Compositions and methods for treating CEACAM-positive cancers

Abstract

The present disclosure provides immune cells comprising a first activating receptor specific for CEA and a second inhibitory receptor, and methods of making and using the same for the treatment of cancer.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 068,244, filed August 20, 2020, the contents of which are incorporated herein by reference.

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

[0003] Incorporation by Reference of Sequence Listing The application for the Sequence Listing Item contains a Sequence Listing that has been submitted in ASCII format via EFS-WEB and is incorporated herein by reference in its entirety. The ASCII copy, created on August 17, 2021, is named A2BI_022_01WO_SeqList_ST25.txt and is 914KB in size. [Background technology]

[0004] Cell therapy is a powerful tool for the treatment of various diseases, especially cancer. In conventional adoptive cell therapy, immune cells are engineered to express specific receptors, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which induce the activation of immune cells against cellular targets through receptor interaction with ligands expressed by the target cells. Identifying suitable target molecules remains challenging because many targets are expressed in normal tissues. This expression can result in toxicity when transplanted cells target normal tissues that express the target molecule. Therefore, there is a need in the art for compositions and methods useful for treating diseases, especially cancer, by adoptive cell therapy. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides compositions and methods for increasing the specificity of immune cells used in adoptive cell therapy. The present disclosure provides immune cells comprising a two-receptor system that increases the specificity of the immune cells for target cells expressing a target antigen. The immune cells comprise a first activator receptor that activates the immune cell in response to binding of the first receptor by the target antigen. The immune cells further comprise a second inhibitory receptor specific for a non-target antigen. This second receptor inhibits activation of the immune cell when the second receptor is bound by the non-target antigen, even when the first receptor is bound by the target antigen.

[0006] The present disclosure provides an immune cell comprising: (a) a first receptor comprising an extracellular ligand-binding domain specific for CEA-cell adhesion molecule 5 (CEA); and (b) a second receptor comprising an extracellular ligand-binding domain specific for a non-target antigen lost in CEA+ cancer cells, wherein the first receptor is an activator receptor that responds to CEA, and the second receptor is an inhibitory receptor that responds to the non-target antigen.

[0007] In some embodiments of the immune cells of the present disclosure, the non-target antigen is lost in the CEA+ cancer cells by loss of heterozygosity.

[0008] In some embodiments of the immune cells of the present disclosure, the extracellular ligand-binding domain of the second receptor specifically binds to an allelic variant of a major histocompatibility complex (MHC) protein. In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to an allelic variant of an HLA-A, HLA-B, or HLA-C protein. In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07. In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A*02. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or CDR sequences with up to one, two, or three substitutions, deletions, or insertions compared to the CDRs of Table 6. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 103-108 or 109-114, or CDR sequences with up to one, two, or three substitutions, deletions, or insertions compared to the CDRs of SEQ ID NOs: 103-108 or 109-114. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises a polypeptide sequence selected from the polypeptide sequences disclosed in Table 5, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises any one of SEQ ID NOs: 91-102, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0009] In some embodiments of the immune cells of the present disclosure, the first receptor is a chimeric antigen receptor (CAR). In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising a set of heavy chain complementarity-determining regions (HC-CDRs) selected from the group consisting of SEQ ID NOs: 55-58, and a light chain variable (VL) portion comprising a set of light chain complementarity-determining regions selected from the group consisting of SEQ ID NOs: 59-63, or CDR sequences having up to one, two, or three substitutions, deletions, or insertions compared to SEQ ID NOs: 55-58 or SEQ ID NOs: 59-63. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising a set of heavy chain complementarity-determining regions (HC-CDRs) comprising SEQ ID NOs: 55-57, and a light chain variable (VL) portion comprising a set of light chain complementarity-determining regions comprising SEQ ID NOs: 59, 61, and 63, or CDR sequences having up to one, two, or three substitutions, deletions, or insertions compared to SEQ ID NOs: 55-57 or SEQ ID NOs: 59, 61, and 63. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising SEQ ID NO: 144, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a light chain variable (VL) portion comprising SEQ ID NO: 148, or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOs: 66-70, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises the scFv sequence of SEQ ID NO: 68, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0010] In some embodiments of the immune cells of the present disclosure, the first receptor is a chimeric antigen receptor (CAR). In some embodiments, the first receptor comprises a hinge domain, a transmembrane domain, and an intracellular domain. In some embodiments, the hinge domain comprises a CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises the sequence of SEQ ID NO: 71, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 75, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain. In some embodiments, the intracellular domain comprises the sequence of SEQ ID NO: 158, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0011] In some embodiments of the immune cells of the present disclosure, the first receptor comprises the sequence of SEQ ID NO: 52, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0012] In some embodiments of the immune cells of the present disclosure, the second receptor comprises a LILRB1 intracellular domain or a functional variant thereof. In some embodiments, the LILRB1 intracellular domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% identical to, or identical to, SEQ ID NO: 131. In some embodiments, the second receptor comprises a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% identical to, or identical to, SEQ ID NO: 135. In some embodiments, the second receptor comprises a LILRB1 hinge domain or a functional variant thereof. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% identical to, or identical to, SEQ ID NO: 134. In some embodiments, the second receptor comprises a LILRB1 intracellular domain, a LILRB1 transmembrane domain, a LILRB1 hinge domain, a functional variant of any of these, or a combination thereof. In some embodiments, the LILRB1 hinge domain, the LILRB1 intracellular domain, and the LILRB1 transmembrane domain comprise SEQ ID NO: 132, or a sequence at least 90%, at least 95%, at least 97%, at least 99% identical to, or identical to, SEQ ID NO: 132.

[0013] In some embodiments of the immune cells of the present disclosure, the second receptor comprises the 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.

[0014] In some embodiments of the immune cells of the present disclosure, the CEA+ cancer cells are pancreatic cancer cells, colorectal cancer cells, lung cancer cells, esophageal cancer cells, gastric cancer cells, head and neck cancer cells, gallbladder cancer cells, diffuse large B-cell cancer cells, or acute myeloid leukemia cancer cells. In some embodiments, the CEA+ cancer cells are lung cancer cells, colorectal cancer cells, or pancreatic cancer cells. In some embodiments, the CEA+ cancer cells are CEA+ / HLA-A*02- cancer cells that do not express HLA-A*02. In some embodiments, the CEA+ / HLA-A*02- cancer cells are derived from CEA+ / HLA-A*02+ cells by loss of heterozygosity at HLA-A, resulting in loss of HLA-A*02. In some embodiments, both the first receptor and the second receptor specifically activate the immune cells in the presence of CEA+ / HLA-A*02- cancer cells with loss of heterozygosity. In some embodiments, both the first receptor and the second receptor do not specifically activate immune cells in the presence of CEA+ cells that have not lost HLA-A*02 by loss of heterozygosity.

[0015] In some embodiments of the immune cells of the present disclosure, the immune cells are T cells. In some embodiments, the T cells are CD8+CD4- T cells.

