain, a transmembrane domain and an intracellular domain.

[0041] In some embodiments, 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%, or is identical to SEQ ID NO: 636.

[0042] In some embodiments, the second receptor comprises a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB 1 transmembrane domain or a functional variant thereof comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 640.

[0043] In some embodiments, the second receptor comprises a LILRB 1 hinge domain or functional variant thereof. In some embodiments, the LILRB 1 hinge domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 639.

[0044] In some embodiments, the second receptor comprises a LILRB 1 intracellular domain, a LILRB 1 transmembrane domain, a LILRB 1 hinge domain, a functional variant of any of these, or combinations thereof. In some embodiments, the LILRB 1 hinge domain, LILRB 1 intracellular domain and LILRB 1 transmembrane domain comprises SEQ ID NO: 643 or a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 643.

[0045] In some embodiments, the second receptor comprises a sequence of SEQ ID NO: 1045, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0046] In some embodiments, 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 kidney cancer cell, an uterine cancer cell, a gastric cancer cell, a pancreatic cancer cell, a lung cancer cell, a colorectal cancer cell or a cholangiocarcinoma cell. 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.

[0047] In some embodiments, the MSLN+ cancer cell is a MSLN+ / HLA-A*03- cancer cell that does not express HLA-A*03. In some embodiments, the MSLN+ / HLA-A*03- cancer cell is derived from a MSLN+ / HLA-A*03+ cell by loss of heterozygosity at HLA-A leading to loss of HLA-A*03.

[0048] In some embodiments, the first receptor and the second receptor together specifically activate the immune cell in the presence of the MSLN+ / HLA-A*03- cancer cell having loss of heterozygosity.

[0049] In some embodiments, the first receptor and the second receptor together do not specifically activate the immune cell in the presence of an MSLN+ cell that has not lost HLA-A*03 by loss of heterozygosity.

[0050] In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8+ CD4- T cell or a CD8- CD4+ T cell.

[0051] In some embodiments, expression and / or function of a MHC Class I gene has been reduced or eliminated. In some embodiments, the MHC Class I gene is beta-2-microglobulin (B2M). In some embodiments, the immune cell further comprises a polynucleotide comprising an interfering RNA, the interfering RNA comprising a sequence complementary to a sequence of a B2M mRNA. In some embodiments, the interfering RNA comprises a sequence selected from the group of sequences as set forth in Table 23, or a sequence having at most 1, 2, 3, or 4 substitutions, insertions or deletions relative thereto. In some embodiments, the interfering RNA is capable of inducing RNAi-mediated degradation of the B2M mRNA. In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises: a first sequence, having from 5’ end to 3’ end a sequence complementary to a sequence of the B2M mRNA; and a second sequence, having from 5’ end to 3’ end a sequence complementary to the first sequence, wherein the first sequence and the second sequence form the shRNA. In some embodiments, the shRNA is encoded by a sequence comprising a sequence of GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 915) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 916), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

[0052] In some embodiments, the one or more modifications reduce the expression and / or eliminate the function of B2M. In some embodiments, the one or more modifications comprise one or more inactivating mutations of the endogenous gene encoding B2M. In some embodiments, the one or more inactivating mutations comprise a deletion, an insertion, a substitution, or a frameshift mutation. In some embodiments, the one or more inactivating mutations are introduced with a nucleic acid guided endonuclease in a complex with at least one guide nucleic acid (gNA) that specifically targets a sequence of the endogenous gene encoding B2M. In some embodiments, the gNA comprises a sequence selected from the group of sequences as set forth in Table 12, or a sequence having at most 1, 2, 3, or 4 substitutions, insertions or deletions relative thereto.

[0053] In some embodiments, the MHC Class I gene is HLA-A*03.

[0054] In some embodiments, the immune cell further comprises a polynucleotide comprising an interfering RNA, comprising a sequence complementary to a sequence of an HLA-A*03 mRNA. In some embodiments, the interfering RNA is capable of inducing RNA interference (RNAi)-mediated degradation of the HLA-A*03 mRNA. In some embodiments, the interfering RNA is a short hairpin RNA (shRNA) comprising: a first sequence, having from 5’ end to 3’ end a sequence complementary to a sequence of the HLA-A*03 mRNA; and a second sequence, having from 5’ end to 3’ end a sequence complementary to the first sequence, wherein the first sequence and the second sequence form the shRNA.

[0055] In some embodiments, the one or modifications reduce the expression and / or eliminate the function of HLA-A*03. In some embodiments, the one or more modifications comprise one or more inactivating mutations of the endogenous gene encoding HLA-A*03. In some embodiments, the one or more inactivating mutations are introduced with a nucleic acid guided endonuclease in a complex with at least one guide nucleic acid (gNA) that specifically targets a sequence of the endogenous gene encoding HLA-A*03.

[0056] In some embodiments, the first receptor comprises a sequence of SEQ ID NO: 1000, and the second receptor comprises a sequence of SEQ ID NO: 1045, or sequences having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the immune cell further comprises an shRNA encoded by a sequence comprising GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 915) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 916) or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the first receptor and second receptor are encoded by a single polynucleotide, and wherein the sequences encoding the first and second receptors are separated by a sequence encoding a selfcleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A selfcleaving polypeptide comprising a sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 657).

[0057] In some embodiments, the immune cell is autologous.

[0058] In some embodiments, the immune cell is allogeneic.

[0059] Also provided herein is a pharmaceutical composition, comprising a therapeutically effective amount of the immune cells of the present disclosure. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, for use as a medicament in the treatment of MSLN+ cancer.

[0060] Also provided herein is a polynucleotide or polynucleotide system, comprising one or more polynucleotides comprising polynucleotide sequences encoding a first receptor, comprising an extracellular ligand binding domain specific to a target antigen; and a second receptor, comprising a humanized extracellular ligand binding domain specific to HLA-A*03, wherein the first receptor is an activator receptor responsive to a target antigen; and wherein the second receptor is an inhibitory receptor responsive to HL A- A* 03.

[0061] Also provided herein is a polynucleotide or polynucleotide system, comprising one or more polynucleotides comprising polynucleotide sequences encoding the first receptor and the second receptor for use in generating the immune cells of the present disclosure. In some embodiments, the system comprises a sequence encoding an shRNA specific to B2M. In some embodiments, the sequences encoding the first receptor, the second receptor and the shRNA specific to B2M areencoded by the same polynucleotide. In some embodiments, the sequence encoding the shRNA specific to B2M comprises GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 915) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 916) or a sequence having at least 80%, at least 90%, or at least 95% identity thereto; the sequence encoding the first receptor comprises a sequence encoding a polypeptide comprising a of SEQ ID NO: 1000, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto; and the sequence encoding the second receptor comprises a sequence encoding a polypeptide comprising a sequence of SEQ ID NO: 1045, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

[0062] The present disclosure also provides a vector, comprising the one or more polynucleotides as described herein.

[0063] The present disclosure also provides a method of killing a cancer cell having loss of heterozygosity at an HLA-A*03 locus, comprising administering to the subject an effective amount of the immune cell as described herein or the pharmaceutical composition as described herein.

[0064] The present disclosure also provides a method of treating cancer in a subject having a MSLN+ tumor having loss of heterozygosity at an HLA-A*03 locus, comprising administering to the subject an effective amount of the immune cell as described herein or the pharmaceutical composition as described herein.

[0065] The present disclosure also provides a method of treating a cancer in a subject comprising: determining HLA-A genotype or expression of normal cells and a plurality of cancer cells of the subject; optionally, determining the expression of target antigen in a plurality of cancer cells of the subject; and administering to the subject an effective amount of the immune cell as described herein or the pharmaceutical composition as described herein if the normal cells express HLA-A*03 and the plurality of cancer cells do not express HLA-A*03, and the plurality of cancer cells express the target antigen. In some embodiments, the subject is a heterozygous HLA-A*03 patient with a malignancy that expresses a target antigen selected from the group consisting of MSLN (MSLN+), EGFR, CEA, and HER2 and has lost HLA-A*03 expression. In some embodiments, the subject is a heterozygous HLA-A*03 patient with recurrent unresectable or metastatic solid tumors that express a target antigen selected from the group consisting of MSLN, EGFR, CEA, and HER2 and have lost HLA-A*03 expression. In some embodiments, the target antigen is MSLN. In some embodiments, the target antigen is EGFR. In some embodiments, the target antigen is CEA. In some embodiments, the target antigen is HER2.

[0066] In some embodiments, the cancer comprises mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, uterine cancer,gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or cholangiocarcinoma. In some embodiments, the cancer has relapsed in a subject, the cancer is refractory to one or more prior administered anti cancer therapies, and / or the cancer is metastatic.

[0067] In some embodiments, the cancer cells comprise MSLN+ / HLA-A*03- cancer cells that do not express HLA-A*03. In some embodiments, the MSLN+ / HLA-A*03- cancer cells are derived from a MSLN+ / HLA-A*03+ cell by loss of heterozygosity at HLA-A leading to loss of HLA-A*03.

[0068] In some embodiments, the first receptor and the second receptor together specifically activate the immune cell in the presence of the MSLN+ / HLA-A*03- cancer cells.

[0069] In some embodiments, the first receptor and the second receptor together do not specifically activate the immune cell in the presence of a MSLN+ cell that has not lost HLA-A*03.

[0070] In some embodiments, administration of the immune cell or the pharmaceutical composition reduces the size of a tumor in the 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.

[0071] In some embodiments, administration of the immune cell or the pharmaceutical composition arrests the growth of a tumor in the subject.

[0072] In some embodiments, administration of the immune cell or the pharmaceutical composition reduces the number of tumors in the subject.

[0073] In some embodiments, administration of the immune cell or the pharmaceutical composition results in selective killing of a cancer cell but not a normal cell in the subject. In some embodiments, at least about 60% of the cells killed are cancer cells, about 65% of the cells killed are cancer cells, about 70% of the cells killed are cancer cells, about 75% of the cells killed are cancer cells, about 80% of the cells killed are cancer cells, about 85% of the cells killed are cancer cells, about 90% of the cells killed are cancer cells, about 95% of the cells killed are cancer cells, or about 100% of the cells killed are cancer cells. In some embodiments, administration of the immune cell or pharmaceutical composition results in the killing of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or all of the cancer cells of the subject. In some embodiments, administration of the immune cell or the pharmaceutical composition results in fewer side effects for the subject than administration of an otherwise equivalent immune cell comprising the first activator receptor but no second inhibitory receptor.

[0074] Also provided herein is a method of making a plurality of immune cells, comprising: providing a plurality of immune cells, and transforming the plurality of immune cells with the polynucleotide system described herein, or the vector described herein.

[0075] Also provided herein is a kit comprising the immune cell of the present disclosure or the pharmaceutical composition of the present disclosure. In some embodiments, the kit further comprises instructions for use.

[0076] Any immune cell, recombinant cell, polypeptide, nucleic acid, composition, pharmaceutical composition, or method disclosed herein is applicable to any herein-disclosed immune cell, recombinant cell, polypeptide, nucleic acid, composition, pharmaceutical composition, or method. In other words, any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIG. 1 is a set plots showing the enrichment of anti-HLA-A*03 binders through multiple rounds of cell sorting of a mammalian scFv display library.

[0078] FIG. 2 is a set of plots showing A*03-pMHC tetramer staining of Jurkat cells transfected with CAR constructs containing the A*03-directed scFvs.

[0079] FIG. 3 is a plot showing Jurkat NF AT luciferase (JNL) cell activation in a solid-state assay.

[0080] FIG. 4 is a set of plots showing Jurkat NF AT luciferase (JNL) cell activation in an mRNA titration assay.

[0081] FIG. 5 is a set of plots showing that Jurkat cells transfected with a CAR construct containing the GAP-A3 hybridoma derived scFv A3-15, but not A3-16, binds A*03-pMHC tetramer.

[0082] FIG. 6 is a set of plots showing that the HuTARG-derived A*03-scFv-LIRl (A3-3) and GAP- A3 hybridoma-derived A*03-scFv-LIRl (A3-15) efficiently block MSLN activator.

[0083] FIG. 7 is a set of plots showing that the HuTARG-derived A*03-scFv-LIRl blocks CEA, MSLN, EGFR, HER2 and HLA-E CAR activators.

[0084] FIG. 8 is a set of plots showing that the GAP-A3 hybridoma-derived A*03-scFv-LIRl blocks CEA, MSLN, EGFR, HER2 and HLA-E CAR activators.

[0085] FIG. 9 is a set of histograms FLAG-epitope tagged HLA-A*03:01 mRNA titrated in HeLa cells, detected with anti-A*03:01 antibody GAP-A3.

[0086] FIG. 10 shows the characterization of MSLN CAR and MSLN A*03 Tmod (i.e. mBA) constructs in Jurkat cell functional assays. mBA GAP -A3 = construct with mouse GAP -A3 blocker and MSLN activator (no shRNA).

[0087] FIG. 11 shows the functional characterization of MSLN CAR and MSLN A*03 Tmod constructs in primary T cells from 5 different donors on MS751 target cells. mBAsh (GAP -A3) = construct with mouse GAP- A3 blocker and MSLN activator (plus P2M shRNA).

[0088] FIG. 12 shows the functional characterization of MSLN CAR and MSLN A*03 Tmod constructs in primary T cells from 5 different donors on HeLa target cells. mBAsh (GAP-A3) = construct with mouse GAP- A3 blocker and MSLN activator (plus P2M shRNA).

[0089] FIG. 13A and FIG. 13B show the Tmod construct mediates selective killing of tumor cells in a xenograft model. FIG. 13A shows a schematic diagram of the dual-flank tumor and normal MS751 xenograft model. FIG. 13B shows xenograft sizes assessed by caliper measurements. Day 0 = xenograft injection. Horizontal dashed line indicated time of T cell injection. mBAsh (GAP- A3) = construct with mouse GAP- A3 blocker and MSLN activator (plus P2M shRNA).

[0090] FIG. 14A shows an alignment of the VH domains of the humanized sequences.

[0091] FIG. 14B shows a phylogenetic tree of the VH domains of the humanized sequences.

[0092] FIG. 15A shows an alignment of the VL domains of the humanized sequences.

[0093] FIG. 15B shows a phylogenetic tree of the VL domains of the humanized sequences.

[0094] FIG. 16A shows an alignment of the VL domains of the humanized sequences.

[0095] FIG. 16B shows a phylogenetic tree of the VL domains of the humanized sequences.

[0096] FIG. 17A shows an alignment of the VL domains of the humanized sequences.

[0097] FIG. 17B shows a phylogenetic tree of the VL domains of the humanized sequences.

[0098] FIG. 18A shows an alignment of the VL domains of the humanized sequences.

[0099] FIG. 18B shows a phylogenetic tree of the VL domains of the humanized sequences.

[0100] FIG. 19 is a plot showing that 10 humanized GAP-A3 hybridoma-derived HLA-A*03 blockers can block MSLN CAR activation to varying degrees.

[0101] FIG. 20 shows that mouse and humanized GAP -A3 hybridoma-derived HLA-A*03 blockers (B) can block a variety of activating (A) CARs. In each graph, the A + B line is lower at lOOngs of A*03:01 mRNA compared to the A.

[0102] FIG. 21 a set of plots showing that the humanized GAP -A3 hybridoma-derived A*03-scFv- LIR1 blocks Ny-ESO-1, CEA, KRAS G12V or KRAS G12D TCRs in Jurkat cell functional assays. TCRs were paired with A*03 blocker (closed circles) or empty vector control (open circles). Jurkat NF AT luciferase cells expressing the TCR + / - blocker were co-cultured with peptide-loaded HLA- A*02(+) or HLA-A* 11(+) HeLa cells transfected with a titration of A*03 :01 mRNA (FIG. 22B). The functional response (RLU) was assessed after a 6 hour co-culture.

[0103] FIG. 22A shows the characterization of MSLN CAR and MSLN A*03 Tmod constructs in Jurkat cell functional assays. MSLN activator CAR was paired with A*03 blocker (MSLN Tmod; closed circles) or empty vector control (MSLN CAR; open circles). Jurkat NF AT luciferase cells expressing the CAR + / - blocker were co-cultured with wild-type, endogenous MSLN(+) HeLa cells transfected with a titration of A*03:01 mRNA (FIG. 22B). The functional response (RLU) was assessed after a 6 hour co-culture. Titrated antigen molecules on the surface were quantified using the QIFIKIT. IC50 (molecules / cell) values are indicated in FIG. 22A. FIG. 22C shows expression of MSLN CAR and MSLN CAR paired with A*03 blocker in Jurkat cells.

[0104] FIG. 23 shows the functional characterization of MSLN CAR and MSLN A*03 Tmod constructs in primary T cells from 1 representative donor on HeLa target cells (left) and MS751 target cells (right). HeLa and MS751 tumor cells express the MSLN antigen. HeLa and MS751 normal cells express both MSLN and A*03 antigens. The selectivity window between tumor cells versus, normal cells for 5 unique donors, including the representative donor, are indicated in the table.

[0105] FIG 24A and FIG. 24B show the Tmod construct mediates selective killing of tumor cells in a xenograft model. FIG. 24A shows a schematic diagram of the dual-flank tumor and normal MS751 xenograft model. FIG. 24B shows xenograft sizes assessed by caliper measurements. Day 0 = xenograft injection. Horizontal dashed line indicates time of T cell injection.

[0106] FIG. 25 shows the functional characterization of mouse and humanized GAP -A3 hybridoma- derived HLA-A*03 blockers expressed with MSLN CAR. GAP -A3 hybridoma-derived HLA-A*03 blockers block activation from the MSLN-directed CAR only in the presence of HLA-A*03 and not other HLA-A, B, and C alleles. Jurkat NF AT Luciferase (JNL) cells with B2M knockout were transfected with empty vector alone (open circle), MSLN CAR alone (dark grey open triangle), MSLN CAR with mouse derived HLA-A*03 blocker (closed black circle), MSLN CAR with humanized HLA-A*03 blocker (closed grey circle), mouse derived HLA-A-*03 activating CAR (open black triangle) or humanized HLA-A*03 activating CAR (open grey triangle).

[0107] FIG. 26 is a set of flow cytometry plots showing various pMHC tetramer staining of Jurkat cells transfected with a CAR construct containing the A* 03 -directed humanized GAP- A3 scFv

[0108] FIG. 27 is a set of flow cytometry plots showing various pMHC tetramer staining of Jurkat cells transfected with a CAR construct containing the A*03-directed mouse GAP-A3 scFv.

[0109] FIG. 28 shows functional characterization of MSLN CAR and MSLN A*03 Tmod constructs in primary T cells from 3 unique donors on HeLa target cells in mixed cultures. HeLa tumor cells express the MSLN antigen, AB normal cells express both MSLN and A*03 antigens, and B-only normal cells express A*03 antigen but not MSLN. Each symbol (e.g., triangle, circle, square) represents cells from a unique donor. In the figure, T cells were transduced with MSLN CAR alone (open bars) or MSLN CAR with humanized HLA-A*03 blocker (closed bars). The transduced primary T cells were then co-cultured with Hela cells expressing MSLN alone (tumor cells), MSLN + HLA- A*03 antigen (AB normal cells) or HLA-A*03 antigen alone (B-only normal cells).

[0110] FIG. 29 shows the functional characterization of MSLN CAR and MSLN A*03 Tmod constructs in HLA-A*03(+) primary T cells on MS751 target cells (top) and HeLa target cells (bottom). MS751 and HeLa tumor cells express the MSLN antigen. MS751 and HeLa normal cells express both MSLN and A*03 antigens. The selectivity window between tumor cells versus normal cells for 4 unique donors, including a representative HLA-A*03(-) donor, are indicated in the tables. Primary T cells expressing the CAR alone are the dark lines. Primary T cells expressing the MSLNCAR and MSLN A* 03 Tmod constructs are in grey. The A tumor cells (MSLN+) percent kill line is marked with closed circles, the normal cells (MSLN+ / HLA-A*03+) percent kill line is marked with open circles and the B only target (HLA-A*03) percent kill line is marked with x.DETAILED DESCRIPTIONDefinitions[OHl] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

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

[0113] As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0114] As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. As also used herein, in any instance or embodiment described herein, “comprising” may be replaced with “consisting essentially of’ and / or “consisting of’ used herein, in any instance or embodiment described.

[0115] As used herein, the term “about” or “approximately” refers to a quantity, 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% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0116] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term“about,” when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.

[0117] As used herein, the phrases “at least one”, “one or more”, and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0118] As used herein, “or” may refer to “and”, “or,” or “and / of ’ and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0119] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

[0120] As used herein, the “administration” of an agent, e.g., an anti-HLA-A*03 antibody or CAR- expressing cell, to a subject or subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and target cell or tissue. Non-limiting examples of route of administration include parenteral, enteral, and topical routes of administration. Administration includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.

[0121] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals. Similarly, the term “subject” or “patient” includes both human andveterinary subjects, for example, humans, non-human primates, dogs, cats, sheep, mice, horses, and cows.

[0122] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as nonmammalian species, such as shark immunoglobulins. The term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M1greater, at least I O4M1greater or at least 105M1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology 3rd, Ed., W.H. Freeman & Co., New York, 1997. The term antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

[0123] “Antibody fragments” or “antigen binding fragments” include proteolytic antibody fragments (such as F(ab')2 fragments, Fab' fragments, Fab'-SH fragments and Fab fragments as are known in the art), recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments, single chain Fv proteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), diabodies, and triabodies (as are known in the art), and camelid antibodies (see, for example, U.S. Pat. Nos. 6,015,695; 6,005,079; 5,874,541; 5,840,526; 5,800,988; and 5,759,808). An scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.

[0124] As used herein, the term “antigen” refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungalantigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.

[0125] As used herein, “binding affinity” refers to the tendency of one molecule to bind (typically non-covalently) with another molecule, such as the tendency of a member of a specific binding pair for another member of a specific binding pair. A binding affinity can be measured as a dissociation constant, which for a specific binding pair (such as an antibody / antigen pair) can be lower than 1 x 105M, lower than 1 x 106M, lower than 1 x 107M, lower than 1 x 108M, lower than 1 x 109M, lower than 1 x 1010M, lower than 1 x 101 1M or lower than 1 x 1012M. In one aspect, binding affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979. In another aspect, binding affinity is measured by a binding constant. In another aspect, binding affinity is measured by an antigen / antibody dissociation rate. In yet another aspect, a high binding affinity is measured by a competition radioimmunoassay.

[0126] The term “specific” or “specificity” refers to the ability of a molecule to bind to a unique epitope. For example, with antibodies or antigen binding domains derived from antibodies, specificity can either be viewed as a measure of the goodness of fit between the antibody-combining site (paratope) and the corresponding antigenic determinant (epitope), or the ability of the antibody to discriminate between similar or even dissimilar antigens.

[0127] The term “human antibody” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis or by somatic mutation in vivo). However, the term “human antibody” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a rabbit, have been grafted onto human framework sequences. Thus, as used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CHI, CH2, CHS), hinge, VL, VH) is substantially non-immunogenic in humans, with only minor sequence changes or variations. Similarly, antibodies designated primate (monkey, baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig, hamster, and the like) and other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies. Further, chimeric antibodies include any combination of the above. Such changes or variations optionally and preferably retain or reduce the immunogenicity in humans or other species relative to non-modified antibodies. Thus, a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linkerpeptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin.

[0128] As used herein, “HLA-A*03 antibody” and “anti-HLA-A*03 antibody” are used interchangeably, and refer to an antibody that specifically binds to an HLA-A*03 polypeptide. Similarly, the term “HLA-A*03”, as mentioned in “HLA-A*03 binding”, “HLA-A*03 targeting”, “HLA-A*03 specific”, “HLA-A*03 expression” or other similar terms here, refers to HLA-A*03 polypeptide. HLA-A*03 antibodies of the disclosure may bind to proteins falling within the HLA- A*03 allele group (for example HLA-A*03 :01, HLA-A*03 :02 and the like), and may not bind, or bind with lower affinity, to proteins falling within other HLA-A allele groups (for example, HLA-A*02, HL A- A* 11, and the like). The person of ordinary skill in the art will recognize that some crossreactivity with other HLA antigens may exist, but that the HLA-A*03 antibodies of the disclosure will still be considered to be specific to HLA-A*03. In some cases, specificity is considered in the context of the subject to be treated with an HLA-A*03 antibody or receptor of the disclosure. When the subject has both an HLA-A*03 allele and a second HLA-A allele not recognized, or only poorly recognized, by the HLA-A*03 antibody or receptor comprising an equivalent antigen binding domain, the HLA- A*03 antibody is specific to the HLA-A*03 allele of the subject.