[0016] In some embodiments of the immune cells of the present disclosure, the expression and / or function of an MHC class I gene is reduced or eliminated. In some embodiments, the MHC class I gene is beta-2-microglobulin (B2M). In some embodiments, the immune cell further comprises a polynucleotide comprising an interfering RNA, wherein the interfering RNA comprises a sequence complementary to a sequence of B2M mRNA. In some embodiments, the interfering RNA comprises a sequence selected from the group of sequences shown in Table 11, or a sequence having up to 1, 2, 3, or 4 substitutions, insertions, or deletions thereto. In some embodiments, the interfering RNA is capable of inducing RNAi-mediated degradation of B2M mRNA. In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises (a) a first sequence having a sequence complementary to a sequence of B2M mRNA from its 5' to 3' end, and (b) a second sequence having a sequence complementary to the first sequence from its 5' to 3' end, wherein the first sequence and the second sequence form the shRNA. In some embodiments, the shRNA is encoded by a sequence comprising the sequence GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 179) or GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 180), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

[0017] In some embodiments of the immune cells of the present disclosure, the expression and / or function of an MHC class I gene is reduced or eliminated. In some embodiments, the MHC class I gene is beta-2-microglobulin (B2M). In some embodiments, the immune cells further comprise one or more modifications to a sequence encoding B2M, wherein the one or more modifications reduce expression and / or eliminate function of B2M. In some embodiments, the one or more modifications comprise one or more inactivating mutations in an endogenous gene encoding B2M. In some embodiments, the one or more inactivating mutations comprise a deletion, insertion, substitution, or frameshift mutation. In some embodiments, the one or more inactivating mutations are introduced with a nucleic acid-guided endonuclease in complex with at least one guide nucleic acid (gNA) that specifically targets a sequence in the endogenous gene encoding B2M. In some embodiments, the gNA comprises a sequence selected from the group of sequence sets shown in Table 10, or a sequence having up to 1, 2, 3, or 4 substitutions, insertions, or deletions thereto.

[0018] In some embodiments of the immune cells of the present disclosure, the expression and / or function of an MHC class I gene is reduced or eliminated. In some embodiments, the MHC class I gene is HLA-A*02. In some embodiments, the immune cells further comprise a polynucleotide comprising an interfering RNA comprising a sequence complementary to a sequence of HLA-A*02 mRNA. In some embodiments, the interfering RNA is capable of inducing RNA interference (RNAi)-mediated degradation of HLA-A*02 mRNA. In some embodiments, the interfering RNA is a short hairpin RNA (shRNA) comprising: (a) a first sequence having a sequence complementary to a sequence of HLA-A*02 mRNA from its 5' to 3' end; and (b) a second sequence having a sequence complementary to the first sequence from its 5' to 3' end, wherein the first sequence and the second sequence form an shRNA. In some embodiments, the shRNA comprises a sequence selected from the group of sequences listed in Table 12.

[0019] In some embodiments of the immune cells of the present disclosure, the expression and / or function of an MHC class I gene is reduced or eliminated. In some embodiments, the MHC class I gene is HLA-A*02. In some embodiments, the immune cells comprise one or more modifications to the sequence of an endogenous gene encoding HLA-A*02, wherein the one or more modifications reduce expression and / or eliminate function of HLA-A*02. In some embodiments, the one or more modifications comprise one or more inactivating mutations in the endogenous gene encoding HLA-A*02. In some embodiments, the one or more inactivating mutations are introduced with a nucleic acid-guided endonuclease in complex with at least one guide nucleic acid (gNA) that specifically targets the sequence of the endogenous gene encoding HLA-A*02. In some embodiments, the gNA comprises a sequence set forth in Table 9.

[0020] In some embodiments of the immune cells of the present disclosure, the first receptor comprises the sequence of SEQ ID NO: 52, and the second receptor comprises the 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. In some embodiments, the immune cells comprise an shRNA encoded by a sequence comprising GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 179), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the first receptor and the second receptor are encoded by a single polynucleotide, and the sequences encoding the first and second receptors are separated by a sequence encoding a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 181).

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

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

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

[0024] The present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the immune cells of the present disclosure for use as a medicament in the treatment of CEA+ cancer.

[0025] The present disclosure provides 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 for CEA-cell adhesion molecule 5 positive (CEA) and (b) a second receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is lost in CEA+ cancer cells, wherein the first receptor is an activator receptor that responds to CEA on CEA+ cancer cells and the second receptor is an inhibitory receptor that responds to the non-target antigen.

[0026] In some embodiments of the polynucleotides or polynucleotide systems of the present disclosure, the polynucleotides or polynucleotide systems comprise one or more polynucleotides comprising polynucleotide sequences encoding a first receptor and a second receptor for use in generating the immune cells of the present disclosure.

[0027] In some embodiments of the polynucleotide or polynucleotide system of the present disclosure, the polynucleotide or polynucleotide system comprises a sequence encoding an shRNA specific for B2M. In some embodiments, the sequences encoding the first receptor, the second receptor, and the B2M-specific shRNA are encoded by the same polynucleotide. In some embodiments, (a) the sequence encoding the B2M-specific shRNA comprises GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 179), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto; (b) the sequence encoding the first receptor comprises SEQ ID NO: 143, 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 SEQ ID NO: 165, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

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

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

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

[0031] The present disclosure provides a method of treating cancer in a subject, the method comprising: (a) determining the HLA-A genotype or expression of normal cells and a plurality of cancer cells of the subject; (b) optionally determining the expression of CEA in a plurality of cancer cells of the subject; and (c) if the normal cells express HLA-A*02, a plurality of cancer cells do not express HLA-A*02, and a plurality of cancer cells are CEA positive, administering an effective amount of an immune cell or pharmaceutical composition of the present disclosure to the subject.

[0032] In some embodiments of the methods of the present disclosure, the subject is a heterozygous HLA-A*02 patient with a malignant tumor that expresses CEA (CEA+) and has lost HLA-A*02 expression. In some embodiments, the subject is a heterozygous HLA-A*02 patient with a recurrent unresectable or metastatic solid tumor that expresses CEA and has lost HLA-A*02 expression. In some embodiments, the cancer comprises pancreatic cancer, colorectal cancer, lung cancer, esophageal cancer, gastric cancer, head and neck cancer, gallbladder cancer, diffuse large B-cell carcinoma, or acute myeloid leukemia. In some embodiments, the cancer comprises lung cancer, colorectal cancer, or pancreatic cancer.

[0033] In some embodiments of the methods of the present disclosure, the cancer cells comprise CEA+ / HLA-A*02 cancer cells that do not express HLA-A*02. In some embodiments, the CEA+ / HLA-A*02- cancer cells are derived from CEA+ / HLA-A*02+ cells by loss of heterozygosity at HLA-A, resulting in loss of HLA-A*02. In some embodiments, the first receptor and the second receptor together specifically activate immune cells in the presence of CEA+ / HLA-A*02- cancer cells. In some embodiments, the first receptor and the second receptor together do not specifically activate immune cells in the presence of CEA+ cells that have not lost HLA-A*02.

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

[0035] In some embodiments of the disclosed methods, administration of the immune cells or pharmaceutical composition results in selective killing of cancer cells but not normal cells in a subject. In some embodiments, at least about 60% of the killed cells are cancer cells, at least about 65% of the killed cells are cancer cells, at least about 70% of the killed cells are cancer cells, at least about 75% of the killed cells are cancer cells, at least about 80% of the killed cells are cancer cells, at least about 85% of the killed cells are cancer cells, at least about 90% of the killed cells are cancer cells, at least about 95% of the killed cells are cancer cells, or about 100% of the killed cells are cancer cells. In some embodiments, administration of the immune cells or pharmaceutical composition results in killing of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or all of the subject's cancer cells.

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

[0037] The present disclosure provides a method of producing a plurality of immune cells, the method comprising: (a) providing a plurality of immune cells; and (b) transforming the plurality of immune cells with a polynucleotide, polynucleotide system, or vector of the present disclosure.