[0129] As used herein, the term “monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0130] As used herein, the term “isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated.

[0131] The terms “subject,” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, 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 mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0132] As used herein “treatment” or “treating,” includes any beneficial or desirable effect, and may include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0133] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of disease prior to onset or recurrence.

[0134] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a virus to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0135] A “therapeutically effective amount” of a virus or cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell 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).

[0136] An “increased” or “enhanced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0137] A “decreased” or “reduced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a “statistically significant” amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0138] By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule / composition. A comparable response is one that is not significantly different or measurable different from the reference response.

[0139] In general, “sequence identity” or “sequence homology” refers to an exact nucleotide-to- nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or moresequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the 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 as 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 may 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 in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0140] As used herein, a “polynucleotide system” refers to one or more polynucleotides. The one or more polynucleotides may be designed to work in concert for a particular application, or to produce a desired transformed cell.

[0141] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0142] The term “MOI” is used herein to refer to multiplicity of infection, which is the ratio of agents (e.g. viral particles) to infection targets (e.g. cells).

[0143] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.

[0144] As used herein, a “target cell” refers to cell that is targeted by an adoptive cell therapy. For example, a target cell can be cancer cell, which can be killed by the transplanted T cells of the adoptivecell therapy. Target cells of the disclosure express a target antigen, as described herein, and do not express a non-target antigen.

[0145] As used herein, a “non-target cell” refers to cell that is not targeted by an adoptive cell therapy. For example, in an adoptive cell targeting cancer cells, normal, healthy, non-cancerous cells are non- target cells. Some, or all, non-target cells in a subject may express both the target antigen and the non- target antigen. Non-target cells in a subject may express the non-target antigen irrespective of whether or not these cells also express the target antigen.

[0146] As used herein, a “non-target allelic variant” refers to an allele of a gene whose product is expressed by non-target cells, but is not expressed 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 subject, but not expressed by cancer cells of the subject. The expression of the non-target allelic variant can be lost in the 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.

[0147] As used herein, “specific to” or “specifically binds to” when used with respect to a ligand binding domain, such as an antigen binding domain, refers to a ligand binding domain that has a high specificity for a named target. Antibody specificity can be viewed as a measure of the goodness of fit between the ligand binding domain and the corresponding ligand, or the ability of the ligand binding domain to discriminate between similar or even dissimilar ligands. In comparison with specificity, affinity is a measure of the strength of the binding between the ligand binding domain and ligand, such that a low-affinity ligand binding domain binds weakly and high-affinity ligand binding domain binds firmly. A ligand binding domain that is specific to a target allele is one that can discriminate between different alleles of a gene. For example, a ligand binding domain that is specific to HLA-A*03 will not bind, or bind only weakly to, other HLA-A alleles such as HLA-A*01 or HLA-A*02. The person of skill in the art will appreciate that a ligand binding domain can be said to be specific to a particular target, and yet still have low levels of binding to one or more additional targets that do not affect its function in the receptor systems described herein.

[0148] As used herein, a “target antigen,” whether referred to using the term antigen or the name of a specific antigen, refers to an antigen expressed by a target cell, such as a cancer cell. Expression of target antigen is not limited to target cells. Target antigens may be expressed by both cancer cells and normal, non-cancer cells in a subject.

[0149] As used herein, a “non-target antigen” (or “blocker antigen”) whether referred to using the term antigen or the name of a specific antigen, refers to an antigen that is expressed by normal, non-cancer cells and is not expressed in cancer cells. This difference in expression allows the inhibitory receptor to inhibit immune cell activation in the presence of non-target cells, but not in the presence of target cells.

[0150] Polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population. A polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion. A polymorphism includes e.g., a simple sequence repeat (SSR) and a single nucleotide polymorphism (SNP), which is a variation, occurring when a single nucleotide of adenine (A), thymine (T), cytosine (C) or guanine (G) is altered.

[0151] As used herein, “affinity” refers to strength of binding of a ligand to a single ligand binding site on a receptor, for example an antigen for the antigen binding domain of any of the receptors described herein. Ligand binding domains can have a weaker interaction (low affinity) with their ligand, or a stronger interaction (high affinity).

[0152] Kd, or dissociation constant, is a type of equilibrium constant that measures the propensity of a larger object to separate reversibly into smaller components, such as, for example, when a macromolecular complex comprising receptor and its cognate ligand separates into the ligand and the receptor. When the Kd is high, it means that a high concentration of ligand is needed to occupy the receptor, and the affinity of the receptor for the ligand is low. Conversely, a low Kd means that the ligand has a high affinity for the receptor.

[0153] As used herein, a receptor that is “responsive” or “responsive to” refers to a receptor comprising an intracellular domain, that when bound by a ligand (i.e. 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 an immune cell expressing the activator receptor. An inhibitory receptor bound to a non-target antigen can generate an inhibitory signal that prevents or reduces an activation of an immune cell expressing the activator receptor. Responsiveness of receptors, and their ability to activate or inhibit immune cells expressing the receptors, can be assayed by any means known in the art and described herein, including, but not limited to, reporter assays and cytotoxicity assays.

[0154] As used herein, “activation” of an immune cell or an immune cell that is “activated” refers to an immune cell that can carry out one or more functions characteristic of an immune response. These functions include proliferation, release of cytokines, and cytotoxicity, i.e. killing of a target cell. Activated immune cells express markers that will be apparent to persons of skill in the art. For example, activated T cells can express one or more of CD69, CD71, CD25 and HLA-DR. An immune cell expressing an activator receptor (e.g. a MSLN CAR) can be activated by the activator receptor when it becomes responsive to the binding of the receptor to a target antigen (e.g. MSLN) expressed by the target cell. A “target antigen” can also be referred to an “activator antigen” and may be isolated or expressed by a target cell. Activation of an immune cell expressing an inhibitory receptor can be prevented when the inhibitory receptor becomes responsive to a non-target antigen (e.g. HLA-A*03), even when the activator receptor is bound to the target activator ligand. A “non-target antigen” canalso be referred to as an “inhibitory ligand” or a “blocker”, and may be isolated or expressed by a target cell.

[0155] Receptor expression on an immune cell can be verified by assays that report the presence of the activator receptors and inhibitory receptors described herein. For example, a population of immune cells can be stained with a labeled molecule (e.g. a fluorophore labeled receptor-specific antibody or a fhiorophore-labeled receptor-specific ligand), and quantified using fluorescence activated cell sorting (FACS) flow cytometry. This method allows a percentage of immune cells in a population of immune cells to be characterized as expressing an activator receptor, an inhibitory receptor, or both receptors. The ratio of activator receptor and inhibitory receptors expressed by the immune cells described herein can be determined by, for example, digital droplet PCR. These approaches can be used to characterize the population of cells for the production and manufacturing of the immune cells, pharmaceutical compositions, and kits described herein. For the immune cells, pharmaceutical compositions, and kits described herein, it is understood that a suitable percentage of immune cells expressing both an activator receptor and an inhibitory receptor is determined specifically for the methods described herein. For example, a suitable percentage of immune cells expressing both an activator receptor and in inhibitory receptor can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. As a further example, between 50% and 99%, between 60% and 95%, between 65% and 95%, between 65% and 90%, between 70% and 90% , between 75% and 90%, between 75% and 85%, between 80% and 99%, between 85% and 99%, between 90% and 99% or between 95% and 99% of immune cells can express both the activator receptor and the inhibitory receptor. For example, a suitable ratio of activator receptor and inhibitory receptor in an immune cell 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 suitable values for the immune cells, pharmaceutical compositions, and kits described herein.

[0156] A responsive receptor expressed by the immune cells described herein can be verified by assays that measure the generation of a signal expected to be generated by the intracellular domain of the receptor. Reporter cell lines, such as Jurkat-Luciferase NF AT cells (Jurkat cells), can be used to characterize a responsive receptor. Jurkat cells are derived from T cells and comprise a stably integrated nuclear factor of activated T-cells (NFAT)-inducible luciferase reporter system. NF AT is a family of transcription factors required for immune cell activation, whose activation can be used as a signaling marker for T cell activation. Jurkat cells can be transduced or transfected with the activator receptors and / or inhibitory receptors described herein. The activator receptor is responsive to the binding of a ligand if the Jurkat cell expresses a luciferase reporter gene, and the level of responsiveness can be determined by the level of reporter gene expression. The presence of luciferasecan be determined using any known luciferase detection reagent, such as luciferin. An inhibitory receptor is responsive to the binding of a ligand if, when co-expressed with an activator receptor in Jurkat cells, it prevents a normally responsive immune cell from expressing luciferase in response to the activator receptor. For example, the responsiveness of an inhibitory receptor can be determined and quantified in a Jurkat cell expressing both an activator and an inhibitor by observing the following: 1) the Jurkat cell expresses luciferase in the presence of activator receptor ligand and absence of inhibitory receptor ligand; and 2) luciferase expression in the Jurkat cell 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 receptors and inhibitory receptors. The sensitivity, potency, and selectivity can be quantified by EC50 or IC50 values using dose-response experiments, where an activator receptor ligand and / or inhibitory receptor ligand is titrated into a culture of Jurkat cells expressing an activator receptor or a specific pair of activator and inhibitory receptors. Alternatively, the EC50 and IC50 values can be determined in a co-culture of immune cells (e.g. Jurkat cells or primary immune cells) expressing an activator receptor or a specific pair of activator and inhibitory receptors and target cells expressing an increasing amount of an activator ligand or inhibitor ligand. An increasing amount of activator ligand or inhibitor ligand can be accomplished in the target cell by, for example, titration of activator ligand or inhibitor ligand encoding mRNA into target cells, or use of target cells that naturally express different levels of the target ligands. Exemplary suitable EC50 and IC50 values for the activator and inhibitory receptors as determined used target cells expressing varying amounts of the target and non-target ligands include an EC50 of 10 transcripts per million (TPM) or less for the activator receptor, for example an EC50 of between 2-10 TPM, and an IC50 of 25 TPM or less for the inhibitory receptor, for example an IC50 of 5-21 TPM.

[0157] Activation of the immune cells described herein that express an activator receptor or specific pairs of activator and inhibitory receptors can be further determined by assays that measure the viability of a target cell following co-incubation with said immune cells. The immune cells, sometimes referred to as effector cells, are co-incubated with target cells that express an activator receptor ligand, an inhibitory receptor ligand, or both an activator and inhibitory receptor ligand. Following coincubation, viability of the target cell is measured using any method to measure viability in a cell culture. For example, viability can be determined using a mitochondrial function assay that uses a tetrazolium salt substrate to measure active mitochondrial enzymes. Viability can also be determined using imaging based methods. Target cells can express a fluorescent protein, such as green fluorescent protein or red fluorescent protein. Reduction in total cell fluorescence indicates a reduction in viability of the target cell. A reduction in viability of the target cell following incubation with immune cells expressing an activator receptor or a specific pair of activator and inhibitory receptors is interpreted astarget cell-mediated activation of the immune cell. A measure of the selectivity of the immune cells can also be determined using this approach. The immune cell expressing a pair of activator and inhibitory receptors is selective if the following is observed: 1) viability is reduced in target cells expressing the activator receptor ligand but not the inhibitory receptor ligand; 2) viability is not reduced in target cells expressing both an activator receptor ligand and an inhibitory receptor ligand. From these measurements, a “specific killing” value can be derived that quantifies the percentage of immune cell activation based on the reduction in viability of target cell as a percentage of a negative control (immune cells that do not express an activator receptor). Further, from these measurements a “selectivity ratio” value can be derived that represents the ratio of the specific killing observed in target cells expressing an activator receptor ligand in the absence of inhibitory receptor ligand to the specific killing observed in target cells expressing both an activator receptor ligand and an inhibitory receptor ligand. This approach can be used to characterize the population of cells for the production and manufacturing of the immune cells, pharmaceutical compositions, and kits described herein. A suitable specific killing value for the immune cells, pharmaceutical compositions, and kits can be, for example, the following criteria: 1) at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% specific killing following a 48 hour co-incubation of immune cells and target cells expressing activator receptor ligand in the absence of inhibitory receptor ligand; and 2) less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, less than or equal to 5%, less than or equal to 3% or less than or equal to 1% specific killing of target cell expressing both an activator receptor ligand and an inhibitory receptor ligand.

[0158] As a further example, a suitable specific killing value for the immune cells, pharmaceutical compositions and kits can be the following criteria: 1) between 30% and 99%, between 40% and 99%, between 50% and 99%, between 55% and 95%, between 60% and 95%, between 60% and 90%, between 50% and 80%, between 50% and 70% or between 50% and 60% of target cells expressing the activator ligand but not the inhibitor ligand are killed; and 2), between 1% and 40%, between 3% and 40%, between 5% and 40%, between 5% and 30%, between 10% and 30%, between 15% and 30% or between 5% and 20% of target cells expressing the activator ligand and the inhibitor ligand are killed. As a still further example, a suitable specific killing value for the immune cells, pharmaceutical compositions, and kits can be, for example, the following criteria: 1) at least 50% specific killing following a 48 hour co-incubation of immune cells and target cells expressing activator receptor ligand in the absence of inhibitory receptor ligand; and 2) less than or equal to 20% specific killing of target cell expressing both an activator receptor ligand and an inhibitory receptor ligand. As a further example, the immune cells are capable of killing at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% of target cellsexpressing the activator ligand and not the inhibitor ligand over a period of 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, or 60 hours, while killing less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3% or less than 1% of target cells expressing the activator and inhibitor ligands over the same time period.

[0159] A suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50% to at least about 95%. A suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. A suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the 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%. A suitable specific killing value of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand for the 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%. The suitable specific killing value for the immune cells, pharmaceutical compositions, and kits can be determined following about 6 hours, about 12 hours, about 18 hours, about 24, 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.

[0160] A suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50% to at least about 95%. A suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. A suitable specific killing value of the target cell expressing an activator ligand in the absence of an inhibitory ligand value for the 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%. A suitable specific killing value of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand for the immunecells, 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%. The suitable specific killing value for the immune cells, pharmaceutical compositions, and kits can be determined following about 6 hours, about 12 hours, about 18 hours, about 24, 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.

[0161] As used herein, the term “functional variant” refers to a protein that has one or more aminoacid substitutions, insertions, or deletions as compared to a parental protein, and which retains one or more desired activities of the parental protein. A functional variant may be a fragment of the protein (i.e. a variant having N- and / or C-terminal deletions) that retain the one or more desired activities of the parental protein.

[0162] All publications and patents mentioned herein are hereby 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 reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.Anti-HLA-A *03 Polypeptides

[0163] The disclosure provides polypeptides comprising antigen binding domains that specifically bind MHC I complexes comprising an HLA-A afrom A*03 alleles of HLA-A (HLA-A*03, encoding a03). The antigen binding domains of the disclosure can be antibodies, or antigen-binding fragments or derivatives thereof, described below. The disclosure further provides antibodies and receptors comprising the polypeptides described herein, and cells and compositions comprising same.

[0164] HLA-A is a highly polymorphic group of human leukocyte antigens (HLA) of serotype A that are coded for by the HLA-A locus. There are currently over 6,425 HLA-A alleles known, which encode at least 3,929 HLA-A proteins (hla.alleles.org / nomenclature / stats.html). HLA-A*03 alleles are a group of HLA-A alleles. The A*03 serotype is determined by antibody recognition of the a3subset of HLA- A a chains. Examples of HLA-A*03 alleles include HLA-A*03:01, HLA-A*03:02, HLA-A*03:03, HLA-A*03:04, HLA-A*03:30, HLA-A*03:33, and many more, all of which are envisaged as within the scope of the instant invention.

[0165] HLA-A*03 binding polypeptides of the disclosure specifically bind to HLA-A*03 HLA-A alleles. In some embodiments, HLA-A*03 binding polypeptides specific to HLA-A*03 bind to HLA-A a3proteins produced by HLA-A*03 alleles and do not bind to HLA-A a chain produced by other HLA-A alleles, or HLA-A a chain produced by other HLA-A alleles with a lower affinity than they bind to HLA-A a3. For example HLA-A*03 binding polypeptides may not bind to, or bind with lesser affinity, HLA-A a chain produced by e.g., HLA-A*01, HLA-A*02, or HLA-A*11. In some embodiments, HLA-A*03 binding polypeptides specific to HLA-A*03 do not bind to HLA alleles of B and / or C serotype.

[0166] An exemplary HLA-A*03 a sequence, HLA-A*03 alpha chain isoform 1, is provided as:1 MAVMAPRTLL LLLSGALALT QTWAGSHSMR YFFTSVSRPG RGE PRFIAVG YVDDTQFVRF61 DSDAASQRME PRAPWIEQEG PEYWDQETRN VKAQSQTDRV DLGTLRGYYN QSEAGSHTIQ121 IMYGCDVGSD GRFLRGYRQD AYDGKDYIAL NEDLRSWTAA DMAAQITKRK WEAAHEAEQL181 RAYLDGTCVE WLRRYLENGK ETLQRTDPPK THMTHHPI SD HEATLRCWAL GFYPAE ITLT241 WQRDGEDQTQ DTELVETRPA GDGTFQKWAA VVVPSGEEQR YTCHVQHEGL PKPLTLRWEL301 SSQPTI PIVG I IAGLVLLGA VITGAVVAAV MWRRKSSDRK GGSYTQAASS DSAQGSDVSL361 TACKV ( SEQ ID NO : 1 )

[0167] The skilled artisan will appreciate that, due to the high degree of polymorphism, additional HLA-A*03 protein sequences may include one or more amino acid substitutions relative to SEQ ID NO: 1, and will still be considered proteins encoded HLA-A*03 alleles.In the present disclosure, in vitro V(D)J recombination was employed to isolate and engineer scFv capable of binding MHC I comprising HLA-A a03with high selectivity (an HLA-A*03 antigen binding domain). These antibodies, and antigen binding domains derived therefrom, are described in more detail below.

[0168] The general structure of antibodies is known in the art. Briefly, an immunoglobulin monomer comprises two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is paired with one of the light chains to which it is directly bound via a disulfide bond. Each heavy chain comprises a constant region (which varies depending on the isotype of the antibody) and a variable region. The variable region comprises three hypervariable regions (or complementarity determining regions) which are designated CDRH1, CDRH2 and CDRH3 and which are supported within framework regions. Each light chain comprises a constant region and a variable region, with the variable region comprising three hypervariable regions (designated CDRL1, CDRL2 and CDRL3) supported by framework regions in an analogous manner to the variable region of the heavy chain.

[0169] The hypervariable regions of each pair of heavy and light chains mutually cooperate to provide an antigen binding site that is capable of binding a target antigen. The binding specificity of a pair of heavy and light chains is defined by the sequence of CDR1, CDR2 and CDR3 of the heavy and light chains. Thus once a set of CDR sequences (i.e. the sequence of CDR1, CDR2 and CDR3 for the heavyand light chains) is determined which gives rise to a particular binding specificity, the set of CDR sequences can, in principle, be inserted into the appropriate positions within any other antibody framework regions linked with any antibody constant regions in order to provide a different antibody with the same antigen binding specificity.

[0170] In some embodiments, the polypeptides comprising HLA-A*03 antigen binding domains comprise one or more complementarity determining regions (CDRs) selected from the group disclosed in Table 1 A, Table IB, Table 2A, or Table 2B below.

[0171] In some embodiments, the HLA-A*03 antigen binding domain comprises a heavy chain (HC) comprising one or more CDRs selected from the HC CDRs as set forth in Table 1 A or Table IB.

[0172] In some embodiments, the antigen binding domain comprises (a) a heavy chain (HC) complementarity determining region 1 (CDR1) sequence selected from the HC CDR1 sequences as set forth in Table 1 A or Table IB; (b) a HC CDR2 sequence selected from the HC CDR2 sequences as set forth in Table 1A or Table IB; and (c) a HC CDR3 sequence selected from the HC CDR3 sequences as set forth in Table 1 A or Table IB.

[0173] In some embodiments, the antigen binding domain comprises a HC CDR1, a HC CDR2, and a HC CDR3 as set forth on one line in Table 1, e.g., the HC CDR1 as set forth in line A3-14 of Table 1, the HC CDR2 as set forth in line A3- 14 of Table 1, and the HC CDR3 as set forth in line A3- 14 of Table 1.

[0174] In some embodiments, the antigen binding domain comprises a HC CDR1 comprising a sequence of NYWMN (SEQ ID NO: 2), a HC CDR2 comprising a sequence of EIRLKSTNYATHYAESVKG (SEQ ID NO: 3), and HC CDR3 comprising a sequence of LITPDY (SEQ ID NO: 4), or CDR sequences having at most 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto.

[0175] In some embodiments, the HLA-A*03 antigen binding domain comprises a light chain (LC) comprising one or more CDRs selected from the LC CDRs as set forth in Table 2A or Table 2B.

[0176] In some embodiments, the antigen binding domain comprises (a) a light chain (LC) complementarity determining region 1 (CDR1) sequence selected from the LC CDR1 sequences as set forth in Table 2A or Table 2B; (b) a LC CDR2 sequence selected from the LC CDR2 sequences as set forth in Table 2A or Table 2B; and (c) a LC CDR3 sequence selected from the LC CDR3 sequences as set forth in Table 2 A or Table 2B.

[0177] In some embodiments, the antigen binding domain comprises a LC CDR1, a LC CDR2, and a LC CDR3 as set forth on one line in Table 2, e.g., the LC CDR1 as set forth in line A3-14 of Table 2, the LC CDR2 as set forth in line A3- 14 of Table 2, and the LC CDR3 as set forth in line A3- 14 of Table 2.

[0178] In some embodiments, the antigen binding domain comprises a LC CDR1 comprising a sequence of KASQDVSTTVA (SEQ ID NO: 33), a LC CDR2 comprising a sequence of SASYRYT (SEQ ID NO: 35), and LC CDR3 comprising a sequence of QQHYSTPPT (SEQ ID NO: 37), or CDR sequences having at most 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto.

[0179] In some embodiments, the HLA-A*03 antigen binding domain comprises (a) a HC comprising one or more CDRs selected from the HC CDRs as set forth in Table 1 A or Table IB; and (b) a LC comprising one or more CDRs selected from the LC CDRs as set forth in Table 2A or Table 2B.

[0180] In some embodiments, the HLA-A*03 antigen binding domain comprises (a) a HC comprising one or more CDRs selected from the HC CDRs as set forth in Table IB; and (b) a LC comprising one or more CDRs selected from the LC CDRs as set forth in Table 2B.

[0181] In some embodiments, the antigen binding domain comprises the antigen binding domain comprises (a) a HC CDR1 sequence selected from the HC CDR1 sequences as set forth in Table 1 A or Table IB (b) a HC CDR2 sequence selected from the HC CDR2 sequences as set forth in Table 1 A or Table IB; (c) a HC CDR3 sequence selected from the HC CDR3 sequences as set forth in Table 1 A or Table IB; (d) a LC CDR1 sequence selected from the LC CDR1 sequences as set forth in Table 2A or Table 2B; (e) a LC CDR2 sequence selected from the LC CDR2 sequences as set forth in Table 2A or Table 2B; and (f) a LC CDR3 sequence selected from the LC CDR3 sequences as set forth in Table 2 A or Table 2B.

[0182] In some embodiments, the antigen binding domain comprises a (a) a heavy chain comprising a HC CDR1, a HC CDR2, and a HC CDR3 as set forth on one line in Table 1, e.g., the HC CDR1 as set forth in line A3-14 of Table 1, the HC CDR2 as set forth in line A3- 14 of Table 1, and the HC CDR3 as set forth in line A3-14 of Table 1 and (b) a light chain comprising a LC CDR1, a LC CDR2, and a LC CDR3 as set forth on one line in Table 2, e.g., the LC CDR1 as set forth in line A3-14 of Table 2, the LC CDR2 as set forth in line A3- 14 of Table 2, and the LC CDR3 as set forth in line A3- 14 of Table 2.

[0183] In some embodiments, the antigen binding domain comprises a HC CDR1 comprising a sequence of NYWMN (SEQ ID NO: 2), a HC CDR2 comprising a sequence of EIRLKSTNYATHYAESVKG (SEQ ID NO: 3), and HC CDR3 comprising a sequence of LITPDY (SEQ ID NO: 4), a LC CDR1 comprising a sequence of KASQDVSTTVA (SEQ ID NO: 33), a LC CDR2 comprising a sequence of SASYRYT (SEQ ID NO: 35), and LC CDR3 comprising a sequence of QQHYSTPPT (SEQ ID NO: 37), or CDR sequences having at most 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto.