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

[0039] [Figure 1] Crystal structure of TNFRSF11A (RANK) bound to TNFRS11 (RANKL), showing that the variant TNFRSF11A epitope is on the protein surface and likely accessible to antibodies. [Figure 2-1] Figure 1 shows the alignment of human integrin alpha-E (ITGAE) (SEQ ID NO: 182) with human integrin alpha-X (ITGAX, P20702, ITAX_human) (SEQ ID NO: 183). SNP variants in ITGAE rs1716R950W (MAF 0.2654 from the 1000 Genomes project) and rs2976230 V1019A / V1019G (MAF 0.282 from the 1000 Genomes project) are shown in boxes. [Figure 2-2] Same as above. [Figure 3] Crystal structure of the inactive conformation of ITGAX, which has 27% identity to ITGAE. The location of the ITGAE SNP is shown as labeled. [Figure 4]This table shows that the addressable colorectal cancer (CRC) patient population that can be treated with a CEA TCR in combination with a RANK blocker receptor is estimated to be between 2,000 and 5,000 patients, depending on which RANK variant is used. In the table, the subtotal of treatable patients above is 5,000 to 11,000, including the percentage of high CEA+ patients, as indicated. Treated patients are calculated as follows: HLA-A*02 retention frequency (0.5) × random loss (0.5) × RANK variant het frequency (0.2–0.5) × cancer RANK LOH frequency = [0.05–0.125] × LOH frequency. [Figure 5] 1 shows the expression of CEA (CEACAM5) in normal tissues. [Figure 6] 1 shows the expression of TNFRSF11A (RANK) in normal tissues. [Figure 7] CEA expression across all TCGA cancers (tumor and normal samples used. Abbreviations: BLCA (bladder cancer), BRCA (breast cancer), CESC (cervical squamous cell carcinoma and adenocarcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal cancer), GBM (glioblastoma multiforme), HNSC (head and neck squamous cell carcinoma), KICH (kidney chromophobe), KIRP (kidney papillary renal cell carcinoma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PAAD (pancreatic adenocarcinoma), PRAD (prostate adenocarcinoma), PCPG (pheochromocytoma and paraganglioma), READ (rectal adenocarcinoma), SARC (sarcoma), SKCM (cutaneous melanoma), THCA (thyroid cancer), THYM (thymoma), STAD (gastric adenocarcinoma), UCEC (uterine endometrial carcinoma). [Figure 8] 1 shows the expression of TNFGSF11A across TCGA cancers (with tumor and normal samples). [Figure 9] 1 is a table showing estimated deaths in the United States by cancer site, statistics from the American Cancer Society. [Figure 10]1 is a series of plots showing that the HLA-A*02 inhibitory receptor can block activation of Jurkat cells by CEA CAR. [Figure 11] FIG. 1 illustrates the bioinformatics search process used to identify potential non-target antigen (blocker) candidate genes. [Figure 12]

[0023] Figure 1 shows a pair of diagrams illustrating the use of loss of heterozygosity (LOH) to distinguish between tumor and normal tissue. Engineered immune cells kill tumors while sparing normal cells. In an exemplary embodiment, the immune cells express a CEA CAR, the activator antigen is CEA, and the blocker antigen is HLA-A*02. Patients with germline heterozygosity for HLA-A*02 in the tumor and clonal LOH of HLA-A*02 are selected. [Figure 13] FIG. 1 shows the molecular composition of an exemplary dual receptor system of the present disclosure, comprising a CEA CAR and an HLA-A*02 scFv LILRB1 inhibitory receptor. [Figure 14] Expression of CEA and HLA-A*02 antigens in HeLa cells. A*02: HLA-A*02. [Figure 15] We demonstrate that CEA activator and HLA-A*02 LILRB1 inhibitory receptor function in Jurkat cells using engineered HeLa cells as targets for cytotoxicity. A*02: HLA-A*02; Tmod: cells express CEA CAR and HLA-A*02 inhibitory receptor; CAR: cells express only CEA CAR. [Figure 16] We demonstrate that the CEA activator and HLA-A*02 LILRB1 inhibitory receptor function in donor T cells from a single donor on HeLa cells. Tmod: cells express CEA CAR and HLA-A*02 inhibitory receptor; CAR: cells express only CEA CAR. [Figure 17]We demonstrate that the CEA activator and HLA-A*02 LILRB1 inhibitory receptor function in T cells from four donors on HeLa cells. Tmod: cells express the CEA CAR and HLA-A*02 inhibitory receptor; CAR: cells express only the CEA CAR. Target cells are HeLa cells expressing only CEA or CEA and HLA-A*02. [Figure 18] Cell surface expression of CEA and HLA-A*02 in HeLa cells by mRNA titration. A*02: HLA-A*02. [Figure 19] Figure 1 shows CEA CAR activator and HLA-A*02 LILRB1 blocker sensitivity measured as a function of the number of CEA surface molecules on HeLa cells using Jurkat effector cells with stably expressed CEA activator and HLA-A*02 blocker receptors. [Figure 20] The sensitivity of primary T cells expressing CEA CAR Tmod (CEA CAR and both HLA-A*02 and LILRB1 inhibitory receptors), CAR alone, and CEA TCR to activators and blockers is shown. Dose-response curves for activators (right) are shown for CEA CAR, CEA CAR with HLA-A*02 blocker (Tmod), and CEA TCR, while dose-response curves for inhibitory receptors (blockers) are only for CEA CAR and CEA CAR with HLA-A*02 blocker (Tmod). A*02: HLA-A*02. [Figure 21] This shows that the combination of CEA CAR and HLA-A*02 inhibitory receptor is predicted to kill tumors while sparing normal tissue. TPM: transcripts per million; A*02: HLA-A*02; LOH: loss of heterozygosity. [Figure 22]The standard curve used to convert molecules / cells to TPM values ​​is shown. Data for the CEA standard curve (left) shows CEA cell surface expression from Bacac et al. 2016, Clin Cancer Res 22, 3286-3297 plotted against mRNA (TPM) from the GTEx database. TPM: transcripts per million. [Figure 23] Surface expression of CEA and HLA-A*02 on H508 and SW1463 cell lines is shown. WT: wild type; KO: indicated gene knocked out. [Figure 24] Cytotoxicity data of CEA Tmod-expressing cells (cells expressing both CEA CAR and HLA-A*02 scFv inhibitory receptor) derived from three HLA-A*02(-) donors are shown, assayed using colorectal cell lines as targets. A*02: HLA-A*02. [Figure 25] Shown is the time course of CEA CAR Tmod and TCR T killing of tumor and normal cells at different E:T ratios using HLA-A*02(+) donor T cells transduced with CEA TCR or Tmod dual receptor systems. [Figure 26] We demonstrate that effector cells expressing the CEA CAR Tmod dual receptor system kill tumor cells as effectively as cells expressing the CEA TCR, but are approximately 70-fold less active in killing CEA(+)HLA-A*02(+) normal H508 target cells. Tumor: CEA(+)HLA-A*02(-) target cells; B-only: target cells expressing only HLA-A*02; Normal: CEA(+)HLA-A*02(+) target cells. [Figure 27] Figure 1 shows the selective cytotoxicity of effector cells expressing the CEA CAR Tmod dual receptor when presented with a 1:1 mixed tumor and normal cell culture. Tumor cells were H508 CEA(+)HLA-A*02(-) cells stably expressing GFP (green). Normal cells were H508 CEA(+)HLA-A*02(+) cells stably expressing RFP (red). T cells were from HLA-A*02(+) donor D12333. Scale bar is 500 microns. [Figure 28] Figure 2 shows a summary of the specific killing of effector cells expressing the CEA CAR and HLA-A*02 inhibitory receptor (Tmod) in a 1:1 mixture of tumor:normal target cells. The genotype of H508 target cells was the same as in Figure 26, and IL-2 was not added. Donor T cells were HLA-A*02(+), except for donor 183534. [Figure 29] Images of serially co-cultured target cells are shown. For cytotoxicity assays, T cells were transduced, enriched with blocking factor antigen, and transferred from one specific type of target cell to the next. Both normal and tumor cells are labeled with GFP, but red pseudocolor is used to visualize tumor cells and green is used for normal cells. The scale bar indicates 500 microns. [Figure 30] Figure 1 shows the time course of CEA CAR Tmod-expressing cells and CEA CAR-expressing cells upon repeated antigen challenge. Horizontal arrows indicate transition from the target cell type (tumor or normal H508). Donor T cells transduced with CEA CAR or Tmod dual receptors were HLA-A*02(+)(D12333). [Figure 31] This shows that the presence of soluble CEA (sCEA; 10 μg / mL) does not significantly affect CEA CAR Tmod cytotoxicity in H508 cells. The genotypes of tumor, normal, and B are as follows: tumor: CEA(+)HLA-A*02(-) target cells; normal: CEA(+)HLA-A*02(+) target cells; B: CEA(-)HLA-A*02(+) target cells. [Figure 32] Cytotoxicity assay using effector T cells expressing CEA CAR Tmod dual receptor and CEA(+) target cell line. E:T is 3:1 for target cell co-culture, and H508 target cells were used. B refers to CEA(-)HLA-A*02(+) target cells only. [Figure 33]Cytotoxicity assay using effector T cells expressing a CEA CAR Tmod dual receptor and a CEA(+) target cell line. E:T is a 3:1 target cell co-culture using SW1463 target cells. B only, CEA(-)HLA-A*02(+) target cells. [Figure 34] We demonstrate that effector T cells expressing CEA Tmod dual receptors (cells transduced with separate activator and blocker lentiviral vectors) enable selective killing of tumor versus normal cells in the colorectal cancer cell line H508. T cells expressing the Tmod receptor were more sensitive to, but more selective for, normal cells than the benchmark CEA TCR. T cells were derived from an HLA-A*02(-) donor (D4809). [Figure 35] Figure 1 shows quantification of reversible cytotoxicity by effector T cells expressing CEA Tmod dual receptors (delivered via two separate lentiviral vectors) in HLA-A*02(-) donor cells (D4809). T cells were first exposed to either tumor or normal cells in round 1, and then to normal or tumor cells, respectively, in round 2, to measure selective tumor-versus-normal cell killing. WT: wild-type; A2KO: HLA-A*02 knockout. [Figure 36]Figure 2 shows a Jurkat cell assay of CEA CAR Tmod dual receptor off-target selectivity using a cell line panel selected to represent >90% of human adult tissue gene expression. Jurkat effector cells expressing Tmod receptors were co-cultured with individual target cell lines listed in Table 26. Positive control cell lines representing tumor cells were transfected with 2 μg of CEA mRNA or naturally expressed CEA. Normal cells are CEA(-)HLA-A*02(+). The horizontal dashed line is placed at the mean + 2x standard deviation (SD) of data from Jurkat cells (expressing Tmod receptors) alone. Co-cultures were of 10,000 (10K) Jurkat cells and 10K target cells in each well. Left bar: Jurkat cells expressing Tmod dual receptors with CEA+HLA-A*02(-) cells; middle bar: Jurkat cells expressing CAR with CEA(-) target cells; right bar: Jurkat cells expressing both receptors with CEA(-)HLA-A*02(+) target cells. Negative controls are shown in gray boxes. [Figure 37] Summary of cytotoxicity data for effector T cells expressing CEA CAR Tmod dual receptors from three HLA-A*02(+) donors. UTD, untransduced. [Figure 38] A summary of selectivity data using primary T effector cells is shown. [Figure 39]