[0184] In each case, the HC CDRs may be paired with any of the LC CDRs, as the heavy chains and light chains share similarity, with routine testing to confirm desired expression and binding activity. However, the preferred pairing between Table 1 and Table 2 in indicated in the “Name” columns ofthe tables, i.e., in preferred embodiments, the HC CDRs in line A3-14 of Table 1 are paired with the LC CDRs in line A3-14 of Table 2 the HC CDRs in line A3-13 of Table 1 are paired with the LC CDRs in line A3- 13 of Table 2, etc.Table 1A and Table IB Heavy Chain CDR sequences for exemplary HLA-A*03 antigen binding domains.Table 1ATable IBTable 2A-2B: Light Chain CDR sequences for exemplary HLA-A *03 antigen binding domains.Table 2ATable 2B

[0185] In some embodiments, the HLA-A*03 antigen binding domain comprises a heavy chain and a light chain. Illustrative variable heavy are provided in Table 3A and Table 3B. Illustrative variable light chain sequences are provided in Table 4 A and Table 4B.

[0186] In some embodiments, the antigen binding domain comprises a variable heavy chain region (VH) comprising a sequence as set forth in Table 3A or Table 3B. In some embodiments, the antigen binding domain comprises a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 3A or Table 3B.

[0187] In some embodiments, the antigen binding domain comprises a variable heavy chain region (VH) comprising a sequence of SEQ ID NOs: 51-56. In some embodiments, the antigen binding domain comprises a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in SEQ ID NOs: 51-56.

[0188] In some embodiments, the antigen binding domain comprises a variable light chain region (VL) comprising a sequence as set forth in Table 4A or Table 4B. In some embodiments, the antigen binding domain comprises a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 4A or Table 4B.

[0189] In some embodiments, the antigen binding domain comprises a variable light chain region (VL) comprising a sequence of SEQ ID NOs: 72-78. In some embodiments, the antigen binding domain comprises a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in SEQ ID NOs: 72-78.

[0190] In some embodiments, the antigen binding domain comprises (a) a VH comprising a sequence as set forth in Table 3A or Table 3B and (b) a VL comprising a sequence as set forth in Table 4A or Table 4B.

[0191] In some embodiments, the antigen binding domain comprises (a) a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 3 A or Table 3B, and (b) a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 4A or Table 4B.

[0192] In some embodiments, the antigen binding domain comprises (a) a VH comprising a sequence of SEQ ID NOs: 51-56 and (b) a VL comprising a sequence of SEQ ID NOs: 72-78.

[0193] In each case, the VH may be paired with any of the VLs, as the heavy chains and light chains share similarity, with routine testing to confirm desired expression and binding activity. However, the preferred pairing between Table 3 and Table 4 in indicated in the “Name” columns of the tables, i.e., in preferred embodiments, the VH designated “A3-14” in Table 3 is paired with the VL designated “A3-14” of Table 4, the VH designated “A3-13” in Table 3 is paired with the VL designated “A3-13” in Table 4, etc.Table 3A-3B: Exemplary VH SequencesTable 3BTable 4A-4B: Exemplary VL SequencesTable 4ATable 4B

[0194] In some embodiments, provided herein is an antibody or antigen binding fragment thereof comprising an antigen binding domain that specifically binds to MHC I comprising an a03chain encoded by an HLA-A*03 allele, as described herein. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a fully human antibody. In some embodiments, the antibody is a humanized antibody. As used herein, a humanized antibodyrefers to an antibody derived from a monoclonal antibody raised initially in a non-human animal, such as a rodent or rabbit. In some embodiments, a humanized antibody refers to non-human CDRs grafted on to a human framework sequences. Certain amino acid residues in this monoclonal antibody, typically from non-antigen recognizing portions of the antibody, are modified to be homologous to corresponding residues in a human antibody of corresponding isotype. Humanization can be performed, for example, using various methods by substituting at least a portion of a rodent or rabbit variable region for the corresponding regions of a human antibody (see, e.g., United States Patent No. 5,585,089, and No. 5,693,762; Jones et al, 1986, Nature 321 :522-525; Riechmann et al, 1988, Nature 332:323-27; and Verhoeyen et al, 1988, Science 239: 1534-1536). In some embodiments, the antibody is a chimeric antibody. In other embodiments, the antibody is an immunoglobulin molecule. In particular examples, the antibody is an IgG.

[0195] Anti-HLA-A*03 antibodies of the disclosure can be monospecific - i.e., contain a single antigen binding domain specific to HLA-A*03. Alternatively, anti-HLA-A*03 antibodies of the disclosure can be multispecific, such as bispecific antibodies. Various formats of multispecific antibodies will be known to persons of ordinary skill in the art, and are envisaged as within the scope of the instant disclosure.

[0196] In some embodiments, the antigen binding domain is an antibody fragment, such as a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single chain variable fragment (scFv), or a disulfide stabilized variable fragment (dsFv).

[0197] The monoclonal antibodies disclosed herein can be of any isotype. The monoclonal antibody can be, for example, an IgM or an IgG antibody, such as IgGl or an IgG2. The class of an antibody can be switched with another (for example, IgG can be switched to IgM), according to well-known procedures. Class switching can also be used to convert one IgG subclass to another, such as from IgGl to IgG2.

[0198] Antibody fragments comprising HLA-A*03 antigen binding domains are also encompassed by the present disclosure, such as single-domain antibodies (e.g., VH domain antibodies), Fab, F(ab')2, and Fv. These antibody fragments retain the ability to selectively bind with the antigen. These fragments include:(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains;(5) Single chain antibody (such as scFv), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule;(6) A dimer of a single chain antibody (scFv2), defined as a dimer of a scFv (also known as a “mini antibody”);(7) VH single-domain antibody, an antibody fragment consisting of a heavy chain variable domain; and(8) A single chain Fab fragment (scFab), which can be formed by the introduction of a polypeptide linker between the Fd and the light chain to result in the formation of a single chain Fab fragment.

[0199] Methods of making these fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).

[0200] In some cases, antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as E. coif) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647).

[0201] Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[0202] In some embodiments, the HLA-A*03 antigen binding domain is a scFv. The scFv can be in either orientation, i.e. a variable heavy chain (Vu)-linker-variable light chain (VL) or a VL-linker-Vu orientation.

[0203] In some embodiments, the scFv comprises a VH comprising a sequence as set forth in Table 3A or Table 3B. In some embodiments, the scFv comprises a VH comprising a sequence having atleast 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 3A or Table 3B.

[0204] In some embodiments, the scFv comprises a VH comprising a sequence of SEQ ID NOs: 51- 56. In some embodiments, the scFv comprises a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in SEQ ID NOs: 51-56.

[0205] In some embodiments, the scFv comprises a VL comprising a sequence as set forth in Table 4A or Table 4B. In some embodiments, the scFv comprises a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 4 A or Table 4B.

[0206] In some embodiments, the scFv comprises a VL comprising a sequence of SEQ ID NOs: 72- 78. In some embodiments, the scFv comprises a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in SEQ ID NOs: 72-78.

[0207] In some embodiments, the scFv comprises (a) a VH comprising a sequence as set forth in Table 3A or Table 3B and (b) a VL comprising a sequence as set forth in Table 4A or Table 4B.

[0208] In some embodiments, the scFv comprises (a) a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to a sequence as set forth in Table 3A or Table 3B, and (b) a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 4 A or Table 4B.

[0209] In some embodiments, the svFv comprises (a) a VH comprising a sequence of SEQ ID NOs: 51-56 and (b) a VL comprising a sequence of SEQ ID NOs: 72-78.

[0210] In each case, the VH may be paired with any of the VLs, as the heavy chains and light chains share similarity, with routine testing to confirm desired expression and binding activity. However, the preferred pairing between Table 3A or Table 3B and Table 4A or Table 4B in indicated in the “Name” columns of the tables, i.e., in preferred embodiments, the VH designated “A3-14” in Table 3B is paired with the VL designated “A3-14” in Table 4B, the VH designated “A3-13” Table 3B is paired with the VL designated “A3-13” in Table 4B, etc.

[0211] Exemplary scFv sequences and the DNA sequences encoding them are as set forth in Table 5A and 5B. In some embodiments, the HLA-A*03 antigen binding domain is an scFv comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to an amino acid sequences as set forth in Table 5A or Table 5B. In some embodiments, the HLA-A*03 antigen binding domain is an scFv comprising, consisting of, or essentially consisting of, an amino acid sequences as set forth in Table 5A or Table 5B. In some embodiments, the HLA-A*03 antigen binding domain is an scFv encoded by a nucleic acid sequences as set forth in Table 5A or Table 5B.Table 5A-5B: Exemplary HLA-A *03 scFv antigen binding domains.Table 5BAntibodies

[0212] The present disclosure provides anti-HLA-A*03 antibodies and antigen-binding fragments thereof.

[0213] The antibodies of the present disclosure can be purified to homogeneity. The separation and purification of the antibodies can be performed by employing conventional protein separation and purification methods.

[0214] By way of example only, the antibody can be separated and purified by appropriately selecting and combining use of chromatography columns, filters, ultrafiltration, salt precipitation, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and the like. Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988).

[0215] Non-limiting examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. In one aspect, chromatography can be performed by employing liquid chromatography such as HPLC or FPLC.Variations

[0216] In some embodiments, one or more amino acid residues in a CDR of the antigen binding domains provided herein are substituted with another amino acid. The substitution may be “conservative” in the sense of being a substitution within the same family of amino acids. The naturally occurring amino acids may be divided into the following four families and conservative substitutions will take place within those families: (1) amino acids with basic side chains: lysine, arginine, histidine; (2) amino acids with acidic side chains: aspartic acid, glutamic acid; (3) amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine; and (4) amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.

[0217] In another aspect, one or more amino acid residues are added to or deleted from one or more CDRs of an antigen binding domain described herein. Such additions or deletions may occur at the N or C termini of the CDR or at a position within the CDR.By varying the amino acid sequence of the CDRs of an antibody by addition, deletion or substitution of amino acids, various effects such as increased binding affinity for the target antigen may be obtained.

[0218] It is to be appreciated that antibodies of the disclosure comprising such varied CDR sequences may still bind HLA-A*03 with similar specificity and sensitivity profiles. This may be tested by way of the binding assays disclosed in the Examples and that are known in the art. When the antigen binding domain comprises an antibody or antibody fragment, the constant regions of antibodies may also be varied. For example, antibodies may be provided with Fc regions of any isotype: IgA (IgAl, IgA2), IgD, IgE, IgG (IgGl, IgG2, IgG3, IgG4) or IgM.

[0219] One of skill will realize that conservative variants of the antigen binding domains and antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions. In some embodiments, the variants will retain the charge characteristics of the residues, for example.

[0220] Amino acid substitutions (such as at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, at most ten, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, or at most 20 amino acid substitutions) can be made in the VH and / or the VL regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: (1) Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).Amino acid substitutions, deletions, and additions, and other such sequence variations, may be performed based on sequence alignment techniques using existing sequence alignment tools.Modifications

[0221] The disclosure provides polypeptides comprising HLA-A*03 antigen binding domains and antibodies, comprising one or more modifications. Modifications include, inter alia detectable labels, conjugates to therapeutic agents and agents that increase stability or bioavailability, and multimerization domains.

[0222] As used herein, the term “detectable label” refers to a molecule or material that can produce a detectable (such as visually, electronically or otherwise) signal that indicates the presence and / or concentration of the label in a sample. When conjugated to a specific binding molecule, the detectablelabel can be used to locate and / or quantify the target to which the specific binding molecule is directed. Thereby, the presence and / or concentration of the target in a sample can be detected by detecting the signal produced by the detectable label. A detectable label can be detected directly or indirectly, and several different detectable labels conjugated to different specific-binding molecules can be used in combination to detect one or more targets. For example, a first detectable label conjugated to an antibody specific to a target can be detected indirectly through the use of a second detectable label that is conjugated to a molecule that specifically binds the first detectable label. Multiple detectable labels that can be separately detected can be conjugated to different specific binding molecules that specifically bind different targets to provide a multiplexed assay that can provide simultaneous detection of the multiple targets in a sample. A detectable signal can be generated by any mechanism including absorption, emission and / or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected through antibody-hapten binding interactions using additional detectably labeled antibody conjugates, and paramagnetic and magnetic molecules or materials. Non-limiting examples of detectable labels include enzymes such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, P-galactosidase or P-glucuronidase; fluorophores such as fluoresceins, luminophores, coumarins, BODIPY dyes, resorufins, and rhodamines (many additional examples of fluorescent molecules can be found in The Handbook— A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, Oreg.); nanoparticles such as quantum dots (obtained, for example, from QuantumDot Corp, Invitrogen Nanocrystal Technologies, Hayward, Calif.; see also, U.S. Pat. Nos. 6,815,064, 6,682,596 and 6,649,138, each of which patents is incorporated by reference herein); metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+; and liposomes, for example, liposomes containing trapped fluorescent molecules. Where the detectable label includes an enzyme, a detectable substrate such as a chromogen, a fluorogenic compound, or a luminogenic compound can be used in combination with the enzyme to generate a detectable signal (A wide variety of such compounds are commercially available, for example, from Invitrogen Corporation, Eugene Oreg.). Non-limiting examples of chromogenic compounds include diaminobenzidine (DAB), 4- nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP / NBT, fast red, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3- ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-P-D- galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-P-galactopyranoside (X-Gal), methylumbelliferyl-P-D-galactopyranoside (MU-Gal), p-nitrophenyl-a- D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-P-D-glucuronide (X-Gluc), 3-amino-9-ethyl carbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue and tetrazolium violet. Alternatively, an enzyme can be used in a metallographic detection scheme. Metallographic detection methods include using an enzyme such as alkaline phosphatase in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redox-active agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, co-pending U.S. patent application Ser. No. 11 / 015,646, filed Dec. 20, 2004, PCT Publication No. 2005 / 003777 and U.S. Patent Application Publication No. 2004 / 0265922; each of which is incorporated by reference herein). Metallographic detection methods include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113, which is incorporated by reference herein). Haptens are small molecules that are specifically bound by antibodies, although by themselves they will not elicit an immune response in an animal and must first be attached to a larger carrier molecule such as a protein to generate an immune response. Examples of haptens include di -nitrophenyl, biotin, digoxigenin, and fluorescein. Additional examples of oxazole, pyrazole, thiazole, nitroaryl, benzofuran, triperpene, urea, thiourea, rotenoid, coumarin and cyclolignan haptens are disclosed in U.S. Provisional Patent Application No. 60 / 856,133, filed Nov. 1, 2006, which is incorporated by reference herein. In some embodiments, the detectable label comprises a non-endogenous hapten (e.g. not biotin), such as, for example, the haptens disclosed in U.S. Pat. Nos. 7,695,929, 8,618,265 and 8,846,320 (incorporated herein by reference), including for example pyrazoles, nitrophenyl compounds, benzofurazans, triterpenes, ureas and thioureas, rotenone and rotenone derivatives, oxazoles and thiazoles, coumarin and coumarin derivatives, and cyclolignans.

[0223] The antigen binding domains or antibodies of the present disclosure may be multimerized to increase the affinity for an antigen. The antibody to be multimerized may be one type of antibody or a plurality of antibodies which recognize a plurality of epitopes of the same antigen. As a method of multimerization of the antibody, binding of the IgG CH3 domain to two scFvmolecules, binding to streptavidin, introduction of a helix-tum-helix motif and the like can be exemplified.

[0224] The antigen binding domain or antibody compositions of the present disclosure may be in the form of a conjugate formed between polypeptide comprising the antigen binding domain or antibody and another agent (immunoconjugate). In one aspect, the polypeptides of the present disclosure are conjugated to radioactive material. In another aspect, the polypeptides of the present disclosure can be bound to various types of molecules such as polyethylene glycol (PEG).

[0225] The antigen binding domains or antibodies specific to HLA-A*03 can be conjugated to a therapeutic agent or effector molecule including, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody. A therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule. One of skill in the art will appreciate that therapeutic agents can include various drugs such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or Diphtheria toxin, encapsulating agents (such as liposomes) which themselves contain pharmacological compositions, radioactive agents such as1251,32P,14C,3H and35S and other labels, target moieties and ligands.

[0226] The choice of a particular therapeutic agent depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the therapeutic agent can be a cytotoxin that is used to bring about the death of a particular target cell (such as a tumor cell). Conversely, where it is desired to invoke a non-lethal biological response, the therapeutic agent can be conjugated to a non- lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.

[0227] With the therapeutic agents and antigen binding domains or antibodies described herein, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same effector moiety or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antigen binding domains, antibodies and conjugates and fusion proteins thereof.

[0228] Effector molecules can be linked to a polypeptide comprising an HLA-A*03 antigen binding domain or antibody using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an HLA-A*03 antigen binding domain or antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine ( — NH2) or sulfhydryl ( — SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0229] In some circumstances, it is desirable to free the effector molecule from the antigen binding domain or antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antigen binding domain or antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.

[0230] In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, label (such as enzymes or fluorescent molecules) drugs, toxins, and other agents to proteins, one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or antigen binding domain.

[0231] The antibodies or antigen binding domains disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). In some cases, the antibody, or an antibody fragment or derivative (such as a VH domain) is fused to a heterologous protein, for example an Fc protein. In some embodiments, the antibody or antibody fragment is fused to a part of a chimeric antigen receptor (CAR) protein.

[0232] In general, the antibodies or antigen binding domain is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0233] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate). Such linkers are commercially available.

[0234] An antibody or antigen binding domain that binds (for example specifically binds) an MHC I complex comprising HLA-A*03 a chain can be labeled with a detectable moiety. Non-limiting examples of detection agents include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, Green fluorescent protein (GFP), Yellow fluorescent protein (YFP). An antibody can also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, P-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labeled with a detectableenzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody may also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be labeled with an enzyme or a fluorescent label.

[0235] An antibody or antigen binding domain may be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels. An antibody may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0236] An antibody or antigen binding domain can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect an antigen by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides:3H,14C,15N,35S,90Y, "Tc,n iIn,125I,131I.An antibody or antigen binding domain can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.

[0237] Toxins can be employed with the antibodies or antigen binding domains described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, Mo.). Contemplated toxins also include variants of the toxins described herein (see, for example, see, U.S. Pat. Nos. 5,079,163 and 4,689,401). In some embodiments, the toxin is Pseudomonas exotoxin (PE) (U.S. Pat. No. 5,602,095). As used herein “Pseudomonas exotoxin” refers to a full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al., J. Biol. Chem. 264: 14256- 14261, 1989).

[0238] PE employed with the antibodies or antigen binding domains described herein can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants ofnative PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE40, PE38, and PE35. For additional description of PE and variants thereof, see for example, U.S. Pat. Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; PCT Publication No. WO 99 / 51643; Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997.

[0239] The antibodies or antigen binding domains described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing HLA-A*03. For example, an antibody of the present disclosure can be attached directly or via a linker to a drug that is to be delivered directly to cells expressing cell-surface HLA-A*03. This can be done for therapeutic, diagnostic or research purposes. Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.

[0240] Alternatively, the molecule linked to an anti- HLA-A*03 antibody can be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Pat. No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).

[0241] Antibodies described herein can also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include magnetic beads, fluorescent dyes (for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (for example,3H,1251,35S,14C, or32P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, and the like) beads.

[0242] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.Receptors

[0243] Also provided herein are compositions and methods for treating cancers using immune cells comprising a two receptor system responsive to differences in gene expression of a ligand between cancer and normal (i.e. healthy or wild type) cells. These differences in expression can be due to loss of heterozygosity in the cancer cells at the HLA-A*03. Alternatively, the differences in expression can be because the gene is not expressed in cancer cells, or is expressed in cancer cells at a lower level than normal cells. The two-receptor system is expressed in immune cells, for example immune cells used in adoptive cell therapy, and targets activity of these immune cells to cancer cells exhibiting loss of heterozygosity or expression differences. In this two receptor system, the first receptor (an activator receptor, sometimes referred to herein as an A module) activates, or promotes activation of the immune cells, while the second receptor (an inhibitory receptor, sometimes referred to herein as a blocker, inhibitor receptor, or B module) acts to inhibit activation of the immune cells by the first receptor. Each receptor contains a ligand-binding domain (LBD) that binds a specific ligand. Signals from the two receptors upon ligand binding are integrated by the immune cell. Differential expression of ligands for the first and second receptors in cancer and normal cells, for example through loss of heterozygosity of the locus encoding the inhibitory ligand in cancer cells, or differences in transcription levels, mediates activation of immune cells by target cancer cells that express the first activator ligand but not the second inhibitory ligand.

[0244] Loss of heterozygosity (LOH) from large-scale chromosomal deletions is a source of genetic difference in tumors. LOH is a common event in tumorigenesis which affects nearly every locus in the genome, with approximately 20% of genes displaying LOH in an average tumor. LOH provides the means to discriminate tumor from normal tissue in a definitive way because tumors can be found in which all malignant cells lack certain germline alleles. One locus that undergoes LOH is the human leukocyte antigen (HLA) locus, which encodes polymorphic, abundant, ubiquitous surface antigens. The two-receptor system described herein employs one receptor to activate T cells exposed to tumor-antigen-positive tumor cells (sometimes referred to as an “activator module”), and a second receptor to prevent activation of the immune cells in the presence of a surface blocker antigen such as HLA-A*03 protein. The dual-receptor system described herein (sometimes referred to herein as “Tmod”) possesses other advantageous properties as a cell therapy, including but not limited to reversible activation / blockade of immune cells, and selectivity in mixtures of tumor and “normal” cells.

[0245] In particular 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. This includes mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer,esophageal cancer, head and neck cancer, kidney cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or cholangiocarcinoma. In some embodiments, the cancer has relapsed in a subject. In some embodiments, the cancer is refractory to one or more prior administered anti cancer therapies. In some embodiments, the cancer is metastatic. In the case of MSLN-positive cancers, the target antigen of the activator receptor is MSLN, or a peptide antigen thereof, in a complex with a major histocompatibility complex class I (MHC-I). MSLN is expressed in normal adipose, fallopian tube, lung and salivary gland tissues, among others. Because of its expression in certain tumors, MSLN is an attractive tumor-specific antigen that could mediate selective killing of MSLN+ tumors if these cancer cells could be specifically targeted with an appropriate therapeutic. However, normal MSLN expression in non-cancer (non-target) cells has prevented the effective use of MSLN for targeted therapies such as adoptive cell therapies. By pairing an MSLN activator receptor with an inhibitory receptor, the methods provided herein increase the specificity of adoptive cell therapies and decrease harmful effects associated with these therapies, such as dose-limited toxicity.

[0246] In some embodiments, the ligand for the activator is a MSLN peptide complexed with MHC class I. In the methods described herein, this MSLN targeted activator receptor is paired with an inhibitory receptor, which increases the safety window of the activator by blocking its cytolytic effect on normal MSLN-positive tissues. However, the activator receptor still directs the targeted killing of tumor cells by immune cells comprising the two-receptor system, as the tumor cells do not express the ligand for the inhibitor, or blocker, receptor. The target for the second, inhibitory receptor is expressed by MSLN positive tissues such as lung, mesothelium and adipose tissues, but not in cancer cells, and the inhibitory receptor recognizes this “non-target antigen” as an inhibitory stimulus. An exemplary target for the second inhibitory receptor is expressed by lung tissue, and is lost from MSLN positive cancer cells due to loss of heterozygosity (LOH) or other mechanisms, leaving a single allelic form in cancer cells that can be distinguished from other alleles via an allelespecific ligand binding domain on the inhibitory receptor.

[0247] The compositions and methods of the disclosure can reduce or eliminate dose-limiting toxicity (DLT) caused by expression of MSLN on normal tissue. The disclosure provides methods of targeting MSLN in cancer cells to treat MSLN positive cancers using adoptive cell therapies by adding a second inhibitory receptor that blocks activation of the adoptive immune cells in the presence of a second ligand (a ligand other than MSLN, termed the non-target antigen or alternatively, blocker antigen). Using the compositions and methods described herein, tumor cells that express MSLN are attacked by the adoptive cells, such as immune cells, expressing the two receptors because these tumor cells express only the activator ligand, MSLN. In contrast, normal cells that express MSLN plus the non-target antigen are protected from the adoptive immune cells.The inhibitory receptor response to the non-target antigen on normal cells prevents activation of immune cells by the MSLN-targeted activator receptor. This dual-targeting approach creates the therapeutic window that will allow a MSLN-directed cell therapy to be dosed safely and effectively in MSLN-positive cancer patients.

[0248] The disclosure provides methods and compositions that allow the use of potent MSLN CAR and TCRs that induce on-target toxicity, and renders these MSLN targeted receptors useful as a therapeutic by mitigating their toxicity.

[0249] In variations, the compositions and methods described herein may be used to kill target cells and / or treat subjects in which expression of HLA-A*03 is partially or completely decreased by causes other than loss of heterozygosity, including but not limited to partial gene deletion, epigenetic silencing, and point mutations or truncating mutations in the sequence encoding the non-target antigen.

[0250] The methods and compositions described in U.S. Patent Application Publication No. 2022 / 0370497, are incorporated herein by reference in their entirety.Activator Receptors

[0251] The disclosure provides a first receptor, comprising a first extracellular ligand binding domain specific to a target antigen comprising a cancer cell-specific antigen, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I). The first receptor is an activator receptor, and mediates activation of an immune cell expressing the first receptor upon binding of the target antigen by the extracellular ligand binding domain of the first receptor. The first receptor is responsive to a target antigen (i.e. activator ligand). For example, when a 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).

[0252] In some embodiments, the first receptor is humanized. As used herein, “humanized” refers to the replacement of a sequence or a subsequence in a transgene that has been isolated or derived from a non-human species with a homologous, or functionally equivalent, human sequence. For example, a humanized antibody can be created by grafting mouse CDRs into human framework sequences, followed by back substitution of certain human framework residues for the corresponding mouse residues from the source antibody.Activator Targets

[0253] In some embodiments, the target antigen for the first receptor is a peptide ligand from any of the activator targets disclosed herein. In some embodiments, the target antigen for the first receptor is a peptide antigen complexed with a major histocompatibility (MHC) class I complex (peptide MHC, or pMHC), for example an MHC complex comprising human leukocyte antigen A*02 allele (HLA- A*02).

[0254] Target cell-specific first activator ligands comprising peptide antigens complexed with pMHC comprising any of human leukocyte antigen (HLA) HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G are envisaged as within the scope of the disclosure. In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-A. HLA-A receptors are heterodimers comprising a heavy a chain and smaller P chain. The a chain is encoded by a variant of HLA-A, while the P chain (P2-microglobulin) is an invariant. There are several thousand HLA-A gene variants, all of which fall within the scope of the instant disclosure. In some embodiments, the MHC-I comprises a human leukocyte antigen A*02 allele (HLA-A*02).

[0255] In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-B. Hundreds of versions (alleles) of the HLA-B gene are known, each of which is given a particular number (such as HLA-B *27).

[0256] In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-C. HLA-C belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). Over one hundred HLA-C alleles are known in the art.

[0257] In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-A. In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-B. In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-C. In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-E. In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-F. In some embodiments, the target antigen for the first receptor comprises a pMHC comprising HLA-G.

[0258] In some embodiments, the target antigen for the first receptor comprises HLA-A. In some embodiments, the target antigen for the first receptor comprises HLA-B. In some embodiments, the target antigen for the first receptor comprises HLA-C. In some embodiments, the target antigen for the first receptor comprises HLA-E. In some embodiments, the target antigen for the first receptor comprises HLA-F. In some embodiments, the target antigen for the first receptor comprises HLA-G. In some embodiments, the target antigen for the first receptor comprises HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G.

[0259] Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure. In some embodiments, the ligand binding domain is an antigen binding domain. Exemplary antigen binding domains include, inter alia, scFv, SdAb, VP-only domains, and TCR antigen binding domains derived from the TCR a and P chain variable domains.

[0260] Any type of antigen binding domain is envisaged as within the scope of the instant disclosure.

[0261] For example, the first extracellular ligand binding domain may be part of a contiguous polypeptide chain including, for example, a VP-only domain, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb, a single chain antibody (scFv) derived from a murine, humanized or human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some aspects, the first extracellular ligand binding domain comprises an antigen binding domain that comprises an antibody fragment. In further aspects, the first extracellular ligand binding domain comprises an antibody fragment that comprises a scFv or an sdAb.

[0262] The term “antibody,” as used herein, refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.

[0263] The terms “antibody fragment” or “antibody binding domain” refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is 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')2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.

[0264] The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a 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.

[0265] “Heavy chain variable region” or “VH” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) with regard to an antibody refers to the fragment of the heavy chain that containsthree CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0266] Unless specified, as used herein a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0267] 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 VH region joined by a linker. In some embodiments, the linker comprises a glycine serine linker, for example GGGGSGGGGSGGGGSGG (SEQ ID NO: 135). In some embodiments, the scFv further comprises a signal sequence at the N terminus of the scFv. Exemplary signal sequences include MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 136), which is encoded by ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGC CAGATGT (SEQ ID NO: 137).

[0268] 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 (“X”) light chains refer to the two major antibody light chain isotypes.

[0269] The term “recombinant antibody” refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0270] The term “VP domain”, “VP-only domain”, “P chain variable domain” or “single variable domain TCR (svd-TCR)” refers to an antigen binding domain that consists 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. The VP-only domain engages antigen using complementaritydetermining regions (CDRs). Each VP-only domain contains three complement determining regions (CDR1, CDR2, and CDR3). Additional elements may be combined provided that the VP domain is configured to bind the epitope in the absence of a second TCR variable domain.

[0271] 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 P chain variable domain (VP).

[0272] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a TCR a chain variable domain and a TCR P chain variable domain.

[0273] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv antigen binding domain.

[0274] In some embodiments, the first, activator target antigen and the second, inhibitor target antigen are not the same.

[0275] In some embodiments, the target antigen for the first receptor is expressed by target cells and is not expressed by non-target cells (i.e. normal cells not targeted by the adoptive cell therapy). In some embodiments, the target cells are cancer cells and the non-target cells are non-cancerous cells.

[0276] In some embodiments, the target antigen for the first receptor has high cell surface expression on the target cells. This high cell surface expression confers the ability to deliver large activation signals. Methods of measuring cell surface expression will be known to the person of ordinary skill in the art and include, but are not limited to, immunohistochemistry using an appropriate antibody against the activator ligand, followed by microscopy or fluorescence activated cell sorting (FACS).

[0277] In some embodiments, the target antigen for the first receptor is encoded by a gene with an essential cellular function. Essential cellular functions are functions required for a cell to live, and include protein and lipid synthesis, cell division, replication, respiration, metabolism, ion transport, and providing structural support for tissues. Selecting activator ligands encoded by genes with essential cellular functions prevents loss of the activator ligand due to aneuploidy in cancer cells, and makes gene encoding the activator ligand less likely to undergo mutagenesis during the evolution of the cancer. In some embodiments, the target antigen for the first receptor is encoded by a gene that is haploinsufficient, i.e. loss of copies of the gene encoding the activator ligand are not tolerated by the cell and lead to cell death or a disadvantageous mutant phenotype.

[0278] In some embodiments, the target antigen for the first receptor is present on all target cells. In some embodiments, the target cells are cancer cells.

[0279] In some embodiments, the target antigen for the first receptor is present on a plurality of target cells. In some embodiments, the target cells are cancer cells. In some embodiments, the target antigen for the first receptor is present on at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% of target cells. In some embodiments, the target antigen for the first receptor is present on at least 95% target cells. In some embodiments, the target antigen for the first receptor is present on at least 99% target cells.

[0280] In some embodiments, the target antigen for the first receptor is present on all cells (ubiquitous activator ligands). Activator ligands can be expressed on all cells, if, for example, the second inhibitor ligand is also expressed on all cells except the target cells.

[0281] In some embodiments, the target antigen for the first receptor is expressed by a plurality of target cells and a plurality of non-target cells. In some embodiments, the plurality of non-target cells expresses both the first, activator ligand and the second inhibitor ligand.

[0282] In some embodiments, and the target antigen for the first receptor and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 : 100 to about 100: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :50 to about 50: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :25 to about 25: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 : 10 to about 10: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :5 to about 5: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :3 to about 3: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 :2 to about 2: 1 of the first ligand to the second ligand. In some embodiments, and the first, activator ligand and second, inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1 : 1.

[0283] The target antigen for the first receptor is recognized by a first ligand binding domain (sometimes referred to herein as the activator LBD).

[0284] Exemplary target antigens include antigens selected from the group consisting of cell adhesion molecules, cell-cell signaling molecules, extracellular domains, molecule involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane proteins, receptors for neurotransmitters and voltage gated ion channels. In some embodiments, the target antigen for the first receptor is transferrin receptor (TFRC) or a peptide antigen thereof. Human transferrin receptor is described in NCBI record No. AAA61153.1, the contents of which are incorporated herein by reference. In some embodiments, TFRC is encoded by a sequence of SEQ ID NO: 138.

[0285] In some embodiments, the target antigen for the first receptor is a cancer cell specific antigen.

[0286] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in a complex with a major histocompatibility complex class I (MHC-I). Any molecule expressed by the target cancer cells and presented by the major histocompatibility complex class I (MHC-I) on the cancer cell surface as a peptide antigen (pMHC) may be a suitable target antigen for the first receptor extracellular ligand binding domain.

[0287] In some embodiments, the cancer cell-specific antigen is Mesothelin (MSLN).

[0288] 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.

[0289] In some embodiments, the cancer cell-specific antigen is MSLN, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I). MSLN is a 40 KDa protein that is normally expressed in mesothelial cells, as well as lung, fallopian tube, salivary gland and adipose tissues. MSLN is expressed in multiple human tumor types, including mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or cholangiocarcinoma. In some embodiments, the cancer has relapsed in a subject. In some embodiments, the cancer is refractory to one or more prior administered anticancer therapies. In some embodiments, the cancer is metastatic.

[0290] All isoforms of MSLN are envisaged as cancer cell-specific antigens of the disclosure. MSLN isoform 1 preprotein is described in NCBI record number NP 005814.2, the contents of which are incorporated by reference herein. In some embodiments, MSLN comprises an amino acid sequence of:1 MALPTARPLL GSCGTPALGS LLFLLFSLGW VQPSRTLAGE TGQEAAPLDG VLANPPNI SS61 LS PRQLLGFP CAEVSGLSTE RVRELAVALA QKNVKLSTEQ LRCLAHRLSE PPEDLDALPL121 DLLLFLNPDA FSGPQACTRF FSRITKANVD LLPRGAPERQ RLLPAALACW GVRGSLLSEA181 DVRALGGLAC DLPGRFVAES AEVLLPRLVS CPGPLDQDQQ EAARAALQGG GPPYGPPSTW241 SVSTMDALRG LLPVLGQPI I RS I PQGIVAA WRQRSSRDPS WRQPERTI LR PRFRREVEKT301 ACPSGKKARE IDESLI FYKK WELEACVDAA LLATQMDRVN AI PFTYEQLD VLKHKLDELY361 PQGYPESVIQ HLGYLFLKMS PEDIRKWNVT SLETLKALLE VNKGHEMS PQ VATLIDRFVK421 GRGQLDKDTL DTLTAFYPGY LCSLS PEELS SVPPSS IWAV RPQDLDTCDP RQLDVLYPKA481 RLAFQNMNGS EYFVKIQS FL GGAPTEDLKA LSQQNVSMDL ATFMKLRTDA VLPLTVAEVQ541 KLLGPHVEGL KAEERHRPVR DWI LRQRQDD LDTLGLGLQG GI PNGYLVLD LSMQEALSGT601 PCLLGPGPVL TVLALLLAST LA (SEQ ID NO: 139).

[0291] In some embodiments, 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 to SEQ ID NO: 139.

[0292] MSLN isoform 2 preprotein is described in NCBI record number NP_037536.2, the contents of which are incorporated by reference herein. In some embodiments, MSLN comprises an amino acid sequence of:1 MALPTARPLL GSCGTPALGS LLFLLFSLGW VQPSRTLAGE TGQEAAPLDG VLANPPNI SS61 LS PRQLLGFP CAEVSGLSTE RVRELAVALA QKNVKLSTEQ LRCLAHRLSE PPEDLDALPL121 DLLLFLNPDA FSGPQACTRF FSRITKANVD LLPRGAPERQ RLLPAALACW GVRGSLLSEA181 DVRALGGLAC DLPGRFVAES AEVLLPRLVS CPGPLDQDQQ EAARAALQGG GPPYGPPSTW241 SVSTMDALRG LLPVLGQPI I RS I PQGIVAA WRQRSSRDPS WRQPERTI LR PRFRREVEKT301 ACPSGKKARE IDESLI FYKK WELEACVDAA LLATQMDRVN AI PFTYEQLD VLKHKLDELY361 PQGYPESVIQ HLGYLFLKMS PEDIRKWNVT SLETLKALLE VNKGHEMS PQ APRRPLPQVA421 TLIDRFVKGR GQLDKDTLDT LTAFYPGYLC SLS PEELSSV PPSS IWAVRP QDLDTCDPRQ481 LDVLYPKARL AFQNMNGSEY FVKIQS FLGG APTEDLKALS QQNVSMDLAT FMKLRTDAVL541 PLTVAEVQKL LGPHVEGLKA EERHRPVRDW I LRQRQDDLD TLGLGLQGGI PNGYLVLDLS 601 MQEALSGTPC LLGPGPVLTV LALLLASTLA (SEQ ID NO: 140).

[0293] In some embodiments, 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 to SEQ ID NO: 140.

[0294] 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 subsequence of SEQ ID NO: 1 and / or SEQ ID NO: 140. In some embodiments, the peptide antigen comprises a sequence identical to a subsequence of SEQ ID NO: 1 and / or SEQ ID NO: 140.

[0295] In some embodiments, the target antigen is MSLN or a peptide antigen thereof, and the activator ligand binding domain comprises a MSLN binding domain.

[0296] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv antigen binding domain. Exemplary MSLN scFv are shown in Table 6 below.Table 6: Exemplary MSLN scFy domains

[0297] 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 to MSLN are shown in Table 6, supra. In Table 6, underlining indicates CDR sequences.

[0298] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an antigen binding domain having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity or at least 99% identity to a sequence of SEQ ID NOS: 142-208, or a sequence as set forth in Table 6. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an antigen binding domain comprising a sequence of SEQ ID NOS: 142-208, as set forth in Table 6.

[0299] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an binding domain having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity or at least 99% identity to a sequence of SEQ ID NO: 164. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an binding domain comprising a sequence of SEQ ID NO: 164.

[0300] 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: 142-208. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv antigen binding domain comprising a sequence of any one of SEQ ID NOs: 142- 208. 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: 142-208.Table 7: Sequences of MSLN complementary determining regions (CDRs)

[0301] In Table 7, the light chain (LC) CDRs paired with the indicated heavy chain (HC) CDRs are indicated in the left column.

[0302] In some embodiments, the extracellular ligand binding domain of the first receptor comprises the HC CDR1, the HC CDR2, and the HC CDR3 as set forth in Table 7 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 7) or sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 7. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the LC CDR1, the LC CDR2, and the LC CDR3 as set forth in Table 7 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 7) or sequences having at most 1, 2, or 3 substitutions, deletions, orinsertion relative to the CDRs of Table 7. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the HC CDR1, the HC CDR2, and the HC CDR3 as set forth in Table 7 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 7). In some embodiments, the extracellular ligand binding domain of the first receptor comprises the LC CDR1, the LC CDR2, and the LC CDR3 as set forth in Table 7 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 7).

[0303] In some embodiments, the extracellular ligand binding domain of the first receptor comprises the HC CDR1, HC CDR2, HC CDR3, LC CDR1, the LC CDR2, and the LC CDR3 as set forth in Table 7 (e.g., the HC CDR1, HC CDR2 and HC CDR3 as set forth in line 1, and the LC CDR 1, the LC CDR2, and the LC CDR 3 in line A) In some embodiments, the extracellular ligand binding domain of the first receptor comprises the HC CDR1, the HC CDR2, and the HC CDR3 as set forth in Table 7 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 7) or sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 7. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the LC CDR1, the LC CDR2, and the LC CDR3 as set forth in Table 7 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 7) or sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 7. In some embodiments, an extracellular ligand binding domain of the first receptor comprises one or more HC CDRs as set forth in Table 7 and one or more LC CDRs as set forth in Table 7. In some embodiments, the extracellular ligand binding domain of the first receptor comprises (i) the HC CDR1, the HC CDR2, and the HC CDR3 as set forth in one line of Table 7 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 2) and (ii) the LC CDR1, the LC CDR2, and the LC CDR3 as set forth in one line of Table 7 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 7). In each case, the HC CDRs may be paired with any of the LC CDRs, as the heavy chains and light chains share similarity, with routine testing to confirm desired expression and binding activity; however, preferred pairing between heavy and light chains of some embodiments are indicated in the right hand column of Table 7.

[0304] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising a sequence of SGDYYWS (SEQ ID NO: 285), a HC CDR2 comprising a sequence of YIYYSGSTYYNPSLKS (SEQ ID NO: 300), and HC CDR3 comprising a sequence of CARED VVKGAFDIW (SEQ ID NO: 379), or CDR sequences having at most 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising a sequence of SGDYYWS (SEQ ID NO: 285), a HC CDR2 comprising a sequence of YIYYSGSTYYNPSLKS (SEQ ID NO: 300), and HC CDR3 comprising a sequence of CARED VVKGAFDIW (SEQ ID NO: 379). In someembodiments, the extracellular ligand binding domain of the first receptor comprises a LC CDR1 comprising a sequence of RASQSISSYLN (SEQ ID NO: 381), a LC CDR2 comprising a sequence of AASSLQS (SEQ ID NO: 385), and a LC CDR3 comprising a sequence of QQSYSTPLT (SEQ ID NO: 388), or CDR sequences having at most 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a LC CDR1 comprising a sequence of RASQSISSYLN (SEQ ID NO: 381), a LC CDR2 comprising a sequence of AASSLQS (SEQ ID NO: 385), and a LC CDR3 comprising a sequence of QQSYSTPLT (SEQ ID NO: 388). In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising a sequence of SGDYYWS (SEQ ID NO: 285), a HC CDR2 comprising a sequence of YIYYSGSTYYNPSLKS (SEQ ID NO: 300), HC CDR3 comprising a sequence of CARED VVKGAFDIW (SEQ ID NO: 379), a LC CDR1 comprising a sequence of RASQSISSYLN (SEQ ID NO: 381), a LC CDR2 comprising a sequence of AASSLQS (SEQ ID NO: 385), and a LC CDR3 comprising a sequence of QQSYSTPLT (SEQ ID NO: 388), or CDR sequences having at most 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising a sequence of SGDYYWS (SEQ ID NO: 285), a HC CDR2 comprising a sequence of YIYYSGSTYYNPSLKS (SEQ ID NO: 300), HC CDR3 comprising a sequence of CARED VVKGAFDIW (SEQ ID NO: 379), a LC CDR1 comprising a sequence of RASQSISSYLN (SEQ ID NO: 381), a LC CDR2 comprising a sequence of AASSLQS (SEQ ID NO: 385), and a LC CDR3 comprising a sequence of QQSYSTPLT (SEQ ID NO: 388).

[0305] 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: 295), LITPYNGASSYNQKFRG (SEQ ID NO: 316) and GGYDGRGFDY (SEQ ID NO: 380). In some embodiments, the heavy chain comprises sequences of GYTMN (SEQ ID NO: 295), LITPYNGASSYNQKFRG(SEQ ID NO: 316) and GGYDGRGFDY (SEQ ID NO: 380). In some embodiments, the scFv comprising a light chain comprising CDRs selected from the sequences of SASSSVSYMH (SEQ ID NO: 384), DTSKLAS (SEQ ID NO: 387) and QQWSGYPLT (SEQ ID NO: 391). In some embodiments, the light chain comprises sequences of SASSSVSYMH (SEQ ID NO: 384), DTSKLAS (SEQ ID NO: 387) and QQWSGYPLT (SEQ ID NO: 391).

[0306] Sequences of exemplary heavy and light chains of antigen binding domains that are specific to MSLN are as set forth in Table 8 and Table 9 below. Light chains paired with heavy chains in preferred embodiments are indicated at right in Table 8.Table 8: Sequences of heavy chain variable fragments (VH)Table 9: Sequences of light chain variable fragments (VL)

[0307] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy region (VH) sequence as set forth in Table 8. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VH sequence that 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 VH as set forth in Table 8. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable light region (VL) sequence as set forth in Table 9. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VL sequence that 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 as set forth as set forth in Table 9.

[0308] In some embodiments, the target antigen is CEA or a peptide antigen thereof, and the activator ligand binding domain comprises a CEA binding domain. In some embodiments, the CEA ligand binding domain comprises an ScFv domain.

[0309] In some embodiments, the extracellular ligand binding domain of the first receptor is an scFv. In some embodiments, the scFv domain binds to CEA. In some embodiments, the scFv is the ligand binding domain of a CAR. Exemplary CAR sequences comprising CEA targeting scFv domains are shown in Table 11 below. In Table 11, CDR sequences are underlined.

[0310] Exemplary scFv that recognize CEA are shown in Table 11 below. Underlining indicates CDR sequences.Table 11. Exemplary scFv that target CEA

[0311] In some embodiments, a CEA scFv comprises a sequence selected from the group consisting of SEQ ID NOs: 454-460, 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, a CEA scFv comprises, or consists essentially of, a sequence selected from the group consisting of SEQ ID NOs: 454-460. Further exemplary anti- CEA antibody sequences are provided in Stewart et al. Cancer Immunol. Immunother. 47:299-306 (1999); WO 1999 / 043817 Al; US 2002 / 0018750 Al; US 2011 / 0104148 Al; US 2016 / 0108131 Al; US20160075795A1; US 2019 / 0185583 Al; US 2020 / 0123270 Al; WO 2020 / 259550 Al;WO 2021 / 053587 Al; WO 2021 / 110647 Al; the contents of which are incorporated by reference herein for the purpose of providing anti-CEA VH, VL, scFv, and / or ligand binding domain sequences.

[0312] Exemplary VH, VL, and CDR regions that recognize CEA are shown in Table 12 below.Table 12

[0313] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 468 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 470 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 variable heavy (VH) portion comprising SEQ ID NO: 468, and a variable light (VL) portion comprising SEQ ID NO: 470. In some embodiments, the extracellular ligand binding domain of the first receptor further comprises a linker between VH and VL portions.

[0314] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 456-460, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFvsequence of SEQ ID NO: 458; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv sequence of SEQ ID NO: 458.

[0315] In some embodiments, the target antigen is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR binding domain. In some embodiments, the EGFR ligand binding domain comprises an ScFv domain.

[0316] Exemplary EGFR scFv are shown in Table 13 below.Table 13. EGFR scFv domainsIll

[0317] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR binding domain. In some embodiments, the EGFR ligand binding domain comprises an scFv domain. In some embodiments, the EGFR ligand binding domain comprises a sequence of any one of SEQ ID NOs: 484-493. In some embodiments, the EGFR ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to any one of SEQ ID NOs: 484-493. In some embodiments, the EGFR ligand binding domain is encoded by a sequence selected from the group of sequences in Table 13. In some embodiments, the EGFR ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence from Table 13. It is understood that the scFv domains of the disclosure that bind EGFR can cross-react with one or more variants of EGFR. For example, an scFv may bind at least one of a normal EGFR, a variant of EGFR, or a mutated EGFR (e.g. EGFRvIII).

[0318] 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: 484-493. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an ScFv antigen binding domain comprising a sequence of any one of SEQ ID NOs: 484- 493. 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: 484-493. Table 14. EGFR Variable Heavy (VH) and Variable Light (VL) domains

[0319] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR ligand binding domain. In some embodiments, the EGFR binding domain comprises a VH and / or a VL domain selected from the group disclosed in Table 14 or a sequence having at least 90% identity, at least 95% identity, at least 97% identity or at least 99% identity thereto. In some embodiments, the EGFR ligand binding domain comprises a VH domain selected from the group consisting of SEQ ID NOs: 504-510. In some embodiments, the EGFR ligand binding domain comprises a VH selected from the group consisting of SEQ ID NOs: 504-510 or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the EGFR ligand binding domain comprises a VL domain selected from the group consisting of SEQ ID NOs: 511-517. In some embodiments, the EGFR ligand binding domain comprises a VL selected from the group consisting of SEQ ID NOs: 511-517 or a sequence having at least 90%, at least 95% or at least 99% identity thereto.Table 15. EGFR antigen binding domain CDRs.