[0023] Figure 1 shows the design of mouse xenograft studies using human T cells expressing CEA CAR or CEA Tmod dual receptors. The xenograft experimental design and tumor volume versus time are shown. [Figure 40]Tumor volume measured by caliper in mouse xenograft studies is shown. Error bars are SEM. N = 7 mice / group (5 mice in saline and UTD, or non-transduced groups); xenograft = H508 colon cancer cell line expressing firefly luciferase; dose = 2E7 human T cells / mouse via tail vein injection. BLI % change = 100 × (BLI on day t - BLI on day 35) / (BLI on day 35). -100% on the lower right y-axis indicates zero bioluminescence signal, i.e., no evidence of any residual tumor cells. Human T cells in mouse blood were detected with hCD3 mAb. [Figure 41] Images of five mice from each group (a subset of those in Figure 40) used to measure bioluminescence (lucerifase) over time are shown. One Tmod mouse (second from the left, day 64) accidentally did not receive the BLI substrate. [Figure 42] Xenograft study results are shown for a T cell dose of 5E6 T cells per mouse. The bottom center panel shows replotted data from the top panel to show tumor volume at higher resolution. UTD: Untransduced; CAR, T cells transduced with CEA CAR only; Tmod, T cells transduced with CEA CAR and HLA-A*02 scFv LILRB1 inhibitory receptor. [Figure 43] Individual tumor data from mouse xenograft studies are shown. Thin light gray lines: individual mice; thick black lines: average; dotted vertical lines: day of T cell injection (day 35). UTD, untransduced T cells; CAR, T cells transduced with CEA CAR; Tmod, T cells transduced with both CEA CAR and HLA-A*02 ScFv LILRB1 inhibitory receptor; saline, mice injected with saline control. [Figure 44]Bioluminescence (BLI) in individual mice from the xenograft study is shown. BLI% was determined as described in Figure 40. UTD, untransduced T cells; CAR, T cells transduced with CEA CAR; Tmod, T cells transduced with both CEA CAR and HLA-A*02 ScFv LILRB1 inhibitory receptor; saline, mice injected with saline control. [Figure 45]

[0023] Figure 1 shows an analysis of cells from the spleens of mice from a xenograft study. Cells were harvested 30 days after T cell injection. [Figure 46] Figure 1 shows how the HLA-A*02 antigen can bind to the HLA-A*02 Tmod blocker receptor in cis in HLA-A*02(+) T cells to prevent blocker receptor binding / function in trans on normal cells. This effect can be detected by labeled HLA-A*02 tetramers and functional assays. [Figure 47] We show that CRISPR using guide RNA (gRNA) against B2M and B2M shRNA reduces HLA expression on the cell surface in HLA-A*02(+) T cells and increases the availability of blocker receptors. [Figure 48] Shown is the effect of B2M shRNA constructs on cis-binding to first generation autologous T cells expressing CEA CAR and HLA-A*02 scFvLILRB1 inhibitory receptor (Tmod). [Figure 49] Cytokine secretion in acute cytotoxicity assays is shown. Tumor cells were CEA(+)HLA-A*02(-)H508 cells, and normal cells were CEA(+)HLA-A*02(+)H508 cells. LD, detection limit = background + 3 times the standard deviation for each assay. [Figure 50] We show that the HLA-A*02 LILRB1 inhibitory receptor is equally sensitive in HLA-A*02(+) and HLA-A*02(-) Jurkat cells when assayed using HeLa target cells. [Figure 51]Figure 1 shows that co-expression of B2M shRNA in T cells expressing the HLA-A*02 scFv LILRB1 inhibitory receptor frees the receptor to bind to the probe on primary T cells. [Figure 52] Cytokine secretion in acute cytotoxicity assays is shown. Tumor, CEA(+)HLA-A*02(-)H508 cells; normal CEA(+)HLA-A*02(+)H508 cells; LD, detection limit = background + 3 times the standard deviation for each assay. [Figure 53] 1 is a table summarizing the properties of dual receptor systems of some embodiments described herein. DETAILED DESCRIPTION OF THE INVENTION