[0320] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain is an EGFR ligand binding domain. In some embodiments, the EGFR binding domain comprises complementarity determining region (CDRs) selected from the group of CDRs disclosed in Table 15. In some embodiments, the EGFR ligand binding domain comprises CDRs having at least 95% sequence identity to CDRs disclosed in Table 15. In some embodiments, the EGFR ligand binding domain comprises CDRs selected from SEQ ID NOs: 518- 559. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 1 (CDR Hl) selected from the group consisting of SEQ ID NOs: 518-524. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 2 (CDR H2) selected from the group consisting of SEQ ID NOs: 525-531. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 3 (CDR H3) selected from the group consisting of SEQ ID NOs: 532- 538. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 1 (CDR LI) selected from the group consisting of SEQ ID NOs: 539-545. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 2 (CDR L2) selected from the group consisting of SEQ ID NOs: 546-552. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 3 (CDR L3) selected from the group consisting of SEQ ID NOs: 553-559. In some embodiments, the EGFR ligand binding domain comprises a CDR Hl selected from SEQ ID NOs: 518-524, a CDR H2 selected from SEQ ID NOs: 525-531, a CDR H3 selected from SEQ ID NOs:4532-538, a CDR LI selected from SEQ ID NOs: 539-545, a CDR L2 selected from SEQ ID NOs: 546-552, and a CDR L3 selected from SEQ ID NOs: 553-559.

[0321] In some embodiments, the target antigen is HER2 or a peptide antigen thereof, and the activator ligand binding domain comprises a HER2 binding domain. In some embodiments, the HER2 ligand binding domain comprises an ScFv domain.

[0322] In some embodiments, the VH and VL regions comprise complement determining regions (CDRs) selected from the group of CDRs disclosed in Table 16.Table 16: HER2 antigen binding domain complement determining regions (CDRs)

[0323] In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of GFNIKDTYIH (SEQ ID NO: 568), ARIYPTNGYTRYADSVKG (SEQ ID NO: 571), and SRWGGDGFYAMD[Y / V] (SEQ ID NO: 574) or (SEQ ID NO: 575); and the VL region comprises one or more CDR sequences selected from the group consisting of RASQDVNTAVA (SEQ ID NO: 560), SASFLY (SEQ ID NO: 562), and QQHYTTPP (SEQ ID NO: 565).

[0324] In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of NYGMN (SEQ ID NO: 569), WINTSTGESTFADDFKG (SEQ ID NO: 572) and WEVYHGYVPY (SEQ ID NO: 576); and the VL region comprises one or more CDR sequences selected from the group consisting of KASQDVYNAVA (SEQ ID NO: 561), SASSRYT (SEQ ID NO: 563), and QQHFRTPFT (SEQ ID NO: 566).

[0325] In some embodiments, the VH region comprises CDR sequences of GFNIKDTYIH (SEQ ID NO: 568), ARIYPTNGYTRYADSVKG (SEQ ID NO: 571), and SRWGGDGFYAMDY(SEQ ID NO: 574) or SRWGGDGFYAMDV (SEQ ID NO: 575); and the VL region comprises CDR sequences of RASQDVNTAVA (SEQ ID NO: 560), SASFLY (SEQ ID NO: 562) and QQHYTTPP (SEQ ID NO: 565).

[0326] In some embodiments, the VH region comprises CDR sequences of NYGMN (SEQ ID NO: 569), WINTSTGESTFADDFKG (SEQ ID NO: 572) and WEVYHGYVPY (SEQ ID NO:576); and the VL region comprises CDR sequences of KASQDVYNAVA (SEQ ID NO: 561), SASSRYT (SEQ ID NO: 563), and QQHFRTPFT (SEQ ID NO: 566).

[0327] In some embodiments, the VH region comprises CDR sequences DTYIH (SEQ ID NO: 570), RIYPTNGYTRYADSVKG (SEQ ID NO: 573), and WGGDGFYAMDV (SEQ ID NO:577); and the VL region comprises CDR sequences of RASQDVNTAVA (SEQ ID NO: 560), SASFLYS (SEQ ID NO: 564), and QQHYTTPPT (SEQ ID NO: 567).

[0328] In some embodiments, the full length VH and VL regions comprise the sequences disclosed in 15. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 17. In some embodiments, the binding domain comprises SEQ ID NO: 578 and SEQ ID NO: 579. In some embodiments, the binding domain comprises SEQ ID NO: 45580 and SEQ ID NO: 581. In some embodiments, the binding domain comprises SEQ ID NO: 582 and SEQ ID NO: 583. In some embodiments, the binding domain comprises SEQ ID NO: 586 and SEQ ID NO: 587. In some embodiments, the binding domain comprises SEQ ID NO: 588 and SEQ ID NO: 589.Table 17: VH and VL Domains for HER2 CAR Constructs

[0329] In some embodiments, the binding domain is a HER2 scFv domain. In some embodiments, the HER2 scFv domain is selected from a sequence listed in Table 18. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 590. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 596. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 591. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 597. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 592. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 598. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 593. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 599. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 594. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 600. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 595.In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 601.Table 18: HER2 ScFv Domains for HER2 CAR Constructs

[0330] In some embodiments, the HER2 scFv domain comprises a sequence of any one of SEQ ID NOS: 590-595, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% or is identical thereto.

[0331] In some embodiments, the target antigen is a pan-HLA ligand, and the activator binding domain is a pan-HLA binding domain, i.e. a binding domain that binds to and recognizes an antigenic determinant shared among products of the HLA A, B and C loci. Various single variable domains known in the art or disclosed herein are suitable for use in embodiments. Such scFvs include, for example and without limitation, the following mouse and humanized pan-HLA scFv antibodies. An exemplary pan-HLA ligand is W6 / 32, which recognizes a conformational epitope, reacting with HLA class I alpha3 and alpha2 domains. Illustrative pan-HLA scFv binding domains derived from W6 / 32 are as set forth in SEQ ID NOs: 392, 393, 394, 395, 396, and 397. Illustrative polynucleotide sequences encoding an-HLA scFv binding domains derived from W6 / 32 are as set forth in SEQ ID NOs: 398, 399, 400, 401, 402, and 403.

[0332] In some embodiments, the target antigen is pan-HLA ligand, and the activator ligand binding domain comprises a pan-HLA ligand binding domain. In some embodiments, the pan-HLA ligand binding domain comprises an ScFv domain. In some embodiments, the pan-HLA ligand binding domain comprises a sequence of SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, or SEQ ID NO: 397. In some embodiments, the pan-HLA ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, or SEQ ID NO: 397. In some embodiments, the pan-HLA ligand binding domain is encoded by a sequence comprising SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, or SEQ ID NO: 403. In some embodiments, the pan-HLA ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, or SEQ ID NO: 403.

[0333] In some embodiments, the target antigen is CD 19 molecule (CD 19) or a peptide antigen thereof, and the activator ligand binding domain comprises a CD 19 ligand binding domain. In some embodiments, the CD 19 ligand binding domain comprises an ScFv domain. In some embodiments, the CD 19 ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 404 or SEQ ID NO: 405. In some embodiments, the CD- 19 ligand binding domain comprises a sequence of SEQ ID NO: 404 or SEQ ID NO: 405. In some embodiments, the CD19 ligand binding domain is encoded by a sequence comprising SEQ ID NO: 406, or SEQ ID NO: 407. In some embodiments, the CD19 ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 406 or SEQ ID NO:407.

[0334] In some embodiments, the target antigen is CD 19 molecule (CD 19) or a peptide antigen thereof, and the activator receptor is a CAR. In some embodiments, the CD 19 CAR comprises asequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 408 or SEQ ID NO: 409. In some embodiments, the CD19 CAR comprises or consists essentially of SEQ ID NO: 408 or SEQ ID NO: 409. In some embodiments, the CD19 CAR is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 410 or SEQ ID NO: 411. In some embodiments, the CD 19 CAR is encoded by a sequence comprising or consisting essentially of SEQ ID NO: 410 or SEQ ID NO: 411.

[0335] In some embodiments, the target antigen is ICAM1 molecule or a peptide antigen thereof, and the activator ligand binding domain comprises an ICAM1 ligand binding domain. In some embodiments, the ICAM1 ligand binding domain comprises an ScFv domain.

[0336] It will be appreciated by the person of ordinary skill that first, activator ligand binding domains for the first receptor may be isolated or derived from any source known in the art, including, but not limited to, art recognized T cell receptors, chimeric antigen receptors and antibody binding domains. For example, the first ligand binding domain may be derived from any of the antibodies disclosed in Table 19, and bind to a first ligand selected from the antigens described in Table 19. Accordingly, the immune cells comprising the two receptor system described can be used to treat any of the diseases or disorders described in Table 19. Selection of an appropriate first, activator receptor ligand binding domain to treat any the cancers described herein will be apparent to those of skill in the art.Table 19: Exemplary AntibodiesChimeric Antigen Receptors (CARs)

[0337] The disclosure provides a first, activator receptor and immune cells comprising same. In some embodiments, the first receptor is a chimeric antigen receptor.

[0338] The term “chimeric antigen receptors (CARs)” as used herein, may refer to artificial receptors derived from T-cell receptors and encompasses engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a tumor associated antigen, for example. Exemplary CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region. In some embodiments, CARs further comprise a hinge domain. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to a CD3 transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides). In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3, 4-1BB, FcR, CD27, CD28, CD137, DAP10, and / or 0X40. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, cytokines, and cytokine receptors.

[0339] In some embodiments, the extracellular ligand binding domain of the first receptor is fused to the extracellular domain of a CAR.

[0340] In some embodiments, the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo. Exemplary hinges can be isolated or derived from IgD and CD8domains, for example IgGl. In some embodiments, the hinge is isolated or derived from CD8a or CD28.

[0341] In some embodiments, the hinge is isolated or derived from CD8a or CD28. In some embodiments, the CD8a hinge 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 a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 602). In some embodiments, the CD8a hinge comprises SEQ ID NO: 602. In some embodiments, the CD8a hinge consists essentially of SEQ ID NO: 602. In some embodiments, the CD8a 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 a sequence ofAC C C GAC GC CAGC GC C GC GAC CAC CAACAC C GGC GC C CAC CAT C GC GT C GCAGC CCCTGTCCCTGCGCC CAGAGGC GT GC CGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT ( SEQ ID NO : 603 ) . In SOme embodiments, the CD8a hinge is encoded by SEQ ID NO: 603.

[0342] 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 is identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 604). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO:604. 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 is identical to a sequence of T G T AC C AT T GAAG TTATGTATCCTCCTCCTTACC T AGAC AAT GAGAAGAG C AAT G GAAC CAT TAT C C ATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 605). In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 605.

[0343] The CARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. For example, a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0344] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions may be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) 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, CD 154, or from an immunoglobulin such as IgG4. Alternatively the transmembrane domain may besynthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0345] In some embodiments of the CARs of the disclosure, the CARs comprise 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 is identical to a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 606). In some embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 606. 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 a sequence of TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG ( SEQ ID NO : 607 ) . In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 607.

[0346] In some embodiments of the CARs of the disclosure, the CARs comprise an IL-2Rbeta transmembrane domain. In some embodiments, the IL-2Rbeta 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 a sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 608). In some embodiments, the IL-2Rbeta transmembrane domain comprises or consists essentially of SEQ ID NO: 608. In some embodiments, the IL-2Rbeta 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 a sequence of ATTCCGTGGC TCGGCCACCT CCTCGTGGGC CTCAGCGGGG CTTTTGGCTT CATCATCTTA GTGTACTTGC TG TC (SEQ ID NO: 609). In some embodiments, the IL-2Rbeta transmembrane domain is encoded by SEQ ID NO: 609.

[0347] The cytoplasmic domain or otherwise the intracellular signaling domain of the CARs of the instant disclosure is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been 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 which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signalingdomain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired functions of the CAR-T cells of the instant disclosure. The term intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.

[0348] Examples of intracellular signaling domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0349] Accordingly, the intracellular domain of CARs of the instant disclosure comprises at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures that there is T-cell receptor (TCR) signaling necessary to activate the effector functions of the CAR T-cell. In some embodiments, the at least one cytoplasmic activation is a CD247 molecule (CD3Q activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX-activating protein of 12 kDa (DAP 12) activation domain.

[0350] In some embodiments, the CD3(^ activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 610).

[0351] In some embodiments, the CD3(^ activation domain comprises or consists essentially of SEQ ID NO: 15. In some embodiments, the CD3(^ 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 a sequence ofAGAGT GAAGT T CAGCAGGAGC GCAGAC GCCCCCGC GT ACAAGCAGGGC CAGAAC CAGCT CT AT AAC GAGCT CAAT CT AGGA C GAAGAGAGGAGT AC GAT GT T T T GGACAAGC GT AGAGGC C GGGAC C CT GAGAT GGGGGGAAAGC C GAGAAGGAAGAAC C CT CAGGAAGGC CT GT ACAAT GAACT GCAGAAAGAT AAGAT GGC GGAGGC CT ACAGT GAGAT T GGGAT GAAAGGC GAGC GC C GG AGGGGCAAGGGGCAC GAT GGC CT T TAG CAGGGACT CAGT ACAGC CAC CAAGGACAC CT AC GAC GC C CT T CACAT GCAGGC C CTGCCCCCTCGC (SEQ ID NO: 611). In some embodiments, the CD3(^ activation domain is encoded by SEQ ID NO: 611).

[0352] It is known that signals generated through the TCR alone are often insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signalingsequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).

[0353] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs, which are known as immunoreceptor tyrosinebased activation motifs or ITAMs. In some embodiments, the ITAM contains a tyrosine separated from a leucine or an isoleucine by any two other amino acids (YxxL / I (SEQ ID NO: 612)). In some embodiments, the cytoplasmic domain contains 1, 2, 3, 4 or 5 ITAMs. An exemplary ITAM containing cytoplasmic domain is the CD3(^ activation domain. Further examples of ITAM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from TCR^, FcRy, FcRp, CD3y, CD35, CD3s, CD3< CD5, CD22, CD79a, CD79b, and CD66d.

[0354] In some embodiments, the CD3(^ activation domain comprising a single ITAM 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 a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 613). In some embodiments, the CD3(^ activation domain comprises SEQ ID NO: 613. In some embodiments, the CD3(^ activation domain comprising a single ITAM consists essentially of an amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 613). In some embodiments, the CD3(^ activation domain comprising a single ITAM 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 a sequence of AGAGTGAAGT TCAGCAGGAG CGCAGACGCC CCCGCGTACC AGCAGGGCCA GAACCAGCTC TATAACGAGC TCAATCTAGG ACGAAGAGAG GAGTACGATG TTTTGCACAT GCAGGCCCTG cccccTCGG (SEQ ID NO: 614). In some embodiments, the CD3(^ activation domain is encoded by SEQ ID NO: 614.

[0355] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise the CD3(^ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the instant disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3(^ chain portion and a co-stimulatory domain. The co-stimulatory domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include the co- stimulatory domain is selected from the group consisting of IL-2RP, Fc Receptor gamma (FcRy), Fc Receptor beta (FcRP), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22molecule (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- IBB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (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 domains, or functional variants thereof. In some embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is isolated or derived from CD28.

[0356] In some embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 413). In some embodiments, the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 19. In some embodiments, the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence ofAGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGC CTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTAC CGGAGC (SEQ ID NO: 615). In some embodiments, the CD28 co-stimulatory domain is encoded by SEQ ID NO: 615.

[0357] In some embodiments, the co-stimulatory domain is isolated or derived from 4- IBB. In some embodiments, the 4-1BB co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 616). In some embodiments, the 4-1BB co-stimulatory domain comprises or consists essentially of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 616). In some embodiments, the 4-1BB co-stimulatory domain s 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 a sequence ofAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGGCCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGG ATGTGAACTG (SEQ ID NO: 617).

[0358] In some embodiments, the intracellular domain of the CAR comprises a CD28 co-stimulatory domain, a 4-1BB costimulatory domain, and a CD3(^ activation domain. In some embodiments, the intracellular domain of the CAR comprises a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR (SEQ ID NO: 618), or a sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity thereto.

[0359] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker. An exemplary linker comprises a sequence of GGGGSGGGGSGGGGSGG (SEQ ID NO: 135).

[0360] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker. Exemplary full length activator receptors of the disclosure are described in Table 20 below.Table 20.

[0361] In some embodiments, the first activator receptor comprises a sequence of SEQ ID NOS: 983- 1044, as set forth in Table 20, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NOS: 983-1044, as set forth in Table 20. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 985, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 994, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 998, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 999, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 1000, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 1011, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 1032, or a sequence having at least 90%, atleast 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 1037, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the first activator receptor comprises a sequence of SEQ ID NO: 1041 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0362] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker.T Cell Receptors (TCRs)

[0363] The disclosure provides a first, activator receptor and immune cells comprising same. In some embodiments, the first receptor is a T cell receptor (TCR).

[0364] As used herein, a “TCR”, sometimes also called a “TCR complex” or “TCR / CD3 complex” refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more of the invariant CD3 chains (zeta, gamma, delta and epsilon), sometimes referred to as subunits. The TCR alpha and beta chains can be disulfide-linked to function as a heterodimer to bind to peptide-MHC complexes. Once the TCR alpha / beta heterodimer engages peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal. In an exemplary TCR complex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3 epsilon and CD3 delta form a heterodimer, CD3 epsilon and CD3 gamma for a heterodimer, and two CD3 zeta form a homodimer.

[0365] Any suitable ligand binding domain may be fused to an extracellular domain, hinge domain or transmembrane of the TCRs described herein. For example, the ligand binding domain can be an antigen binding domain of an antibody or TCR, or comprise an antibody fragment, a VP only domain, a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb).

[0366] In some embodiments, the ligand binding domain is fused to one or more extracellular domains or transmembrane domains of one or more TCR subunits. The TCR subunit can be TCR alpha, TCR beta, CD3 delta, CD3 epsilon, CD3 gamma or CD3 zeta. For example, the ligand binding domain can be fused to TCR alpha, or TCR beta, or portions of the ligand binding can be fused to two subunits, for example portions of the ligand binding domain can be fused to both TCR alpha and TCR beta.

[0367] TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma and CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta, CD3 epsilon, orCD3 zeta, or fragments or derivative thereof, can be fused to one or more domains capable of providing a stimulatory signal of the disclosure, thereby enhancing TCR function and activity.

[0368] TCR transmembrane domains isolated or derived from any source are envisaged as within the scope of the disclosure. The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein.

[0369] In some embodiments, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex has bound to a target. A transmembrane domain of particular use in this disclosure may include at least the transmembrane region(s) of e.g, 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.

[0370] In some embodiments, the transmembrane domain can be attached to the extracellular region of a polypeptide of the TCR, e.g., the antigen binding domain of the TCR alpha or beta chain, via a hinge, e.g., a hinge from a human protein. For example, the hinge can be a human immunoglobulin (Ig) hinge, e.g, an IgG4 hinge, or a CD8a hinge. In some embodiments, the hinge is isolated or derived from CD 8 a or CD28.

[0371] In some embodiments, the extracellular ligand binding domain is attached to one or more transmembrane domains of the TCR. In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain, a TCR beta transmembrane domain, or both. In some embodiments, the transmembrane comprises a CD3 zeta transmembrane domain.

[0372] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was 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).

[0373] In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0374] When present, the transmembrane domain may be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain may be a membrane anchor domain. Without limitation, a natural or artificial transmembrane domain may comprise a hydrophobic a-helix of about 20 amino acids, often with positive charges flanking the transmembrane segment. The transmembrane domain may have one transmembrane segment or more than one transmembrane segment. Prediction oftransmembrane domains / segments may be made using publicly available prediction tools (e.g. TMHMM, Krogh et al. Journal of Molecular Biology 2001; 305(3):567-580; or TMpred, Hofmann & Stoffel Biol. Chem. Hoppe-Seyler 1993; 347: 166). Non-limiting examples of membrane anchor systems include platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (added post- translationally to a signal sequence) and the like.

[0375] 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 a sequence of: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 620). 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 a sequence of GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTG CGGCTGTGG (SEQ ID NO: 621).

[0376] 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 a sequence of: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 622). 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 a sequence of ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGC CCTCGTGCTG (SEQ ID NO: 623).

[0377] TCRs of the disclosure can comprise one or more intracellular domains. Exemplary TCRs comprising intracellular domains for use in the instant disclosure are described in PCT / US2020 / 045250 filed on 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 a 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 a first, second and third intracellular domain capable of providing a stimulatory signal. The intracellular domains capable of providing a stimulatory signal are selected from the group consisting of a CD28 molecule (CD28) domain, a LCK proto-oncogene, Src family tyrosine kinase (Lek) domain, a TNF receptor superfamily member 9 (4- IBB) domain, a TNF receptor superfamilymember 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, Src family tyrosine kinase (Fyn) domain, a zeta chain of T cell receptor associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) alpha, TCRbeta, CD3 delta, CD3 gamma and CD3 epsilon intracellular domains.

[0378] In some embodiments, an intracellular domain comprises at least one intracellular signaling domain. An intracellular signaling domain generates a signal that promotes a function a cell, for example an immune effector function of a TCR containing cell, e.g., a TCR-expressing T-cell. In some embodiments, the intracellular domain of the first receptor of the disclosure includes at least one intracellular signaling domain. For example, the intracellular domains of CD3 gamma, delta or epsilon comprise signaling domains.

[0379] In some embodiments, the extracellular domain, transmembrane domain and intracellular domain are isolated or derived from the same protein, for example T-cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, CD3 epsilon or CD3 zeta.

[0380] Examples of intracellular domains for use in activator receptors of the disclosure include the cytoplasmic sequences of the TCR alpha, TCR beta, CD3 zeta, and 4-1BB, and the intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0381] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the proteins responsible for primary stimulation, or antigen dependent stimulation.

[0382] 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.

[0383] In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain. In some embodiments, a TCR alpha intracellular domain comprises Ser-Ser. In some embodiments, a TCR alpha intracellular domain is encoded by a sequence of TCCAGC.

[0384] 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 is identical to a sequence of: MAMVKRKDSR (SEQ ID NO: 624). In some embodiments, the TCR beta intracellular domain comprises, or consists essentially of SEQ ID NO: 25. In some embodiments, the TCRbeta intracellular domain is encoded by a sequence ofATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 625).

[0385] 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 and CD3 epsilon intracellular domain, and one additional stimulatory intracellular domain, for example a co-stimulatory domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.

[0386] Exemplary co-stimulatory intracellular signaling domains include those derived from proteins responsible for co-stimulatory signals, or antigen independent stimulation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA, a Toll ligand receptor, as well as DAP10, DAP12, CD30, LIGHT, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1 (CDl la / CD18) 4-1BB (CD 137, TNF receptor superfamily member 9), and CD28 molecule (CD28). A co-stimulatory protein can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, 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 with CD83, CD4, and the like. The co-stimulatory domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional variant thereof.

[0387] In some embodiments, the stimulatory domain comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises a CD28 or 4- IBB co-stimulatory domain. CD28 and 4-1BB are well characterized co-stimulatory molecules required for full T cell activation and known to enhance T cell effector function. For example, CD28 and 4-1BB have been utilized in chimeric antigen receptors (CARs) to boost cytokine release, cytolytic function, and persistence over the first-generation CAR containing only the CD3 zeta signaling domain. Likewise, inclusion of co- stimulatory domains, for example CD28 and 4- IBB domains, in TCRs can increase T cell effector function and specifically allow co-stimulation in the absence of co-stimulatory ligand, which is typically down-regulated on the surface of tumor cells. In some embodiments, the stimulatory domain comprises a CD28 intracellular domain or a 4-1BB intracellular domain.Inhibitory Receptors

[0388] The disclosure provides a second receptor, comprising an extracellular ligand binding domain specific to HLA-A*03 that has been lost in a cancer cell. The HLA-A*03 can be lost in the cancer cell through any mechanism, such as, without limitation, epigenetic changes that effect non-target allelicvariant expression, mutations to the gene encoding the HLA-A*03, disruption of cellular signaling that regulates expression of the HLA-A*03, chromosome loss, partial or complete deletion of the genomic locus, gene silencing through modification of nucleic acids or heterochromatin, or loss of expression through other mechanisms. In variations of the compositions and methods disclosed herein, the cells or subject treated may exhibit a loss of expression of the HLA-A*03 because of non-genetic changes. Accordingly the disclosure provides compositions and methods for killing cells and / or treating subject lacking expression of the non-target antigen from any cause, including but not limited to, loss of heterozygosity.

[0389] The non-target antigen can be a HLA-A*03 protein, or an antigen peptide thereof in a complex with a major histocompatibility complex class I (MHC-I), where the non-target antigen comprises a polymorphism. Because the non-target antigen is polymorphic, loss of a single copy of the gene encoding the non-target antigen, which may occur through loss of heterozygosity in a cancer cell, yields a cancer cell that retains the other polymorphic variant of gene, but has lost the non-target antigen.

[0390] In some embodiments, the second receptor is humanized.

[0391] The disclosure provides a second receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can discriminate between single amino-acid variant alleles of a non- target antigen. This ability to discriminate between allelic variants of a non-target antigen allows the second receptor to inhibit activation of immune cells comprising the second receptor in the presence of non-target cells that express that the allele recognized by the ligand binding domain. However, activation of immune cells is not inhibited in the presence of target cells that have lost the allele, for example cancer cells that have lost one allele of a gene through loss of heterozygosity.

[0392] The disclosure provides a second receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can discriminate between different levels of expression of a non-target antigen. This allows the second receptor to inhibit activation of immune cells comprising the second receptor in the presence of non-target cells that express the ligand for the second receptor, but to allow activation of immune cells in the presence of cancer cells that express low levels, or have no expression, of the ligand for the second receptor.Inhibitor Ligands

[0393] In some embodiments, the HLA-A*03 is not expressed by the target cells, and is expressed by non-target cells. In some embodiments, the HLA-A*03 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 the non-target antigen.

[0394] In some embodiments, the HLA-A*03 is lost in the cancer cells due to loss of heterozygosity.

[0395] Non-target major histocompatibility complex class I MHC-I (or pMHC-I) antigens comprising any of HLA-A are envisaged as within the scope of the disclosure. In some embodiments, the nontarget 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. In some embodiments, the HLA-A allele comprises HLA-A*03.

[0396] In some embodiments, the ligand binding domain of the second, inhibitory receptor comprises an scFv. In some embodiments, the scFv binds to HLA-A*03, and comprises a sequence selected from the group of sequences as set forth in Table 5A or Table 5B, 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.

[0397] Exemplary heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR-L1, CDR-L2 and CDR-L3, respectively) for HLA-A* 03 ligand binding domains are shown in Table 1 A, IB, 2A, and 2B.

[0398] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-A*03. In some embodiments, the extracellular ligand binding domain of the second receptor comprises HLA-A*03 complementarity determining regions (CDRs) CDR-L1, CDR- L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed Table 1A-1B and Table 2A-2B; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the HLA-A*03 CDRs of Table 1A-1B and Table 2A-2B.

[0399] In further embodiments of any of the ligand binding domains, each CDR sequence may have 1, 2, 3 or more substitutions, insertions, or deletions. CDR sequences may tolerate substitutions, deletions, or insertions. Using sequence alignment tools, routine experimentation, and known assays, those of skill in the art may generate and test variant sequences having 1, 2, 3, or more substitutions, insertions, or deletions in CDR sequences without undue experimentation.

[0400] In some embodiments, the non-target antigen comprises HLA-A*03, and the extracellular ligand binding domain of the second receptor comprises an HLA-A*03 ligand binding domain. In some embodiments, the HLA-A*03 ligand binding domain comprises an scFv domain comprising a sequence selected from the group of sequences as set forth in Table 5 A or Table 5B, or a sequence at least 90%, at least 95% or at least 99% identical to thereto. In some embodiments, the HLA-A*03 scFv comprises HLA-A*03 CDR sequences as set forth in Table 5A or Table 5B.

[0401] In some embodiments, the non-target antigen comprises HLA-A*03, and the ligand binding domain of the second receptor comprises an HLA-A*03 ligand binding domain. In some embodiments, the ligand binding domain binds HLA-A*03 independent of the peptide in a pMHC complexcomprising HLA-A*03. In some embodiments, the HLA-A*03 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*03 ligand binding domain comprises a sequence of any one of SEQ ID NOs: 83-94. In some embodiments, the HLA-A*03 ligand binding domain comprises a sequence at least 90%, at least 95%, at least 97% or at least 99% identical to a sequence of any one of SEQ ID NOs:83-94.

[0402] In some embodiments, the HLA-A*03 antigen binding domain comprises a heavy chain and a light chain. Exemplary variable heavy and light chain sequences are provided in Table 3 A-3B and 4A- 4B, respectively.

[0403] In some embodiments, the antigen binding domain comprises a variable heavy chain region (VH) comprising a sequence as set forth in Table 3A or Table 3B. In some embodiments, the antigen binding domain comprises a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 3A or Table 3B.

[0404] In some embodiments, the antigen binding domain comprises a variable heavy chain region (VH) comprising a sequence of SEQ ID NOs: 51-57. In some embodiments, the antigen binding domain comprises a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in SEQ ID NOs: 51-57.

[0405] In some embodiments, the antigen binding domain comprises a variable light chain region (VL) comprising a sequence as set forth in Table 4A or Table 4B. In some embodiments, the antigen binding domain comprises a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 4A or Table 4B.

[0406] In some embodiments, the antigen binding domain comprises a variable light chain region (VL) comprising a sequence of SEQ ID NOs: 72-78. In some embodiments, the antigen binding domain comprises a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in SEQ ID NOs: 72-78.

[0407] In some embodiments, the antigen binding domain comprises (a) a VH comprising a sequence as set forth in Table 3A or Table 3B and (b) a VL comprising a sequence as set forth in Table 4A or Table 4B.

[0408] In some embodiments, the antigen binding domain comprises (a) a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 3 A or Table 3B, and (b) a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 4A or Table 4B.

[0409] In some embodiments, the antigen binding domain comprises (a) a VH comprising a sequence of SEQ ID NOs: 51-57 and (b) a VL comprising a sequence of SEQ ID NOs: 72-78.

[0410] In each case, the VH may be paired with any of the VLs, as the heavy chains and light chains share similarity, with routine testing to confirm desired expression and binding activity. However, thepreferred pairing between Table 3 and Table 4 in indicated in the “Name” columns of the tables, i.e., in preferred embodiments, the VH designated “A3-14” in Table 4 is paired with the VL designated “A3-14” of Table 5, the VH designated “A3-13” in Table 4 is paired with the VL designated “A3-13” in Table 5, etc.

[0411] In some embodiments, the VH and VL are separated by a linker, for example GGGGSGGGGSGGGGSGG (SEQ ID NO: 135). In some embodiments, the VH and VL are ordered, from N to C terminal, VH, linker and VL. In some embodiments, the VH and VL are ordered, from N to C terminal, VL, linker and VH.

[0412] In some embodiments, the HLA-A*03 extracellular ligand binding domain comprises the complementarity determined regions (CDRs) of any one of SEQ ID NOS: 2-4 or 33-38. In some embodiments, the extracellular ligand binding domain comprises a sequence at least 95% identical to any one of SEQ ID NOs: 83-94. In some embodiments, the extracellular ligand binding domain comprises a sequence identical to any one of SEQ ID NOs: 83-94. In some embodiments, the heavy chain of the antigen binding domain comprises the heavy chain CDRs of any one of SEQ ID NOs: 51- 56, and wherein the light chain of the antigen binding domain comprises the light chain CDRs of any one of SEQ ID NOs: 72-78. In some embodiments, the HLA-A*03 antigen binding domain comprises a heavy chain and a light chain, and the heavy chain comprises CDRs selected from SEQ ID NOs: 33- 37 and the light chain comprises CDRs selected from SEQ ID NOs: 33-37. 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: 33- 37 and SEQ ID NOs: 2-4.

[0413] In some embodiments, the HLA-A*03 antigen binding domain comprises a heavy chain and a light chain, and the heavy chain comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOS: 51-56, and the light chain comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOS: 72-78.

[0414] In some embodiments, the heavy chain comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOS:51-56, and wherein the light chain of comprises a sequence identical to the light chain portion of any one of SEQ ID NOS: 72-78.Differentially Expressed Inhibitor Ligands

[0415] The disclosure provides inhibitor ligands (non-target antigens, e.g. HLA-A*03) that are differentially expressed between cancer cells and normal cells.

[0416] Activation of the inhibitory receptor is mediated by the presence of HLA-A*03 on the surface of a cell. A cell that expresses HLA-A*03 will activate the inhibitory receptor based on the level of expression of the HLA-A*03. In some embodiments, the HLA-A*03 is expressed by both target and non-target cells. However, in these embodiments, the HLA-A*03 is expressed by non-target cells at ahigher level than the target cells. The higher levels of HLA-A*03 expressed by the non-target cells activate the inhibitory receptor, thereby preventing activation of the immune cell. In contrast, the lower levels of HLA-A*03 expressed by the target are not sufficient to activate the inhibitory receptor, leading to activation of the immune cell.

[0417] In alternative embodiments, the HLA-A*03 is expressed by non-target cells but not by target cells. In the absence of expression of the HLA-A*03, the target cells activate the target receptor, thereby activating the immune cells.

[0418] Differential expression can be determined by any techniques known in the art used to measure expression. These include, inter alia, techniques for measuring mRNA and / or protein levels of a target gene in a cell. Methods of measuring protein levels in samples include immunohistochemistry, enzyme-linked immunosorbent assays (ELISA), and analytical methods such as liquid chromatography-mass spectrometry (LC-MS). Methods of measuring mRNA levels include real time quantitative reverse transcription PCR (qRT-PCR), as well as high throughput sequencing. Expression differences can be observed between, for example, a normal cell and a diseased cell, for example a cancer cell.

[0419] Activation of the inhibitory receptor by HLA-A*03 can occur according to various modalities known in the art. Activation of the inhibitory receptor by HLA-A*03 can be determined by methods known in the art. For example, the level of downstream intracellular signaling in a cell expressing the inhibitory receptor can be measured through the use of a reporter gene.

[0420] Without wishing to be bound by theory, whether or not expression of HLA-A*03 inhibits activation of an immune cell via activation of the inhibitory receptor can occur according to the ratio of the HLA-A*03 to the inhibitor receptor. The expression levels of the HLA-A*03 and the inhibitory receptor, and the ratio thereof, 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 the HLA-A*03 on target and non-target cells can be used to predict selective targeting of the immune cells expressing the inhibitory receptor. Low or no expression of the HLA-A*03 on a target or non-target cell can indicate, for example, that the inhibitory receptor will not be activated in an immune cell of the disclosure.

[0421] Alternatively, or in addition, and without wishing to be bound by theory, inhibition of immune cell activation by HLA-A*03 via activation of the inhibitory receptor can depend on the affinity of the HL A- A* 03 for the inhibitory receptor. Methods of measuring affinity are known in the art, and include, inter alia, enzyme-linked immunosorbent assay or radioimmunoassay methods.

[0422] Alternatively, or in addition, and without wishing to be bound by theory, inhibition of immune cell activation by HLA-A*03 via activation of the inhibitory receptor can occur according to cross talk between the inhibitory receptor and the activator receptor, leading to down-regulation of the activityof the activator receptor. For example, activation of the inhibitory receptor by HLA-A*03 can lead to reduced expression of the activator receptor on the surface of the immune cell.

[0423] In some embodiments, HLA-A*03 is expressed at a lower level in a target cell than a normal cell. In some embodiments, the HLA-A*03 is expressed by healthy cells, i.e. cells that are not cancer cells. In some embodiments, tHLA-A*03 expression level is at least about 10 times less, at least about 30 times less, at least about 50 times less, at least about 70 times less, at least about 90 times less, at least about 100 times less, at least about 110 times less, at least about 150 times less, at least about 200 times less, at least about 250 times less, at least about 300 times less, at least about 350 times less, at least about 400 times less, at least about 450 times less, at least about 500 times less, at least about 600 times less, at least about 700 times less, at least about 800 times less, at least about 900 times less or at least about 1000 times less in the target cell than in the non-target cell. In some embodiments, the HLA-A*03 expression level is about 10 times less, about 30 times less, about 50 times less, about 70 times less, about 90 times less, about 100 times less, or about 110 times less than the plurality of healthy cells. In some embodiments, the HLA-A*03 expression level is at least about 5 times less in the plurality of cancer cells than in the plurality of healthy cells. In some embodiments, the HLA-A*03 expression level is at least about 5 times less in a target cell than a non-target cell. In some embodiments, the target cells are a plurality of cancer cells that have low or no expression of HLA- A*03.Inhibitory Chimeric Antigen Receptors

[0424] The 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 HLA-A*03 or a peptide derivative thereof in a MHC-I complex.

[0425] The term “inhibitory receptor” as used herein refers to a ligand-binding domain that is fused to an intracellular signaling domain capable of transducing an inhibitory signal that inhibits or suppresses the immune activity of an immune cell. Inhibitory receptors have immune cell inhibitory potential, and are distinct and distinguishable from CARs, which are receptors with immune cell activating potential. For example, CARs are activating receptors as they include intracellular stimulatory and / or costimulatory domains. Inhibitory receptors are inhibiting receptors that contain intracellular inhibitory domains.

[0426] As used herein “inhibitory signal” refers to signal transduction or changes in protein expression in an immune cell resulting in suppression of an immune response (e.g., decrease in cytokine production or reduction of immune cell activation). Inhibition or suppression of an immune cell can selective and / or reversible, or not selective and / or reversible.

[0427] Inhibitory receptors of the disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure. Inhibitory receptors are responsive to non-target antigens (e.g. HLA-A*03). For example, when a non-target antigen (e.g. HLA-A*03) binds to or contacts the inhibitory receptor, the inhibitory receptor is responsive and activates an inhibitory signal in the immune cell expressing the inhibitory receptor upon binding of the non-target antigen by the extracellular ligand binding domain of the inhibitory receptor.

[0428] Inhibitory receptors of the disclosure may comprise an extracellular ligand binding domain. Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure.

[0429] In some embodiments, the ligand binding domain is an antigen binding domain. Exemplary antigen binding domains include, inter alia, scFv, SdAb, VP-only domains, and TCR antigen binding domains derived from the TCR a and P chain variable domains.

[0430] Any type of antigen binding domain is envisaged as within the scope of the instant disclosure.

[0431] In some embodiments, the extracellular ligand binding domain of the second receptor is an scFv.

[0432] In some embodiments, the extracellular ligand binding domain of the second receptor binds to and recognizes HLA-A*03. In some embodiments, the extracellular ligand binding domain of the second receptor is an scFv.

[0433] In some embodiments, the extracellular ligand binding domain of the second receptor is fused to the extracellular domain of an inhibitory receptor.

[0434] In some embodiments, the inhibitory receptors of the present disclosure comprise an extracellular hinge region. Exemplary hinges can be isolated or derived from IgD and CD8 domains, for example IgGl. In some embodiments, the hinge is isolated or derived from CD8a or CD28.

[0435] The inhibitory receptors of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the inhibitory receptor. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0436] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions may be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) 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, CD 154, or from an immunoglobulin such as IgG4. Alternatively the transmembrane domain may besynthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular domain of the inhibitory receptor. A glycine-serine doublet provides a particularly suitable linker.

[0437] The disclosure provides an inhibitory receptor comprising an intracellular domain. The intracellular domain of the inhibitory receptors of the instant disclosure is responsible for inhibiting activation of the immune cells comprising the inhibitory receptor, which would otherwise be activated in response to activation signals by the first receptor. In some embodiments, the inhibitory intracellular domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immune inhibitory receptors expressed on the surface of T cells, and play a pivotal role in attenuating or terminating T cell responses.

[0438] In some embodiments, an inhibitory intracellular domain is isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1. In some embodiments, the TRAIL receptor comprises TR10A, TRI OB or TR10D.

[0439] In some embodiments, an inhibitory intracellular domain is isolated from phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1). In some embodiments, an inhibitory intracellular domain is isolated from leukocyte immunoglobulin like receptor Bl (LILRB1).

[0440] In some embodiments, the inhibitory domain is isolated or derived from a human protein, for example a human TRAIL receptor, CTLA-4, PD-1, PAG1 or LILRB1 protein.

[0441] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region or a combination thereof.

[0442] In some embodiments, the inhibitory domain is isolated or derived from killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin like receptor Bl (LIR1, also called LIR-1 and LILRB1), programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin like receptor KI (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).

[0443] In some embodiments, the inhibitory domain is isolated or derived from a human protein.

[0444] 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 an intracellular domain of an inhibitory receptor. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of an inhibitory receptor.

[0445] In some embodiments, the second, inhibitory receptor comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain or a portion thereof isolated or derived isolated or derived from the same protein, for example an ITEM containing protein. In some embodiments, the second, inhibitory receptor comprises a hinge region isolated or derived from isolated or derived from the same protein as the intracellular domain and / or transmembrane domain, for example an ITEM containing protein.

[0446] In some embodiments, the second receptor is a TCR comprising an inhibitory domain (an inhibitory TCR). In some embodiments, the inhibitory TCR comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon or a portion thereof a TCR. 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.

[0447] In some embodiments, the second receptor is a TCR comprising an inhibitory domain (an inhibitory TCR). In some embodiments, the inhibitory domain is isolated or derived from LILRB1. LILRB 1 Inhibitory receptors

[0448] The disclosure provides a second, inhibitory receptor comprising a LILRB 1 inhibitory domain, and optionally, a LILRB 1 transmembrane and / or hinge domain, or functional variants thereof. The inclusion of the LILRB 1 transmembrane domain and / or the LILRB 1 hinge domain in the inhibitory receptor may increase the inhibitory signal generated by the inhibitory receptor compared to a reference inhibitory receptor having another transmembrane domain or another hinge domains. The second, inhibitory receptor comprising the LILRB 1 inhibitory domain may be a CAR or TCR, as described herein. Any suitable ligand binding domain, as described herein, may be fused to the LILRB 1-based second, inhibitory receptors.

[0449] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB 1), also known as Leukocyte immunoglobulin-like receptor Bl, as well as ILT2, LERI, MIR7, PIRB, CD85J, ILT-2 LIR- 1, MIR-7 and PIR-B, is a member of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB 1 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 LILRB 1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces anegative signal that inhibits stimulation of an immune response. LILRB1 is thought to regulate inflammatory responses, as well as cytotoxicity, and to play a role in limiting auto-reactivity. Multiple transcript variants encoding different isoforms of LILRB1 exist, all of which are contemplated as within the scope of the instant disclosure.

[0450] In some embodiments of the inhibitory receptors described herein, the inhibitory receptor comprises one or more domains isolated or derived from LILRB1. In some embodiments of the receptors having one or more domains isolated or derived from LILRB1, the one or more domains of LILRB 1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO:645. In some embodiments, the one or more domains of LILRB 1 comprise an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 645. In some embodiments, the one or more domains of LILRB 1 consist of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 645. In some embodiments, the one or more domains of LILRB 1 consist of an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 645.

[0451] In some embodiments of the receptors having one or more domains isolated or derived from LILRB 1, the one or more domains of LILRB 1 are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 646.

[0452] In some embodiments of the receptors having one or more domains of LILRB 1, the one or more domains of LILRB 1 are encoded by a polynucleotide sequence that is identical to a sequence or subsequence of SEQ ID NO: 646.

[0453] In various embodiments, an inhibitory receptor is provided, comprising a polypeptide, wherein the polypeptide comprises one or more of: an LILRB 1 hinge domain or functional variant thereof; an LILRB 1 transmembrane domain or a functional variant thereof; and an LILRB 1 intracellular domain or an intracellular domain comprising at least one, or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0454] As used herein an “immunoreceptor tyrosine-based inhibitory motif’ or “ITIM” refers to a conserved sequence of amino acids with a consensus sequence of S / I / V / LxYxxI / V / L (SEQ ID NO: 647), or the like, that is found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-possessing inhibitory receptors interact with their ligand, the ITIM motif is phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or the inositol-phosphatase called SHIP.

[0455] 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 3 ITIMs, at least 4 ITIMs, at least 5 ITIMs or at least 6 ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0456] 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: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0457] In further particular embodiments, the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0458] In some embodiments, the intracellular domain comprises both ITIMs NLYAAV (SEQ ID NO:626) and VTYAEV (SEQ ID NO: 627). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 630. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 630.

[0459] In some embodiments, the intracellular domain comprises both ITIMs VTYAEV (SEQ ID NO:627) and VTYAQL (SEQ ID NO: 628). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 631. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 631.

[0460] In some embodiments, the intracellular domain comprises both ITIMs VTYAQL (SEQ ID NO:628) and SIYATL (SEQ ID NO: 629). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 632. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 632.

[0461] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO:626), VTYAEV (SEQ ID NO: 627), and VTYAQL (SEQ ID NO: 628). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 633. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 633.

[0462] In some embodiments, the intracellular domain comprises the ITIMs VTYAEV (SEQ ID NO:627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 634. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 634.

[0463] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 635. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 635.

[0464] In some embodiments, the intracellular domain comprises a sequence at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 636). In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to the LILRB1 intracellular domain (SEQ ID NO: 636).

[0465] LILRB1 intracellular domains or functional variants thereof of the 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.

[0466] In particular embodiments, the intracellular domain comprises two, three, four, five, or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0467] In particular embodiments, the intracellular domain comprises at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0468] In particular embodiments, the intracellular domain comprises three immunoreceptor tyrosinebased inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0469] In particular embodiments, the intracellular domain comprises four immunoreceptor tyrosinebased inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0470] In particular embodiments, the intracellular domain comprises five immunoreceptor tyrosinebased inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0471] In particular embodiments, the intracellular domain comprises six immunoreceptor tyrosinebased inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0472] In particular embodiments, the intracellular domain comprises at least seven immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0473] The LILRB1 protein has four immunoglobulin (Ig) like domains termed DI, D2, D3 and D4. In some embodiments, the LILRB1 hinge domain comprises an LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1 D3D4 domain comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to SEQ ID NO: 637. In some embodiments, the LILRB1 D3D4 domain comprises or consists essentially of SEQ ID NO: 637.

[0474] In some embodiments, the polypeptide comprises the LILRB1 hinge domain or functional variant thereof. In embodiments, the LILRB1 hinge domain or functional variant thereof comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or identical to SEQ ID NO: 639, SEQ ID NO: 637, or SEQ ID NO: 638. In embodiments, the LILRB1 hinge domain or functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 639, SEQ ID NO: 637, or SEQ ID NO: 638.

[0475] In some embodiments, the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO: 639, SEQ ID NO: 637, or SEQ ID NO: 638.

[0476] In some embodiments, the LILRB1 hinge domain consists essentially of a sequence identical to SEQ ID NO: 639, SEQ ID NO: 637, or SEQ ID NO:638.

[0477] In some embodiments, the transmembrane domain is a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB 1 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% to SEQ ID NO: 640. In some embodiments, the LILRB 1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 640. In some embodiments, the LILRB 1 transmembrane domain comprises a sequence identical to SEQ ID NO:640. In embodiments, the LILRB 1 transmembrane domain consists essentially of a sequence identical to SEQ ID NO: 640.

[0478] In some embodiments, the transmembrane domain can be attached to the extracellular region of the second, inhibitory receptor, e.g., the antigen binding domain or ligand binding domain, via a hinge, e.g., a hinge 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 an LILRB 1 hinge.

[0479] In some embodiments, the second, inhibitory receptor comprises an inhibitory domain. In some embodiments, the second, inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory domain is isolated or derived from LILR1B.Inhibitory Receptors Comprising Combinations of LILRB1 Domains

[0480] In some embodiments, the LILRB 1-based inhibitory receptors of the disclosure comprise more than one LILRB 1 domain or functional equivalent thereof. For example, in some embodiments, the inhibitory receptor comprises an LILRB 1 transmembrane domain and intracellular domain, or an LILRB 1 hinge domain, transmembrane domain and intracellular domain.

[0481] In particular embodiments, the inhibitory receptor comprises an LILRB 1 hinge domain or functional variant thereof, and the LILRB 1 transmembrane domain or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence 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: 641. In some embodiments, the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 641. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 641.

[0482] In further embodiments, the inhibitory receptor comprises: the LILRB 1 transmembrane domain or a functional variant thereof, and an LILRB 1 intracellular domain and / or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629). In some embodiments, the polypeptide comprises the LILRB 1 transmembrane domain or a functional variant thereof, and an LILRB 1 intracellular domain and / or an intracellular domain comprising at least two ITIM, wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 626), VTYAEV (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0483] In some embodiments, the inhibitory receptor comprises a LILRB 1 transmembrane domain and intracellular domain. In some embodiments, the polypeptide comprises a sequence 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: 642. In some embodiments, the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 642. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 642.

[0484] In preferred embodiments, the inhibitory receptor comprises: an LILRB 1 hinge domain or functional variant thereof; an LILRB 1 transmembrane domain or a functional variant thereof; and an LILRB 1 intracellular domain and / or an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from LYAAV (SEQ ID NO: 626), VTYAE (SEQ ID NO: 627), VTYAQL (SEQ ID NO: 628), and SIYATL (SEQ ID NO: 629).

[0485] In some embodiments, the inhibitory receptor comprises a sequence at least 95% identical to SEQ ID NO: 643 or SEQ ID NO: 643, or at least 99% identical to SEQ ID NO: 643 or SEQ ID NO: 644, or identical to SEQ ID NO: 643 or SEQ ID NO: 644.