[0040] Provided herein are compositions and methods for treating cancer using immune cells containing two receptor systems that respond to differences in gene expression of ligands between cancer and normal wild-type cells. These expression differences may be due to loss of heterozygosity in cancer cells. Alternatively, the expression differences may be due to gene expression not being expressed in cancer cells or being expressed at a lower level in cancer cells than in normal cells. The two-receptor system is expressed in immune cells, such as immune cells used in adoptive cell therapy, and targets the activity of these immune cells to cancer cells that exhibit loss of heterozygosity or expression differences. In this two-receptor system, the first receptor (an activator receptor, sometimes referred to herein as the A module) activates or promotes the activation of the immune cell, while the second receptor (an inhibitory receptor, sometimes referred to herein as the blocker or inhibitor receptor, or B module) acts to inhibit the activation of the immune cell by the first receptor. Each receptor contains a ligand-binding domain (LBD) that binds to a specific ligand. Upon ligand binding, signals from the two receptors are integrated by the immune cell. For example, differential expression of ligands for the first and second receptors in cancer and normal cells due to loss of heterozygosity or differences in transcription levels of a gene locus encoding the inhibitory ligand in the cancer cells mediates immune cell activation by target cancer cells that express the first activator ligand but not the second inhibitory ligand.

[0041] In certain embodiments of the compositions and methods provided herein, immune cells comprising the two-receptor system described herein are used to treat CEA-cell adhesion molecule 5 (CEA)-positive cancers, including CEA-positive cancers of the gastrointestinal (GI) tract. In the case of CEA-positive cancers, the target antigen of the activator receptor is CEA or its peptide antigen in complex with major histocompatibility complex class I (MHC-I). CEA is primarily expressed in normal adults in GI tissue as a surface protein that can be cleaved from the membrane and released in a soluble form. Due to its selective expression in GI tumors, it has long been considered an attractive tumor-specific antigen that could mediate selective killing of GI tumors if CEA-positive cancer cells could be specifically targeted with appropriate therapeutic agents. Furthermore, the CEA gene product is an attractive target for cancer due to its high expression in virtually all colorectal tumors (and many subsets of other solid tumors) and its restricted expression in adult tissues. However, normal CEA expression in non-cancer (non-target) cells prevents its effective use in targeted therapies, such as adoptive cell therapy. Several treatments for CEA have been clinically tested and have been found to induce colitis as a dose-limiting toxicity (DLT). In 2011, a clinical trial using a mouse TCR against a CEA peptide (i.e., pMHC) complexed with HLA-A*02 was discontinued in a phase 1 trial (n=3) due to localized toxicity to the colon (Parkhurst et al. Molecular Therapy 2011 19(3):P620-626, Parkhurst et al. Clin Cancer Res. 2009 Jan 1;15(1):169-180). DLT occurred at a significantly lower dose of 2–4E8 cells / patient.

[0042] HLA heterozygous gene loss in a subset of tumors can be exploited to protect patients from on-target, off-tumor toxicity. By pairing activator receptors with inhibitory receptors, the methods provided herein increase the specificity of adoptive cell therapies and reduce adverse effects associated with these therapies, such as dose-limiting toxicity. Immune cells containing a CEA activator receptor and an HLA-A*02-specific inhibitory receptor selectively killed A*02(-) tumor cells in vitro and in vivo. These immune cells were as potent as clinically active CEA TCR-T cells but highly selective for tumor cells lacking HLA-A*02. CEA CARs paired with inhibitory receptors are potential solid tumor therapeutics whose activity is directed by deleted genes in tumor cells so that normal tissues can be protected from CEA-mediated cytotoxicity.

[0043] In some embodiments, the ligand for the activator is an MHC complex containing a CEA peptide complexed with MHC class I, e.g., HLA-A*02. In the methods described herein, this CEA-targeted activator receptor is paired with an inhibitory receptor, thereby increasing the safety window of the activator by blocking its cytolytic activity against normal CEA-positive tissues. Without wishing to be bound by theory, these tissues are thought to be primarily in the gastrointestinal tract. However, because tumor cells do not express the inhibitory, or blocking, receptor ligand, the activator receptor still induces targeted killing of tumor cells by immune cells comprising a two-receptor system. The target for the second inhibitory receptor is expressed by gastrointestinal (GI) tissue but not 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 on the surface of normal GI epithelial cells and is lost from GI tumor cells by loss of heterozygosity (LOH) or other mechanisms, leaving a single allele form in the cancer cell that can be distinguished from other alleles via the allele-specific ligand-binding domain on the inhibitory receptor. Exemplary targets of inhibitory receptors include, but are not limited to, major histocompatibility complex (MHC) proteins such as human leukocyte antigen A (HLA-A), HLA-B, HLA-C, and other HLAs. HLAs are encoded by variant genes such as HLA-A*01, HLA-A*02, HLA-A*03, HLA-C*07, and others, which can be lost from CEA-positive cancer cells through loss of heterozygosity. Alternatively, further exemplary targets of inhibitory receptors include, but are not limited to, TNF receptor superfamily member 11a (TNFRSF11A, also known as RANK), integrin subunit alpha E (ITGAE), cholinergic receptor nicotinic beta 1 subunit (ACHRB, or CHRNB), transient receptor potential cation channel subfamily V member 1 (TRPV1), and scavenger receptor class F member 1 (SREC, or SCARF).Each of these has a common nonsynonymous variant form with amino acid changes in its extracellular domain that are accessible to antibodies, which can be used as a B module target for cellular integrators designed to safely treat patients with GI cancers with engineered T cells activated by activator receptors such as CEA or CEA pMHC-responsive activator receptors.

[0044] The compositions and methods of the present disclosure can reduce or eliminate dose-limiting toxicities (DLTs) caused by CEA expression on normal GI tissues. Without wishing to be bound by theory, it is believed that CEA expression, while limited, is sufficiently high in the GI tract to induce adverse events of sufficient severity that it has prevented further development of CEA as a target for adoptive cell therapy or immunotherapy in clinical settings. The present disclosure provides methods for targeting CEA in cancer cells to treat CEA-positive cancers using adoptive cell therapy by adding a second inhibitory receptor that blocks the activation of adoptive immune cells in the presence of a second ligand (a ligand other than CEA, referred to as a non-target antigen, or alternatively, a blocker antigen). Using the compositions and methods described herein, CEA-expressing tumor cells are attacked by adoptive immune cells expressing two receptors because these tumor cells express only the activator ligand, CEA. In contrast, normal cells expressing CEA and a non-target antigen (alternatively referred to as a "blocker antigen") are protected from the adoptive immune cells. Inhibitory receptor responses to non-target antigens on normal cells prevent immune cell activation by CEA-targeted activator receptors. This dual-targeting approach creates a therapeutic window that allows for the safe and effective administration of CEA-directed cell therapy in patients with CEA-positive cancers.

[0045] The present disclosure provides methods and compositions that enable the use of potent CEA CARs and TCRs to induce on-target toxicity, making these CEA-targeted receptors useful as therapeutics by mitigating their toxicity. None of the existing clinically tested therapies, including cellular and large molecule therapies, provide a mechanism for protecting normal CEA-positive tissues.

[0046] Alternatively, the compositions and methods described herein can be used to kill target cells and / or treat subjects in which expression of a non-target antigen is partially or completely reduced due to causes other than loss of heterozygosity, including, but not limited to, partial gene deletion, epigenetic silencing, and point or truncating mutations in the sequence encoding the non-target antigen.