[0486] In some embodiments, the polypeptide comprises a sequence at least 99% identical to SEQ ID NO: 641, or at least 99% identical to SEQ ID NO: 641, or identical to SEQ ID NO: 641.

[0487] In some embodiments, the polypeptide comprises a sequence at least 99% identical to SEQ ID NO: 642, or at least 99% identical to SEQ ID NO: 642, or identical to SEQ ID NO: 642.Table 21: Polypeptide sequences for illustrative LILRBl-based inhibitory receptors

[0488] Exemplary inhibitory receptors of the disclosure comprise the scFv specific to any of HLA- A*03, the sequences of which are as set forth in Table 5, fused to the N terminus a LILRB1 hinge, transmembrane and intracellular domain. In some embodiments, the LILRB1 hinge comprises a sequence of SEQ ID NO: 639, the LILRB1 transmembrane domain comprises a sequence of SEQ ID NO: 640, and the LILRB1 intracellular domain comprises a sequence of SEQ ID NO: 636. For example, the second, inhibitory receptor comprises an scFv sequence of Table 5 fused to the N terminus of SEQ ID NO: 643.

[0489] In some embodiments, the non-target antigen comprises HLA-A*03, and the second inhibitory receptor comprises a sequence of: EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWM NWVRQAPGKGLEWVGEIRLKSTNYATHYAESVKGRFTISRDDSKNTL YLQM NSLKTEDTAVYYCTTLITPDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSSVSASVGDRVTITCKASQ DVSTTVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPPTFGGGTKVEIKYGSQ SSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQ GKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVK HSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAI HGSGEGRGSLLTCGDVEENPGPMEFGLSWLFLVAILKGVQCQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIR QPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVVKGAFDIWGQGTMVTVSSGG GGSGGGGSGGGGSGGDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 1045), 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 a sequence of SEQ ID NO: 1045.Polynucleotides and Vectors

[0490] The disclosure provides polynucleotides encoding the sequence(s) of the first and second receptors of the disclosure. The disclosure provides immune cells comprising the polynucleotides and vectors described herein.

[0491] In some embodiments, the sequence of the first and / or second receptor is operably linked to a promoter. In some embodiments, the sequence encoding the first receptor is operably linked to a first promoter, and the sequence encoding the second receptor is operably linked to a second promoter.

[0492] The disclosure provides vectors comprising the polynucleotides described herein.

[0493] 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, for example a B2M shRNA.

[0494] In some embodiments, both receptors are encoded by a single vector. In some embodiments the vector comprises an shRNA, for example a B2M shRNA.

[0495] In some embodiments, the first and second receptors are encoded by a single vector. Methods of encoding multiple polypeptides using a single vector will be known to persons of ordinary skill in the art, and include, inter alia, encoding multiple polypeptides under control of different promoters, or, if a single promoter is used to control transcription of multiple polypeptides, use of sequences encoding internal ribosome entry sites (IRES) and / or self-cleaving peptides. Exemplary self-cleaving peptides include T2A, P2A, E2A and F2A self-cleaving peptides. In some embodiments, the T2A selfcleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 653). In some embodiments, the P2A self-cleaving peptide comprises a sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 654). In some embodiments, the E2A self-cleaving peptide comprises a sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 655). In some embodiments, the F2A self-cleaving peptide comprises a sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 656). In some embodiments, the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 657). Any of the foregoing can also include an N terminal GSG linker. For example, a T2A self-cleaving peptide can also comprise a sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 658), which can be encoded by a sequence ofGGATCCGGAGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTG GCCCC (SEQ ID NO: 659).

[0496] In some embodiments, the vector is an expression vector, i.e. for the expression of the first and / or second receptor in a suitable cell.

[0497] Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0498] The expression of natural or synthetic nucleic acids encoding receptors is typically achieved by operably linking a nucleic acid encoding the receptor or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0499] The polynucleotides encoding the receptors can be cloned into a number of types of vectors. For example, the polynucleotides 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.

[0500] Further, the expression vector may be provided to cells, such as immune cells, in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0501] A number of viral 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 in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0502] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 basepairs (bp) upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0503] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-la). However, other constitutive promoter sequences may 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, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, a U6 promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0504] In order to assess the expression of a receptor, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0505] Reporter genes are used for identifying potentially transfected or transduced cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0506] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0507] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising 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 the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0508] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0509] Chemical means for introducing a polynucleotide into a host cell 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).

[0510] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.Immune Cells

[0511] The disclosure provides immune cells comprising the receptors, vectors and polynucleotides described herein.

[0512] In some embodiments, the immune cells comprise: (a) first receptor, comprising a first extracellular ligand binding domain specific to a target antigen selected from: (i) a cancer cell-specific antigen, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I); or (ii) MSLN, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I); and (b) a second receptor, comprising a second extracellular ligand bindingspecific to HLA-A*03. 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.

[0513] As used herein, the term “immune cell” refers to a cell involved in the innate or adaptive (acquired) immune systems. Exemplary innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, Natural Killer (NK) cells, polymophonuclear leukocytes such as neutrophils eosinophils and basophils and mononuclear cells such as monocytes, macrophages and mast cells. Immune cells with roles in acquired immunity include lymphocytes such as T-cells and B-cells.

[0514] As used herein, a “T-cell” refers to a type of lymphocyte that originates from a bone marrow precursor that develops in the thymus gland. There are several distinct types of T-cells which develop upon migration to the thymus, which include, 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 the ordinarily skilled artisan based on their expression of markers. Methods of distinguishing between T-cell types will be readily apparent to the ordinarily skilled artisan.

[0515] In some embodiments, the first receptor and the second receptor together specifically activate the immune cell in the presence of the target cell.

[0516] In some embodiments, the immune cell is CD4+, CD8+, a gamma delta T cell, an invariant T cells, an iNK cell , a NK cell, a macrophages, or combinations thereof. In some embodiments, the immune cell is a gamma delta (y5) T cell. In some embodiments, the immune cell is an invariant T cell. In some embodiments, the immune cell is an invariant natural killer T cell (iNKT cell). In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is CD8-. In some embodiments, the immune cell is CD8+. In some embodiments, the immune cell is CD4+. In some embodiments, the immune cell is CD4-. In some embodiments, the immune cell is CD8- / CD4+. In some embodiments, the immune cell is a CD8+ CD4- T cell.

[0517] In some embodiments, the immune cell is non-natural. In some embodiments, the immune cell is isolated.

[0518] Methods transforming populations of immune cells, such as T cells, with the vectors of the instant disclosure will be readily apparent to the person of ordinary skill in the art. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi), according to manufacturer’s instructions. T cells can be cultured at a density of 1 x 10A6 cells / mL in X-Vivo 15 media 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). After 2 days, T cells can be transduced with viral vectors, such as lentiviral vectors using methods known in the art. In someembodiments, the viral vector is transduced at a multiplicity of infection (MOI) of 5. Cells can then be cultured in IL-2 or other cytokines such as combinations of IL-7 / 15 / 21 for an additional 5 days prior to enrichment. Methods of isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells, will be readily apparent to the person of ordinary skill in the art. Although this method outlines a potential approach it should be noted that these methodologies are rapidly evolving. For example excellent viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of growth to generate a >99% CD3+ highly transduced cell population.

[0519] Methods of activating and culturing populations of T cells comprising the TCRs, CARs, inhibitory receptors, receptors or vectors encoding same, will be readily apparent to the person of ordinary skill in the art.

[0520] Whether prior to or after genetic modification of T cells to express a TCR, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10040846; and U.S. Pat. Appl. Pub. No. 2006 / 0121005.

[0521] In some embodiments, T cells of the instant disclosure are expanded and activated in vitro. Generally, the T cells of the instant disclosure are expanded in vitro by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti- CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can 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(l-2):53-63, 1999).

[0522] In some embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In some embodiments, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be insolution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present disclosure.

[0523] In some embodiments, the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of 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 antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In some embodiments, the ratio of CD3 :CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present disclosure, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the disclosure, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1.

[0524] Ratios of particles to cells from 1 :500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. In some embodiments, a ratio of 1 : 1 cells to beads is used. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present disclosure. In particular, ratios will vary depending on particle size and on cell size and type.

[0525] In further embodiments of the present disclosure, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0526] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached to contact the T cells. In one embodiment the cells (for example, CD4+ T cells) and beads (for example, DYNABEADS CD3 / CD28 T paramagnetic beads at a ratio of 1 : 1) are combined in a buffer. Again, those of ordinary skill in the art can readily appreciateany cell concentration may be used. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. In some embodiments, cells that are cultured at a density of IxlO6cells / mL are used.

[0527] In some embodiments, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the beads and T cells are cultured together for 2-3 days. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl- cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F- 12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. 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).

[0528] The T cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0529] In some embodiments, the T cells comprising TCRs, CARs and inhibitory receptors of the disclosure are autologous. Prior to expansion and genetic modification, a source of T cells is obtained from a 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, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T cell lines available in the art, may be used. In certain embodiments of the present disclosure, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation.

[0530] In some embodiments, cells from the circulating blood of an individual 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, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0531] In some embodiments, immune cells such as T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. 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 time period sufficient for positive selection of the desired T cells.

[0532] Enrichment of an immune cell population, such as a T cell population, by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immune- adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD 11b, CD 16, HLA-DR, and CD8.

[0533] For isolation of a desired population of immune cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads.

[0534] In some embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10° C or at room temperature.

[0535] T cells for stimulation, or PBMCs from which immune cells such as T cells are isolated, can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspendedin a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80° C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.

[0536] The disclosure provides an immune cell expressing the activator and / or blocker receptors described herein, wherein the immune cell has reduced expression and / or function the major histocompatibility (MHC) class I complex.

[0537] In some embodiments, the immune cell is autologous. For example, the immune cells is isolated or derived from same subject who will receive the cell as part of a therapeutic regimen. It can be advantageous to modify autologous immune cells to have reduced expression and / or function of MHC class I with the blocker receptor is specific to an MHC class I antigen. Without wishing to be bound by theory, modification of autologous immune cells to have reduced expression and / or function of MHC class I reduces binding of the blocker receptor by MHC class I expressed by the immune cells, either in cis or in trans.

[0538] In some embodiments, the immune cell is allogeneic. Allogeneic immune cells can be derived from a donor other than the subject to which the immune cells will be administered. Allogeneic immune cells have been commonly referred to in cell therapy as “off-the-shelf’ or “universal” because of the possibility for allogeneic cells to be prepared and stored for use in subjects of a variety of genotypes.

[0539] Any suitable methods of reducing expression and / or function the MHC class I complex are envisaged as within the scope of the instant disclosure, and include, inter alia, expression of interfering RNAs that knock down one or more RNAs encoding MHC class I components, or modifications of genes encoding MHC class I components.

[0540] The major histocompatibility complex (MHC) is a locus on the vertebrate genome that encodes a set of polypeptides required for the adaptive immune system. Among these are MHC class I polypeptides that include HLA-A, HLA-B, and HLA-C and alleles thereof. 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 alleles thereof form heterodimers with P2 microglobulin (B2M) and present in complex 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 said polypeptide. In some embodiments, the immune cells of the disclosure are inactivated by aninhibitor ligand comprising an MHC class I polypeptide, e.g. HLA-A and alleles thereof. HLA-A alleles can be, for example HLA-A*03, and / or any gene that encodes protein identical or similar to HLA-A*03 protein. Thus, to prevent autocrine signaling / binding as described herein, it is desirable to eliminate or reduce expression of polypeptides encoded by HLA-A and alleles thereof in the immune cells.Immune Cells with Reduced MHC Class I Polypeptide Expression

[0541] 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. MHC- I complex. The MHC-I complex is required for presentation of antigens on the cell surface. The MHC -I complex is disrupted and non-functional when the B2M is deleted (Wang D et al. Stem Cells Transl Med. 4: 1234-1245 (2015)). Furthermore, the B2M gene can be disrupted with high efficiency using gene editing techniques known in the art (Ren et al. Clin. Cancer Res. 23 :2255-2266 (2017)). Reducing or eliminating B2M can reduce, or eliminate functional MHC I on the surface of the immune cell.

[0542] The disclosure provides gene editing systems for editing an endogenous target gene in an immune cell. The disclosure provides interfering RNAs specific to sequences of target genes. Gene editing systems such as CRISPR / Cas systems, TALENs and zinc fingers can be used to generate double strand breaks, which, through gene repair mechanisms such as homology directed repair or non-homologous end joining (NHEJ), can be used to introduce mutations. NHEJ after resection of the ends of the break, or improper end joining, can be used to introduce deletions. In some embodiments, the target gene comprises a gene encoding a subunit of the MHC-I complex.

[0543] Target gene sequences includ...

Claims

CLAIMSWhat is claimed is:

1. An immune cell comprising: a. a first receptor, comprising an extracellular ligand binding domain specific to a target antigen; and b. a second receptor, comprising a humanized extracellular ligand binding domain specific to HLA-A*03, wherein the first receptor is an activator receptor responsive to a target antigen; and wherein the second receptor is an inhibitory receptor responsive to HL A- A* 03.2 The immune cell of claim 1, wherein the HLA-A*03 is lost on a target cell through loss of heterozygosity.3 The immune cell of any one of claims 1-2, 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 Table 1A or Table IB and Table 2A or Table 2B; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 1 A or Table IB and Table 2A or Table 2B.4 The immune cell of any one of claims 1-2, wherein the humanized 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: 2-4 or 33-38 or a sequence disclosed in Table 1 A or Table 2A; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of SEQ ID NOS: 2-4 or 33-38 or a sequence disclosed in Table 1 A or Table 2 A.5 The immune cell of any one of claims 1-4, wherein the humanized 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: 2-4 or 33-38.6 The immune cell of any one of claims 1-5, wherein the humanized extracellular ligand binding domain of the second receptor comprises any one of SEQ ID NO: 83-94 or a sequence disclosed in Table 5A, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.7 The immune cell of any one of claims 1-5, wherein the humanized extracellular ligand binding domain of the second receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 51- 57 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto; wherein the extracellular ligand binding domain of the second receptor comprises a variable light (VL) portion comprising SEQ ID NO: 72-78 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto; and / or wherein the humanized extracellularligand binding domain of the second receptor comprises SEQ ID NO: 83-94, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

8. The immune cell of any one of claims 1-7, wherein the target antigen is a cancer cell-specific antigen.

9. The immune cell of claim 8, wherein the cancer cell-specific antigen is selected from the group consisting of EGFR, CEA, MSLN, and HER2.

10. The immune cell of claim 9, wherein the cancer cell-specific antigen is MSLN.

11. The immune cell of claim 9, wherein the cancer cell-specific antigen is EGFR.

12. The immune cell of claim 9, wherein the cancer cell-specific antigen is CEA.

13. The immune cell of claim 9, wherein the cancer cell-specific antigen is HER2.

14. The immune cell of any one of claims 1-13, wherein the first receptor is a chimeric antigen receptor (CAR).

15. The immune cell of any one of claims 1-9, 10, or 14, 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 Table 7; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 7.

16. The immune cell of any one of claims 1-9, 10, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a sequence as set forth in Table 8 and a variable light (VL) portion comprising a sequence as set forth in Table 9; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

17. The immune cell of any one of claims 1-9, 10, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 917- 978 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 979-982 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

18. The immune cell of any one of claims 1-9, 10, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 143-208 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

19. The immune cell of any one of claims 1-9, 10, or 14, wherein the extracellular ligand binding domain of the first receptor comprises an scFv sequence of SEQ ID NO: 164; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

20. The immune cell of any one of claims 1-9, 11, or 14, 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 Table 13; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 14.

21. The immune cell of any one of claims 1-9, 11, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a sequence as set forth in Table 12 and a variable light (VL) portion comprising a sequence as set forth in Table 12; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

22. The immune cell of any one of claims 1-9, 11, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 504- 510 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 511-517 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

23. The immune cell of any one of claims 1-9, 11, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 484-493 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

24. The immune cell of any one of claims 1-9, 12, or 14, 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 Table 12 or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 12.

25. The immune cell of any one of claims 1-9, 12, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion and a variable light (VL) portion comprising a sequence as set forth in Table 12; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

26. The immune cell of any one of claims 1-9, 12, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 468 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 470 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

27. The immune cell of any one of claims 1-9, 12, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 454-460 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

28. The immune cell of any one of claims 1-9, 13, or 14, 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 Table 16; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 16.

29. The immune cell of any one of claims 1-9, 13, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion and a variable light (VL) portion comprising a sequence as set forth in Table 16; or a sequence having at least 80%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

30. The immune cell of any one of claims 1-9, 13, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 578 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 579 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

31. The immune cell of any one of claims 1-9, 13, or 14, wherein the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 590-595 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

32. The immune cell of any one of claims 1-31 wherein the first receptor comprises a hinge domain, a transmembrane domain and an intracellular domain.

33. The immune cell of claim 32, wherein the hinge domain comprises a CD8a hinge domain.

34. The immune cell of claim 33, wherein the CD8a hinge domain comprises a sequence of SEQ ID NO: 602, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

35. The immune cell of any one of claims 14-34, wherein the transmembrane domain comprises a CD28 transmembrane domain.

36. The immune cell of claim 35, wherein the CD28 transmembrane domain comprises a sequence of SEQ ID NO: 606, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

37. The immune cell of any one of claims 32-36, wherein the intracellular domain comprises a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, and a CD3(^ activation domain.

38. The immune cell of claim 37, wherein the intracellular domain comprises a sequence of SEQ ID NO: 618, or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

39. The immune cell of any one of claims 1-38, wherein the first receptor comprises a sequence of SEQ ID NO: 1000, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

40. The immune cell of any one of claims 1-39, wherein the second receptor comprises a hinge domain, a transmembrane domain and an intracellular domain.

41. The immune cell of any one of claims 1-40, wherein the second receptor comprises an LILRB1 intracellular domain or a functional variant thereof.

42. The immune cell of claim 41, wherein the LILRB1 intracellular domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99%, or is identical to SEQ ID NO: 636.

43. The immune cell of any one of claims 1-42, wherein the second receptor comprises an LILRB1 transmembrane domain or a functional variant thereof.

44. The immune cell of claim 43, wherein the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 640.

45. The immune cell of any one of claims 1-44, wherein the second receptor comprises an LILRB1 hinge domain or functional variant thereof.

46. The immune cell of claim 45, wherein the LILRB1 hinge domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 639.

47. The immune cell of any one of claims 1-46, wherein the second receptor comprises an LILRB1 intracellular domain, an LILRB1 transmembrane domain, and / or an LILRB1 hinge domain, a functional variant of any of these, or combinations thereof.

48. The immune cell of claim 47, wherein the LILRB1 hinge domain, the LILRB1 intracellular domain and the LILRB1 transmembrane domain comprises SEQ ID NO: 643 or a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 643.

49. The immune cell of any one of claims 1-48, wherein the second receptor comprises a sequence of SEQ ID NO: 1045, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

50. The immune cell of any one of claims 1-49, wherein the target antigen is on a MSLN+ cancer cell selected from 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 kidney cancer cell, an uterine cancer cell, a gastric cancer cell, a pancreatic cancer cell, a lung cancer cell, a colorectal cancer cell or a cholangiocarcinoma cell.

51. The immune cell of claim 50, wherein 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.

52. The immune cell of any one of claims 1-51, wherein the target antigen is on a MSLN+ / HLA- A*03- cancer cell that does not express HLA-A*03.

53. The immune cell of claim 52, wherein the MSLN+ / HLA-A*03- cancer cell is derived from a MSLN+ / HLA-A*03+ cell by loss of heterozygosity at HLA-A leading to loss of HLA-A*03.

54. The immune cell of any one of claims 1-53, wherein the first receptor and the second receptor together specifically activate the immune cell in the presence of a MSLN+ / HLA-A*03- cancer cell having loss of heterozygosity.

55. The immune cell of any one of claims 1-54, wherein the first receptor and the second receptor together do not specifically activate the immune cell in the presence of an MSLN+ cell that has not lost HLA-A*03 by loss of heterozygosity.

56. The immune cell of any one of claims 1-55, wherein the immune cell is a T cell.

57. The immune cell of claim 56, wherein the T cell is a CD8+ CD4- T cell or a CD8- CD4+ T cell.

58. The immune cell of any one of claims 1-57, wherein expression and / or function of a MHC Class I gene has been reduced or eliminated.

59. The immune cell of claim 58, wherein the MHC Class I gene is beta-2 -microglobulin (B2M).

60. The immune cell of claim 59 further comprising a polynucleotide comprising an interfering RNA, the interfering RNA comprising a sequence complementary to a sequence of a B2M mRNA.

61. The immune cell of claim 60, wherein the interfering RNA comprises a sequence selected from the group of sequences as set forth in Table 23, or a sequence having at most 1, 2, 3, or 4 substitutions, insertions or deletions relative thereto.

62. The immune cell of claim 60 or 61, wherein the interfering RNA is capable of inducing RNAi- mediated degradation of the B2M mRNA.

63. The immune cell of claim 62, wherein the interfering RNA is a short hairpin RNA (shRNA).

64. The immune cell of claim 63, wherein the shRNA comprises: a. a first sequence, having from 5’ end to 3’ end a sequence complementary to a sequence of the B2M mRNA; and b. a second sequence, having from 5’ end to 3’ end a sequence complementary to the first sequence, wherein the first sequence and the second sequence form the shRNA.

65. The immune cell of claim 63 or 64 wherein the shRNA is encoded by a sequence comprising a sequence of GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 915) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 916), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

66. The immune cell of claim 59, comprising one or more modifications to a sequence encoding B2M, wherein the one or more modifications reduce the expression and / or eliminate the function of B2M.

67. The immune cell of claim 66, wherein the one or more modifications comprise one or more inactivating mutations of the endogenous gene encoding B2M.

68. The immune cell of claim 67, wherein the one or more inactivating mutations comprise a deletion, an insertion, a substitution, or a frameshift mutation.

69. The immune cell of any one of claims 67 or 68, wherein the one or more inactivating mutations are introduced with a nucleic acid guided endonuclease in a complex with at least one guide nucleic acid (gNA) that specifically targets a sequence of the endogenous gene encoding B2M.

70. The immune cell of claim 69, wherein the gNA comprises a sequence selected from the group of sequences as set forth in Table 23 or a sequence having at most 1, 2, 3, or 4 substitutions, insertions or deletions relative thereto.

71. The immune cell of claim 58, wherein the MHC Class I gene is HLA-A*03.

72. The immune cell of claim 71, further comprising a polynucleotide comprising an interfering RNA, comprising a sequence complementary to a sequence of an HLA-A*03 mRNA.

73. The immune cell of claim 72, wherein the interfering RNA is capable of inducing RNA interference (RNAi)-mediated degradation of the HLA-A*03 mRNA.

74. The immune cell of claim 73 wherein the interfering RNA is a short hairpin RNA (shRNA) comprising: a. a first sequence, having from 5’ end to 3’ end a sequence complementary to a sequence of the HL A- A* 03 mRNA; and b. a second sequence, having from 5’ end to 3’ end a sequence complementary to the first sequence, wherein the first sequence and the second sequence form the shRNA.

75. The immune cell of claim 71, comprising one or more modifications to a sequence of an endogenous gene encoding HLA-A*03, wherein the one or modifications reduce the expression and / or eliminate the function of HLA-A*03.

76. The immune cell of claim 75, wherein the one or more modifications comprise one or more inactivating mutations of the endogenous gene encoding HLA-A*03.

77. The immune cell of claim 75 or 76, wherein the one or more inactivating mutations are introduced with a nucleic acid guided endonuclease in a complex with at least one guide nucleic acid (gNA) that specifically targets a sequence of the endogenous gene encoding HLA-A*03.

78. The immune cell of any one of claims 1-77, wherein the first receptor comprises a sequence of SEQ ID NO: 164 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto, and the second receptor comprises a sequence of SEQ ID NO: 1259, or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

79. The immune cell of claim 78, comprising an shRNA encoded by a sequence comprising GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 915) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 916) or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

80. The immune cell of claim 78 or 79, wherein the first receptor and second receptor are encoded by a single polynucleotide, and wherein the sequences encoding the first and second receptors are separated by a sequence encoding a self-cleaving polypeptide.

81. The immune cell of claim 80, wherein the self-cleaving polypeptide comprises a T2A selfcleaving polypeptide comprising a sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 657).

82. The immune cell of any one of claims 1-81, wherein the immune cell is autologous.

83. The immune cell of any one of claims 1-81, wherein the immune cell is allogeneic.

84. A pharmaceutical composition, comprising a therapeutically effective amount of the immune cells of any one of claims 1-83.

85. The pharmaceutical composition of claim 84, further comprising a pharmaceutically acceptable carrier, diluent or excipient.