[0047] definition Before setting forth the present disclosure in more detail, it may be helpful to an understanding of the present disclosure to provide definitions of certain terms used herein.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of a particular embodiment, preferred embodiments of the compositions, methods, and materials are described herein. For purposes of this disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0049] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0050] As used herein, the term "isolated" means material that is substantially or essentially free from components that normally accompany it in its natural state. In certain embodiments, the terms "obtained" or "derived" are used synonymously with isolated.

[0051] The terms "subject," "patient," and "individual" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Also encompassed are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro. As used herein, "subject," "patient," or "individual" includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (such as cats or dogs). Non-human primates and preferably human patients are included.

[0052] As used herein, "treatment" or "treating" includes any beneficial or desired effect, and may include even minimal amelioration of symptoms. "Treatment" does not necessarily indicate a complete eradication or cure of a disease or condition, or its associated symptoms.

[0053] As used herein, "prevent" and similar words such as "prevented," "preventing," and the like refer to an approach for preventing, inhibiting, or reducing the likelihood of disease symptoms. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the occurrence or recurrence of disease symptoms. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease before onset or recurrence.

[0054] As used herein, the term "amount" refers to an "effective amount" or "effective dose" of a virus to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0055] A "therapeutically effective amount" of a virus or cells can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient).

[0056] An "increased" or "augmented" amount of physiological response, e.g., electrophysiological activity or cellular activity, is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points greater than 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) above the level of activity in untreated cells.

[0057] A "reduced" or "diminished" amount of physiological response, e.g., electrophysiological activity or cellular activity, is typically a "statistically significant" amount and can include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points greater than 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) above the level of activity in untreated cells.

[0058] "Maintain," or "preserve," or "maintain," or "no change," or "no substantial change," or "no substantial reduction" generally refers to a physiological response that is equivalent to the response elicited by either a vehicle or a control molecule / composition. An equivalent response is one that is not significantly or measurably different from the reference response.

[0059] Generally, "sequence identity" or "sequence homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, an advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and is discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences, for example with the blastp program.This program can also use an SEG filter to mask off segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Desired sequence identity ranges are approximately 80% to 100%, and integer values ​​therebetween. Typically, the percent identity between the disclosed and claimed sequences is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0060] As used herein, a "polynucleotide system" refers to one or more polynucleotides that can be designed to work together for a particular use or to produce a desired transformed cell.

[0061] The term "exogenous" is used herein to refer to any molecule, including nucleic acids, proteins or peptides, small molecules, etc., that originate outside an organism. In contrast, the term "endogenous" refers to any molecule that originates within an organism (i.e., is naturally produced by the organism).

[0062] The term "MOI" is used herein to refer to the multiplicity of infection, which is the ratio of agent (e.g., virus particles) to infected target (e.g., cell).

[0063] As used herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers). The term "about," when immediately preceding a number or numerical value, means that the number or numerical value is within a range of plus or minus 10%.

[0064] As used herein, "target cell" refers to the cell that is targeted by adoptive cell therapy.For example, target cell can be cancer cell, which can be killed by the transferred T cell of adoptive cell therapy.The target cell of the present disclosure expresses the target antigen as described herein and does not express non-target antigen.

[0065] As used herein, "non-target cells" refers to cells that are not targeted by adoptive cell therapy. For example, in adoptive cell therapy targeting cancer cells, normal, healthy, non-cancerous cells are non-target cells. Some or all of the non-target cells in a subject may express both the target antigen and the non-target antigen. The non-target cells in a subject may express the non-target antigen regardless of whether these cells also express the target antigen.

[0066] As used herein, a "non-target allelic variant" refers to an allele of a gene whose product is expressed by non-target cells but not by target cells. For example, a non-target allelic variant is an allele of a gene that is expressed by normal, non-cancer cells of a subject but not by cancer cells of the subject. Expression of a non-target allelic variant can be lost in cancer cells by any mechanism, including, but not limited to, loss of heterozygosity, mutation, or epigenetic modification of the gene encoding the non-target allelic variant.

[0067] As used herein, when used with respect to a ligand-binding domain, such as an antigen-binding domain, "specific for" or "specifically binds to" refers to a ligand-binding domain that has high specificity for a named target. Antibody specificity can be considered a measure of the goodness of fit between the ligand-binding domain and the corresponding ligand, or the ability of the ligand-binding domain to discriminate between similar or even dissimilar ligands. Compared to specificity, affinity is a measure of the strength of binding between the ligand-binding domain and the ligand, such that a low-affinity ligand-binding domain binds weakly and a high-affinity ligand-binding domain binds strongly. A ligand-binding domain that is specific for a target allele is one that can discriminate between different alleles of the gene. For example, a ligand-binding domain specific for HLA-A*02 will not bind, or only weakly bind, other HLA-A alleles, such as HLA-A*01 or HLA-A*03. Those skilled in the art will understand that while a ligand-binding domain can be said to be specific for a particular target, there will still be low levels of binding to one or more additional targets that do not affect its function in the receptor system described herein.

[0068] As used herein, "target antigen" refers to an antigen expressed by a target cell, such as a cancer cell, whether referred to using the term antigen or the name of a specific antigen. Expression of the target antigen is not limited to the target cell. The target antigen can be expressed by both cancer cells and normal, non-cancerous cells in a subject.

[0069] As used herein, a "non-target antigen" (or "blocker antigen") refers to an antigen that is expressed by normal, non-cancerous cells but not in cancer cells, whether referred to using the term antigen or the name of the specific antigen. This difference in expression allows inhibitory receptors to inhibit immune cell activation in the presence of non-target cells, but not in the presence of target cells.

[0070] Polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population. Polymorphisms can include one or more base changes, insertions, repeats, or deletions. Polymorphisms include, for example, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), which are mutations that occur when a single nucleotide, adenine (A), thymine (T), cytosine (C), or guanine (G), is changed.

[0071] As used herein, "affinity" refers to the strength of binding of a ligand to an antigen for a single ligand-binding site on a receptor, e.g., the antigen-binding domain of any of the receptors described herein. Ligand-binding domains can have a weaker interaction (low affinity) or a stronger interaction (high affinity) with their ligand.

[0072] The Kd, or dissociation constant, is a type of equilibrium constant that measures the tendency of a larger entity to reversibly dissociate into smaller components, such as when a macromolecular complex containing a receptor and its cognate ligand dissociates into the ligand and receptor. A high Kd indicates a high concentration of ligand is required to occupy the receptor, indicating a low affinity of the receptor for the ligand. Conversely, a low Kd indicates a high affinity of the ligand for the receptor.

[0073] As used herein, a receptor "responsive to" or "responsive to" refers to a receptor comprising an intracellular domain that, upon binding of a ligand (i.e., an antigen), generates a signal corresponding to the known function of the intracellular domain. An activator receptor bound to a target antigen can generate a signal that causes activation of immune cells that express the activator receptor. An inhibitory receptor bound to a non-target antigen can generate an inhibitory signal that prevents or reduces activation of immune cells that express the activator receptor. The responsiveness of receptors and their ability to activate or inhibit immune cells that express the receptor can be assayed by any means known in the art and described herein, including, but not limited to, reporter assays and cytotoxicity assays.

[0074] As used herein, an "activated" or "activated" immune cell is an immune cell that can perform one or more functions characteristic of an immune response. These functions include proliferation, cytokine release, and cytotoxicity, i.e., killing of target cells. Activated immune cells express markers that will be apparent to those skilled in the art. For example, activated T cells can express one or more of CD69, CD71, CD25, and HLA-DR. Immune cells expressing an activator receptor (e.g., a CEA CAR) can be activated by the activator receptor when the receptor becomes responsive to binding of the receptor to a target antigen (e.g., CEA) expressed by the target cell. A "target antigen" can also be referred to as an "activator antigen" and can be isolated or expressed by the target cell. Activation of immune cells expressing an inhibitory receptor can be prevented when the inhibitory receptor becomes responsive to binding of a non-target antigen (e.g., HLA-A*02), even when the activator receptor is bound to a target activator ligand. "Non-target antigens" may also be referred to as "inhibitory ligands" or "blocking factors" and may be isolated or expressed by target cells.