86. The pharmaceutical composition of claim 84 or 85, for use as a medicament in the treatment of an MSLN+ cancer.

87. A polynucleotide or polynucleotide system, comprising one or more polynucleotides comprising polynucleotide sequences encoding: a. a first receptor, comprising an extracellular ligand binding domain specific to a target antigen; and / or b. a second receptor, comprising a humanized extracellular ligand binding domain specific to HLA-A*03, wherein the first receptor is an activator receptor responsive to a target antigen; and wherein the second receptor is an inhibitory receptor responsive to HLA-A*03.

88. A polynucleotide or polynucleotide system, comprising one or more polynucleotides comprising polynucleotide sequences encoding the first receptor and the second receptor for use in generating the immune cells of any one of claims 1-83.

89. The polynucleotide or polynucleotide system of claim 87 or 88, further comprising a sequence encoding an shRNA specific to B2M.

90. The polynucleotide or polynucleotide system of claim 89, wherein the sequences encoding the first receptor, the second receptor, and the shRNA specific to B2M are encoded by the same polynucleotide.

91. The polynucleotide or polynucleotide system of claims 89 or 90, whereina. the sequence encoding the shRNA specific to B2M comprises GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 915) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 916) or a sequence having at least 80%, at least 90%, or at least 95% identity thereto; b. the sequence encoding the first receptor comprises a sequence encoding a polypeptide comprising a of SEQ ID NO: 1000, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto; and c. the sequence encoding the second receptor comprises a sequence encoding a polypeptide comprising a sequence of SEQ ID NO: 1045, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

92. A vector, comprising the one or more polynucleotides of any one of claims 87-91.

93. A method of killing a cancer cell having loss of heterozygosity at an HLA-A*03 locus, comprising administering to the subject an effective amount of the immune cell of any one of claims 1-83 or the pharmaceutical composition of any one of claims 84-86.

94. A method of treating cancer in a subject having a MSLN+ tumor having loss of heterozygosity at an HLA-A*03 locus, comprising administering to the subject an effective amount of the immune cell of any one of claims 1-83 or the pharmaceutical composition of any one of claims 84-86.

95. A method of treating a cancer in a subject comprising: a. determining an HLA-A genotype or expression of HLA-A*03 in normal cells and in a plurality of cancer cells of the subject; b. optionally, determining the expression of target antigen in a plurality of cancer cells of the subject; and c. administering to the subject an effective amount of the immune cell of any one of claims 1-84 or the pharmaceutical composition of any one of claims 84-86 if the normal cells express HLA-A*03 and the plurality of cancer cells do not express HLA- A*03, and the plurality of cancer cells express the target antigen.

96. The method of claim 94 or 95, wherein the subject is heterozygous for HLA-A*03 and comprises cancers cells that expresses a target antigen selected from the group consisting of MSLN (MSLN+), EGFR, CEA, and HER2 and that have lost HLA-A*03 expression.

97. The method of claim 94 or 95, wherein the subject is heterozygous for HLA-A*03 and has recurrent unresectable or metastatic solid tumors comprising cancer cells that express a target antigen selected from the group consisting of MSLN, EGFR, CEA, and HER2 and that have lost HLA-A*03 expression.

98. The method of any one of claims 94-97, wherein the target antigen is MSLN.

99. The method of any one of claims 94-97, wherein the target antigen is EGFR.

100. The method of any one of claims 94-97, wherein the target antigen is CEA.

101. The method of any one of claims 94-97, wherein the target antigen is HER2.

102. The method of any one of claims 94-101, wherein the cancer is selected from mesothelioma cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, uterine cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, or cholangiocarcinoma.

103. The method of any one of claims 102, wherein the cancer has relapsed in a subject, the cancer is refractory to one or more prior administered anti cancer therapies, and / or the cancer is metastatic.

104. The method of any one of claims 94-98, wherein the cancer cells comprise MSLN+ / HLA- A*03- cancer cells that do not express HLA-A*03.

105. The method of claim 104, wherein the MSLN+ / HLA-A*03- cancer cells are derived from a MSLN+ / HLA-A*03+ cell by loss of heterozygosity at HLA-A leading to loss of HLA-A*03.

106. The method of any one of claims 94-105, wherein the first receptor and the second receptor together specifically activate the immune cell in the presence of the MSLN+ / HLA-A*03- cancer cells.

107. The method of any one of claims 94-106, wherein the first receptor and the second receptor together do not specifically activate the immune cell in the presence of a MSLN+ cell that has not lost HLA-A*03.

108. The method of any one of claims 94-107, wherein administration of the immune cell of any one of claims 1-83 or the pharmaceutical composition of any one of claims 84-87 reduces the size of a tumor in the subject.

109. The method of claim 108, wherein the tumor is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

110. The method of claim 108, wherein the tumor is eliminated.

111. The method of claim 108 or claim 109, wherein administration of the immune cell or the pharmaceutical composition arrests the growth of a tumor in the subject.

112. The method of any one of claims 94-111, wherein administration of the immune cell or the pharmaceutical composition reduces the number of tumors in the subject.

113. The method of any one of claims 94-112, wherein administration of the immune cell or the pharmaceutical composition results in selective killing of a cancer cell but not a normal cell in the subject.

114. The method of claim 111, wherein at least about 60% of the cells killed are cancer cells, about 65% of the cells killed are cancer cells, about 70% of the cells killed are cancer cells, about 75% of the cells killed are cancer cells, about 80% of the cells killed are cancer cells, about 85% of the cells killed are cancer cells, about 90% of the cells killed are cancer cells, about 95% of the cells killed are cancer cells, or about 100% of the cells killed are cancer cells.

115. The method of claim 113, wherein administration of the immune cell or pharmaceutical composition results in the killing of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or all of the cancer cells of the subject.

116. The method of any one of claims 94-115, wherein administration of the immune cell or the pharmaceutical composition results in fewer side effects for the subject than administration of an otherwise equivalent immune cell comprising the first activator receptor but no second inhibitory receptor.

117. A method of making a plurality of immune cells, comprising: a. providing a plurality of immune cells, and b. transforming the plurality of immune cells with the polynucleotide system of any one of claim 87-91, or the vector of claim 92.

118. A kit comprising the immune cell of any one of claims 1-83 or the pharmaceutical composition of any one of claims 84-86.

119. The kit of claim 118, further comprising instructions for use.

120. A polypeptide, comprising an antigen binding domain that specifically binds to a major histocompatibility complex class I (MHC I) protein comprising a human leukocyte antigen a chain encoded by an HLA-A*03 allele (HLA-A*03).

121. The polypeptide of claim 120, wherein the antigen binding domain is a human antibody or an antigen-binding fragment thereof.

122. The polypeptide of claim 120, wherein the antigen binding domain comprises a single chain variable fragment (scFv), a single chain Fab (scFab), a single domain antibody (sdAb), a fragment antigen binding (Fab), a F(ab’)2, or a Fab’.

123. The polypeptide of claim 122, wherein the antigen binding domain is a scFv.

124. The polypeptide of claim 123, wherein the scFv comprises a variable heavy chain (VH)- linker-variable light chain (VL) or a VL-linker-VH orientation.

125. The polypeptide of any one of claims 120-124, wherein the antigen binding domain comprises (a) a heavy chain (HC) complementarity determining region 1 (CDR1) sequence selected from the HC CDR1 sequences as set forth in Table 1 A or Table IB, (b) a HC CDR2 sequence selected from the HC CDR2 sequences as set forth in Table 1 A or Table IB, and (c) a HC CDR3 sequence selected from the HC CDR3 sequences as set forth in Table 1 A or Table IB.

126. The polypeptide of any one of claims 120-125 wherein the antigen binding domain comprises a HC CDR1, a HC CDR2, and a HC CDR3 as set forth on one line in Table 1 A.

127. The polypeptide of any one of claims 120-126, wherein the antigen binding domain comprises (a) a light chain (LC) complementarity determining region 1 (CDR1) sequence selected from the LC CDR1 sequences as set forth in Table 2 A or Table 2B, (b) a LC CDR2 sequence selected from the LC CDR2 sequences as set forth in Table 2A or Table 2B, and (c) a LC CDR3 sequence selected from the LC CDR3 sequences as set forth in Table 2A or Table 2B.

128. The polypeptide of any one of claims 120-127, wherein the antigen binding domain comprises a LC CDR1, a LC CDR2, and a LC CDR3 as set forth on one line in Table 2A.

129. The polypeptide of any one of claims 120-128, wherein the antigen binding domain comprises a VH comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 3A or Table 3B.

130. The polypeptide of any one of claims 120-129, wherein the antigen binding domain comprises a variable heavy chain region (VH) comprising a sequence as set forth in Table 3A or Table 3B.

131. The polypeptide of any one of claims 120-130, wherein the antigen binding domain comprises a VL comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity a sequence as set forth in Table 4 A or Table 4B.

132. The polypeptide of any one of claims 120-131, wherein the antigen binding domain comprises a variable light chain region (VL) comprising a sequence as set forth in Table 4A or Table 4B.

133. The polypeptide of any one of claims 120-132, wherein the antigen binding domain comprises an scFv comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to a sequence as set forth in Table 5.

134. The polypeptide of any one of claims 120-133, wherein the antigen binding domain comprises, consists of, or essentially consists of, an amino acid sequences as set forth in Table 5.

135. The polypeptide of any one of claims 120-134, wherein the polypeptide comprises a monoclonal antibody.

136. The polypeptide of any one of claims 120-135, wherein the polypeptide comprises a multispecific antibody.

137. A receptor comprising the polypeptide of anyone of claims 120-136.

138. The receptor of claim 137, wherein the receptor provides an activating signal to a cell.

139. The receptor of claim 138, wherein the cell is an immune cell.

140. The receptor of claim 138 or 139, wherein the receptor is a chimeric antigen receptor (CAR) or a T Cell Receptor (TCR).

141. The receptor of claim 140, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and one or more intracellular domains, and wherein the extracellular antigen-binding domain comprises the polypeptide.

142. The receptor of claim 141, wherein the transmembrane domain comprises a transmembrane domain isolated or derived from CD8a molecule (CD8a), CD4 molecule (CD4), CD28 molecule (CD28), TNF receptor superfamily member 9 (CD137 or 4-1BB), CD80 molecule (CD80), CD86 molecule (CD86), cytotoxic T-lymphocyte associated protein 4 (CD 152), programmed cell death 1 (PD-1), CD247 molecule (CD3Q, or Fc fragment of IgE receptor Ig (FcRy).

143. The receptor of claim 141 or 142, wherein the one or more intracellular domains comprise an intracellular signaling domain isolated or derived from an immune effector cell protein.

144. The receptor of claim 143, wherein the intracellular signaling domain comprises an intracellular signaling domain isolated or derived from CD3(^.

145. The receptor of any one of claims 140-144, wherein the CAR comprises a co-stimulatory domain.

146. The receptor of claim 145, wherein the co-stimulatory domain comprises a co-stimulatory domain isolated or derived from CD27 molecule (CD27), CD28, CD137, TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), CD40 ligand (CD40L), CD3(^, integrin subunit beta 2 (LFA-1), inducible T cell costimulator (ICOS), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C), CD276 molecule (B7-H3), or hematopoietic cell signal transducer (DAP 10).

147. The receptor of any one of claims 141-146, wherein the one or more intracellular domains comprise intracellular domains isolated or derived from CD28, 4-1BB, or CD3(^.

148. The receptor of any one of claims 140-147, wherein the CAR comprises a hinge domain between the extracellular domain and the transmembrane domain.

149. The receptor of claim 148, wherein the hinge domain is isolated or derived from CD4, CD8a, IgGl, IgG2, or IgG4.

150. The receptor of any one of claims 140-149, wherein the CAR comprises a signal peptide.

151. The receptor of claim 150, wherein the signal peptide comprises a sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 123).

152. The receptor of any one of claims 148-151, wherein the hinge, transmembrane, and intracellular domains of the CAR comprise a sequence at least 90%, at least 95%, at least 99%, or 100% identical to TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 124).

153. The receptor of claim 140, wherein the TCR comprises an extracellular antigen-binding domain comprising the polypeptide.

154. The receptor of claim 153, wherein the extracellular antigen binding domain is fused to one or more of a TCRa, TCRP, CD3(^, CD35, CD3s, or CD3y subunits of the TCR.

155. The receptor of claim 137, wherein the receptor provides an inhibitory signal to a cell.

156. The receptor of claim 155, wherein the cell is an immune cell.

157. The receptor of claim 155 or 156, wherein the receptor is an inhibitory CAR or a TCR.

158. The receptor of any one of claims 155-157, comprising an extracellular domain comprising the antibody or antigen-binding fragment thereof and an inhibitory intracellular domain.

159. The receptor of claim 158, wherein the inhibitory intracellular domain is isolated or derived from leukocyte immunoglobulin like receptor Bl (LILRB1).

160. The receptor of any one of claims 155-159, comprising a transmembrane domain.

161. The receptor of claim 160, wherein the transmembrane domain is isolated or derived from TCRa, TCRb, CD8alpha, CD28, or LILRBl.

162. The receptor of claim 161, further comprising an extracellular hinge domain.

163. The receptor of claim 162, wherein the extracellular hinge domain comprises a hinge domain isolated or derived from CD8alpha, CD28, or LILRB1.

164. The receptor of any one of claims 162-163, wherein the extracellular hinge, transmembrane, and intracellular domains comprise a sequence at least 90%, at least 95%, at least 99%, or 100% identical toYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV VIGIL VAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSP AADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYA TLAIH (SEQ ID NO: 130).

165. A nucleic acid encoding the polypeptide of any one of claims 120-136.

166. A vector comprising the nucleic acid of claim 165.

167. A nucleic acid encoding the receptor of any one of claims 137-164.

168. A vector comprising the nucleic acid of claim 167.

169. A recombinant cell comprising the nucleic acid of claim 165 or the vector of claim 166.

170. A recombinant cell comprising the nucleic acid of claim 167 or the vector of claim 168.

171. A recombinant immune cell expressing the receptor of any one of claims 137-164.

172. The recombinant immune cell of claim 171, wherein the immune cell is a T cell, B cell, macrophage or an NK cell.

173. A pharmaceutical composition comprising the polypeptide of any one of claims 120-136 or the receptor of any one of claims 137-164, and a pharmaceutically acceptable carrier, diluent, or excipient.

174. A pharmaceutical composition comprising a plurality of the recombinant immune cells of claim 52 or 53, and a pharmaceutically acceptable carrier, diluent, or excipient.

175. A method for treating a cancer a subject in need thereof, the method comprising administering a therapeutically effective amount of the recombinant immune cell of claim 171 or 172, or the pharmaceutical composition of claim 54 to the subject, wherein cells of the cancer have lost expression of HLA-A*03 due to loss of heterozygosity.

176. The method of claim 175, further comprising: a. determining if the subject is heterozygous for an HLA-A*03 allele; b. isolating a plurality of cancer cells from the subject; c. detecting the presence or absence of HLA-A*03 on the cancer cells using the polypeptide of any one of claims 120-136 or using the antigen binding domain thereof; and d. administering the recombinant immune cell or the pharmaceutical composition when the plurality of cancer cells do not express HLA-A*03.

177. The method of claim 175 or 176, wherein the cancer comprises a liquid tumor or a solid tumor.

178. A method for determining whether cancer cells express HL A- A* 03, comprising: a. providing a plurality of cancer cells; and b. detecting the presence of absence of HL A- A* 03 on the cancer cells using the polypeptide of any one of claims 120-136 or using the antigen binding domain thereof.

179. The method of claim 178, wherein the detecting at step (b) comprises immunohistochemistry.

180. A method of making a recombinant immune cell, comprising: a. providing a plurality of immune cells; and b. transforming the plurality of immune cells with the nucleic acid of claim 167 or the vector of claim 168.

181. A method of making a polypeptide, comprising: a. contacting the nucleic acid of claim 165 or the vector of claim 166 with a cell; b. culturing the cell under conditions whereby the polypeptide is expressed by the cell; and c. purifying the polypeptide.

182. A kit comprising the polypeptide of any one of claims 120-136, the receptor of any one of claims 137-164, the nucleic acid of claim 165 or 167, the vector of claim 166 or 168, the recombinant cell of claim 169 or 170, the recombinant immune cell of claim 171 or 172, or the pharmaceutical composition of claim 173 or 174.

183. A polypeptide comprising a humanized antigen binding domain that specifically binds to a major histocompatibility complex class I (MHC I) protein comprising a human leukocyte antigen a chain encoded by an HLA-A*03 allele (HLA-A*03), wherein the complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 comprise a sequence of SEQ ID NOs: 2-4 and SEQ ID NOs: 33, 35, and 37, and / or wherein the variable heavy (VH) portion comprises SEQ ID NO: 51-56 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto and / or the variable light (VL) portion comprises SEQ ID NO: 72-78 or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

184. The polypeptide of claim 183, wherein the antigen binding domain comprises a single chain variable fragment (scFv), a single chain Fab (scFab), a single domain antibody (sdAb), a fragment antigen binding (Fab), a F(ab’)2, or a Fab’.

185. The polypeptide of claim 184, wherein the antigen binding domain is a scFv.

186. The polypeptide of claim 185, wherein the scFv comprises a variable heavy chain (VH)- linker-variable light chain (VL) or a VL-linker-VH orientation.

187. The polypeptide of any one of claims 183-186, wherein the antigen binding domain comprises an scFv comprising a sequence having at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to a sequence of SEQ ID NO: 83-92.

188. The polypeptide of any one of claims 183-187, wherein the antigen binding domain comprises, consists of, or essentially consists of, an amino acid sequences of SEQ ID NO: 83-92.

189. The polypeptide of any one of claims 183-188, wherein the polypeptide comprises a monoclonal antibody.

190. The polypeptide of any one of claims 183-189, wherein the polypeptide comprises a multispecific antibody.

191. A receptor comprising the polypeptide of any one of claims 183-190.

192. The receptor of claim 191, wherein the receptor provides an activating signal to a cell.

193. The receptor of claim 192, wherein the cell is an immune cell.

194. The receptor of claim 191 or 192, wherein the receptor is a chimeric antigen receptor (CAR) or a T Cell Receptor (TCR).

195. The receptor of claim 194, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and one or more intracellular domains, and wherein the extracellular antigen-binding domain comprises the polypeptide.

196. The receptor of claim 195, wherein the transmembrane domain comprises a transmembrane domain isolated or derived from CD8a molecule (CD8a), CD4 molecule (CD4), CD28 molecule (CD28), TNF receptor superfamily member 9 (CD137 or 4-1BB), CD80 molecule (CD80), CD86 molecule (CD86), cytotoxic T-lymphocyte associated protein 4 (CD 152), programmed cell death 1 (PD-1), CD247 molecule (CD3Q, or Fc fragment of IgE receptor Ig (FcRy).

197. The receptor of claim 195 or 196, wherein the one or more intracellular domains comprise an intracellular signaling domain isolated or derived from an immune effector cell protein.

198. The receptor of claim 197, wherein the intracellular signaling domain comprises an intracellular signaling domain isolated or derived from CD3(^.

199. The receptor of any one of claims 194-198, wherein the CAR comprises a co-stimulatory domain.

200. The receptor of claim 199, wherein the co-stimulatory domain comprises a co-stimulatory domain isolated or derived from CD27 molecule (CD27), CD28, CD137, TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), CD40 ligand (CD40L), CD3(^, integrin subunit beta 2 (LFA-1), inducible T cell costimulator (ICOS), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C), CD276 molecule (B7-H3), or hematopoietic cell signal transducer (DAP 10).

201. The receptor of any one of claims 195-200, wherein the one or more intracellular domains comprise intracellular domains isolated or derived from CD28, 4-1BB, or CD3(^.

202. The receptor of any one of claims 194-201, wherein the CAR comprises a hinge domain between the extracellular domain and the transmembrane domain.

203. The receptor of claim 202, wherein the hinge domain is isolated or derived from CD4, CD8a, IgGl, IgG2, or IgG4.

204. The receptor of any one of claims 194-203, wherein the CAR comprises a signal peptide.

205. The receptor of claim 204, wherein the signal peptide comprises a sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 123).

206. The receptor of any one of claims 202-205, wherein the hinge, transmembrane, and intracellular domains of the CAR comprise a sequence at least 90%, at least 95%, at least 99%, or 100% identical to TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 124).

207. The receptor of claim 194, wherein the TCR comprises an extracellular antigen-binding domain comprising the polypeptide.

208. The receptor of claim 207, wherein the antigen binding domain is fused to one or more of a TCRa, TCRP, CD3< CD35, CD3s or CD3y subunits of the TCR.

209. The receptor of claim 191, wherein the receptor provides an inhibitory signal to a cell.

210. The receptor of claim 209, wherein the cell is an immune cell.

211. The receptor of claim 209 or 210, wherein the receptor is an inhibitory CAR or a TCR.

212. The receptor of any one of claims 209-211, comprising an extracellular domain comprising the antibody or antigen-binding fragment thereof and an inhibitory intracellular domain.

213. The receptor of claim 212, wherein the inhibitory intracellular domain is isolated or derived from leukocyte immunoglobulin like receptor Bl (LILRB1).

214. The receptor of any one of claims 209-213, comprising a transmembrane domain.

215. The receptor of claim 214, wherein the transmembrane domain is isolated or derived from TCRa, TCRb, CD8alpha, CD28, or LILRBl.

216. The receptor of claim 215, further comprising an extracellular hinge domain.

217. The receptor of claim 216, wherein the extracellular hinge domain comprises a hinge domain isolated or derived from CD8alpha, CD28 or LILRB1.

218. The receptor of any one of claims 213-217, wherein the extracellular hinge, transmembrane, and intracellular domains comprise a sequence at least 90%, at least 95%, at least 99%, or 100% identical toYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV VIGIL VAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSP AADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSG EFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAH4 (SEQ ID NO: 130).

219. A nucleic acid encoding the polypeptide of any one of claims 183-190.

220. A vector comprising the nucleic acid of claim 219.

221. A nucleic acid encoding the receptor of any one of claims 191-218.

222. A vector comprising the nucleic acid of claim 221.

223. A recombinant cell comprising the nucleic acid of claim 219 or the vector of claim 220.

224. A recombinant cell comprising the nucleic acid of claim 221 or the vector of claim 222.

225. A recombinant immune cell expressing the receptor of any one of claims 191-218.

226. The recombinant immune cell of claim 225, wherein the immune cell is a T cell, B cell, macrophage or an NK cell.

227. A pharmaceutical composition comprising the polypeptide of any one of claims 183-190 or the receptor of any one of claims 191-218, and a pharmaceutically acceptable carrier, diluent, or excipient.

228. A pharmaceutical composition comprising a plurality of the recombinant immune cells of claim 223 or 224, and a pharmaceutically acceptable carrier, diluent, or excipient.

229. A method for treating a cancer a subject in need thereof, the method comprising administering a therapeutically effective amount of the recombinant cells of claim 223 or 224 or the pharmaceutical composition of claim 228 to the subject, wherein cells of the cancer have lost expression of HLA-A*03 due to loss of heterozygosity.

230. The method of claim 229, further comprising: a. determining if the subject is heterozygous for an HLA-A*03 allele; b. isolating a plurality of cancer cells from the subject; c. detecting the presence or absence of HLA-A*03 on the cancer cells using the polypeptide of any one of claims 183-190 or using the antigen binding domain thereof; and d. administering the recombinant cells or the pharmaceutical composition when the plurality of cancer cells do not express HLA-A*03.

231. The method of claim 229 or 230, wherein the cancer comprises a liquid tumor or a solid tumor.

232. A method for determining whether cancer cells express HL A- A* 03, comprising: a. providing a plurality of cancer cells; and b. detecting the presence of absence of HL A- A* 03 on the cancer cells using the polypeptide of any one of claims 183-190 or using the antigen binding domain thereof.

233. The method of claim 232, wherein the detecting at step (b) comprises immunohistochemistry.

234. A method of making a recombinant immune cell, comprising: a. providing a plurality of immune cells; and b. transforming the plurality of immune cells with the nucleic acid of claim 221 or the vector of claim 222.

235. A method of making a polypeptide, comprising: a. contacting the nucleic acid of claim 219 or the vector of claim 220 with a cell; b. culturing the cell under conditions whereby the polypeptide is expressed by the cell; and c. purifying the polypeptide.

236. A kit comprising the polypeptide of any one of claims 183-190, the receptor of any one of claims 191-218, the nucleic acid of claim 219 or 221, the vector of claim 220 or 222, the recombinant cell of claim 223 or 224, the recombinant immune cell of claim 225 or 226, or the pharmaceutical composition of claim 227 or 228.

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