[0075] Receptor expression on immune cells can be verified by assays that report the presence of activator and inhibitory receptors described herein. For example, a population of immune cells can be stained with a labeling molecule (e.g., a fluorophore-labeled receptor-specific antibody or a fluorophore-labeled receptor-specific ligand) and quantified using fluorescence-activated cell sorting (FACS) flow cytometry. This method allows the percentage of immune cells in a population of immune cells to be characterized as expressing activator receptors, inhibitory receptors, or both receptors. The ratio of activator and inhibitory receptors expressed by the immune cells described herein can be determined, for example, by digital droplet PCR. These approaches can be used to characterize populations of cells for the production and manufacture of the immune cells, pharmaceutical compositions, and kits described herein. It should be understood that for the immune cells, pharmaceutical compositions, and kits described herein, the appropriate percentage of immune cells expressing both activator and inhibitory receptors is determined specifically for the methods described herein. For example, a suitable percentage of immune cells expressing both activator receptors and inhibitory receptors may 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%. For example, a suitable percentage of immune cells expressing both activator receptors and inhibitory receptors may be up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or up to 95%. For example, a suitable ratio of activator receptors to inhibitory receptors on immune cells may be about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5. It is understood that purification, enrichment, and / or depletion steps can be used on populations of immune cells to meet the preferred values ​​for the immune cells, pharmaceutical compositions, and kits described herein.

[0076] Responsive receptors expressed by immune cells described herein can be verified by assays measuring the generation of signals predicted by the intracellular domain of the receptor. Responsive receptors can be characterized using reporter cell lines such as Jurkat-luciferase NFAT cells (Jurkat cells). Jurkat cells are derived from T cells and contain a stably integrated nuclear factor of activated T cells (NFAT)-inducible luciferase reporter system. NFAT is a family of transcription factors required for immune cell activation, and its activation can be used as a signaling marker for T cell activation. Jurkat cells can be transduced or transfected with activating and / or inhibitory receptors described herein. Activating receptors respond to ligand binding when Jurkat cells express a luciferase reporter gene, and the level of responsiveness can be determined by the level of reporter gene expression. The presence of luciferase can be determined using any known luciferase detection reagent, such as luciferin. When co-expressed with an activator receptor in Jurkat cells, an inhibitory receptor responds to ligand binding and prevents normally responding immune cells from expressing luciferase in response to the activator receptor. For example, the responsiveness of an inhibitory receptor can be determined and quantified in Jurkat cells expressing both an activator and an inhibitor by observing: 1) the Jurkat cells express luciferase in the presence of an activator receptor ligand and in the absence of an inhibitory receptor ligand; and 2) luciferase expression in Jurkat cells is reduced or eliminated in the presence of both an activator receptor ligand and an inhibitory receptor ligand. This approach can be used to determine the sensitivity, potency, and selectivity of activator receptors and specific pairs of activator receptors and inhibitory receptors. Sensitivity, potency, and selectivity can be quantified by EC50 or IC50 values ​​using dose-response experiments in which activator receptor ligands and / or inhibitory receptor ligands are titrated into cultures of Jurkat cells expressing a particular pair of activator receptors or activator and inhibitory receptors.Alternatively, EC50 and IC50 values ​​can be determined in co-cultures of immune cells (e.g., Jurkat cells or primary immune cells) expressing an activator receptor or a particular pair of activator and inhibitory receptors with target cells expressing increasing amounts of the activator or inhibitory ligand. Increased amounts of the activator or inhibitory ligand can be achieved in the target cells, for example, by titrating mRNA-encoding the activator or inhibitory ligand into the target cells or by using target cells that naturally express different levels of the target ligand. Exemplary suitable EC50 and IC50 values ​​for activator and inhibitory receptors when determining target cells expressing various amounts of target and non-target ligands include an EC50 of 260 or less transcripts per million (TPM) for activator receptors, e.g., an EC50 of 10 to 260 TPM, and an IC50 of 10 or less TPM for inhibitory receptors, e.g., an IC50 of 1 to 5 TPM.

[0077] Activation of immune cells described herein expressing an activator receptor or a specific pair of an activator receptor and an inhibitory receptor can be further determined by an assay measuring the viability of target cells after co-incubation with the immune cells. Immune cells, sometimes referred to as effector cells, are co-incubated with target cells expressing an activator receptor ligand, an inhibitory receptor ligand, or both an activator receptor and an inhibitory receptor ligand. After co-incubation, the viability of the target cells is measured using any method that measures viability in cell culture. For example, viability can be determined using a mitochondrial function assay that measures active mitochondrial enzymes using tetrazolium substrate. Viability can also be determined using imaging-based methods. The target cells can express a fluorescent protein, such as green fluorescent protein or red fluorescent protein. A decrease in total cell fluorescence indicates a decrease in target cell viability. A decrease in target cell viability after incubation with immune cells expressing an activator receptor or a specific pair of an activator receptor and an inhibitory receptor is interpreted as target cell-mediated activation of the immune cells. A measure of immune cell selectivity can also be determined using this approach. Immune cells expressing a pair of activator and inhibitory receptors are selective if the following are observed: 1) there is a decrease in viability of target cells that express the activator receptor ligand but not the inhibitory receptor ligand, and 2) there is no decrease in viability of target cells that express both the activator and inhibitory receptor ligands. From these measurements, a "specific killing" value can be derived, which quantifies the percentage of immune cell activation based on the decrease in target cell viability as a percentage of the negative control (immune cells that do not express the activator receptor). Furthermore, from these measurements, a "selectivity ratio" value can be derived, which represents the ratio of the specific killing observed in target cells expressing the activator receptor ligand in the absence of the inhibitory receptor ligand to the specific killing observed in target cells expressing both the activator and inhibitory receptor ligands.This approach can be used to characterize populations of cells for the production and manufacture of the immune cells, pharmaceutical compositions, and kits described herein.

[0078] Suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits can be, for example, the following criteria: 1) at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% specific killing after 48 hours of co-incubation of immune cells and target cells expressing an activator receptor ligand in the absence of an inhibitory receptor ligand, and 2) no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 3%, or no more than 1% specific killing of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand.

[0079] As further examples, suitable specific kill values ​​for immune cells, pharmaceutical compositions, and kits may be the following criteria: 1) 30% to 99%, 40% to 99%, 50% to 99%, 55% to 95%, 60% to 95%, 60% to 90%, 50% to 80%, 50% to 70%, or 50% to 60% of target cells that express an activator ligand but not an inhibitory ligand are killed, and 2) 1% to 40%, 3% to 40%, 5% to 40%, 5% to 30%, 10% to 30%, 15% to 30%, or 5% to 20% of target cells that express an activator ligand and an inhibitory ligand are killed.

[0080] By way of still further example, suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits may be, for example, the following criteria: 1) at least 50% specific killing after 48 hours of co-incubation of immune cells and target cells expressing an activator receptor ligand in the absence of an inhibitory receptor ligand, and 2) no more than 20% specific killing of target cells expressing both an activator receptor ligand and an inhibitory receptor ligand. As a further example, the immune cells may kill at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% of target cells that express an activator ligand and do not express an inhibitory ligand over a 6-hour, 12-hour, 18-hour, 24-hour, 30-hour, 36-hour, 42-hour, 48-hour, 54-hour, or 60-hour period, while killing less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, or less than 1% of target cells that express an activator ligand and an inhibitory ligand over the same period.

[0081] Suitable specific killing values ​​for target cells expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50% to at least about 95%. Suitable specific killing values ​​for target cells expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Suitable specific killing values ​​for target cells expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, or at most about 95%. Suitable specific killing values ​​for target cells expressing both activator and inhibitory receptor ligands for immune cells, pharmaceutical compositions, and kits may be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. Suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits may be determined after about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, or about 72 hours of co-incubation of immune cells with target cells.

[0082] Suitable specific killing values ​​for target cells expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50% to at least about 95%. Suitable specific killing values ​​for target cells expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Suitable specific killing values ​​for target cells expressing an activator ligand in the absence of an inhibitory ligand value for immune cells, pharmaceutical compositions, and kits can be, for example, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, or at most about 95%. Suitable specific killing values ​​for target cells expressing both activator and inhibitory receptor ligands for immune cells, pharmaceutical compositions, and kits may be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. Suitable specific killing values ​​for immune cells, pharmaceutical compositions, and kits may be determined after about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, or about 72 hours of co-incubation of immune cells with target cells.

[0083] As used herein, the term "functional variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein and that retains one or more desired activities of the parent protein. A functional variant can be a fragment of a protein (i.e., a variant with an N-terminal and / or C-terminal deletion) that retains one or more desired activities of the parent protein.

[0084] All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be considered as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general knowledge anywhere in the world.

[0085] activator receptor The present disclosure provides a first receptor comprising a first extracellular ligand-binding domain specific for a target antigen, including a cancer cell-specific antigen or a peptide antigen thereof, in complex with major histocompatibility complex class I (MHC-I). The first receptor is an activator receptor, and upon binding of the target antigen by the extracellular ligand-binding domain of the first receptor, mediates activation of an immune cell expressing the first receptor. The first receptor responds to the target antigen (i.e., an activator ligand). For example, when the target antigen binds to or contacts the first receptor, the first receptor is responsive and activates an immune cell expressing the first receptor upon binding of the target antigen by the extracellular ligand-binding domain of the first receptor. In some embodiments, the first receptor is a chimeric antigen receptor (CAR). In some embodiments, the first receptor is a T cell receptor (TCR).

[0086] In some embodiments, the first receptor is humanized. As used herein, "humanization" refers to replacing a sequence or subsequence in a transgene isolated from 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 murine CDRs onto human framework sequences, followed by back-substitution of certain human framework residues for the corresponding murine residues from the original antibody.

[0087] activator target In some embodiments, the target antigen of the first receptor is a cancer cell-specific antigen.Any cell surface molecule expressed by target cancer cell can be a suitable target antigen for the first receptor ligand binding domain.For example, cell adhesion molecules, intercellular signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane receptors, receptors for neurotransmitters, or voltage-dependent ion channels can be used as target antigens.

[0088] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in complex with major histocompatibility complex class I (MHC-I). Any molecule expressed by a target cancer cell and presented as a peptide antigen (pMHC) by major histocompatibility complex class I (MHC-I) on the cancer cell surface can be a suitable target antigen for the first receptor extracellular ligand-binding domain.

[0089] In some embodiments, the cancer cell-specific antigen is CEA cell adhesion molecule 5 (CEA) in complex with major histocompatibility complex class I (MHC-I), or a peptide antigen thereof.

[0090] The major histocompatibility complex class I (MHC-I) is a protein complex that presents antigens to cells of the immune system and induces an immune response. The human leukocyte antigens (HLA) that correspond to MHC-I are HLA-A, HLA-B, and HLA-C.

[0091] Cancer cell-specific pMHC antigens, including any of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G, are contemplated within the scope of the present disclosure. In some embodiments, the cancer cell-specific antigen comprises HLA-A. HLA-A receptors are heterodimers comprising a heavy α chain and a smaller β chain. The α chain is encoded by a variant of HLA-A, while the β chain (β2-microglobulin) is invariant. Thousands of variant HLA-A genes exist, all of which are within the scope of the present disclosure. In some embodiments, the MHC-I comprises the human leukocyte antigen A*02 allele (HLA-A*02).

[0092] In some embodiments, the cancer cell-specific antigen comprises HLA-B. Hundreds of versions (alleles) of the HLA-B gene are known, each of which is given a specific number (e.g., HLA-B27).

[0093] In some embodiments, the cancer cell-specific antigen comprises HLA-C. HLA-C belongs to the HLA class I heavy chain paralogs. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). More than 100 HLA-C alleles are known in the art.

[0094] In some embodiments, the cancer cell-specific antigen is a colorectal cancer antigen, hi some embodiments, the colorectal cancer antigen comprises CEA in complex with major histocompatibility complex class I (MHC-I), or a peptide antigen thereof.

[0095] In some embodiments, the cancer cell-specific antigen is CEA cell adhesion molecule 5 (CEA) in complex with major histocompatibility complex class I (MHC-I), or a peptide antigen thereof. CEA is a 180 kDa glycoprotein tumor-associated protein expressed by various cancer cells. CEA is a GPI-anchored adhesion molecule composed of repeated immunoglobulin domains. It is used as a biomarker for colon cancer, both as a diagnostic and a surrogate for treatment response. CEA-expressing cancers include adenocarcinoma, colorectal cancer, and select other epithelial cancers, including colorectal adenocarcinoma. However, CEA is also expressed in a variety of normal epithelial cells throughout the gastrointestinal tract, for example, in highly differentiated epithelial cells in the upper third of colonic crypts (see Figure 7 for CEA expression).

[0096] All isoforms of CEA are contemplated as cancer cell-specific antigens in the present disclosure. CEA isoform 1 is described in NCBI record number NP_001278413.1, the contents of which are incorporated herein by reference. In some embodiments, CEA comprises the following amino acid sequence: [ka] In some embodiments, CEA comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1. CEA isoform 2 is described in NCBI accession number NP_001295327.1, the contents of which are incorporated herein by reference. In some embodiments, CEA comprises the following amino acid sequence: [ka] In some embodiments, CEA comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:15.

[0097] In some embodiments, the cancer cell-specific antigen is a peptide antigen derived from CEA. 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. In some embodiments, the peptide antigen comprises a sequence identical to a subsequence of SEQ ID NO: 1. Exemplary CEA peptide antigens include amino acids 691-699 of SEQ ID NO: 1 (IMIGVLVGV), amino acids 605-613 of SEQ ID NO: 1 (YLSGANLNL), and amino acids 694-702 of SEQ ID NO: 1 (GVLVGVALI). In some embodiments, the CEA peptide antigen comprises or consists essentially of amino acids 691-699 of SEQ ID NO: 1 (IMIGVLVGV). 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: 15. In some embodiments, the peptide antigen comprises a sequence identical to a subsequence of SEQ ID NO: 15. In some embodiments, the CEA peptide antigen is complexed with MHC-I. In some embodiments, the MHC-I comprises the human leukocyte antigen A*02 allele (HLA-A*02).

[0098] Extracellular Ligand Binding Domain The present disclosure provides a first receptor comprising a first extracellular ligand-binding domain specific for a target antigen. In some embodiments, the target antigen comprises a cancer cell-specific antigen.

[0099] In some embodiments, the cancer cell-specific antigen is CEA or a CEA-derived peptide antigen complexed with MHC-I, and the ligand-binding domain of the first receptor recognizes and binds to the CEA antigen.

[0100] Any type of ligand-binding domain capable of modulating receptor activity in a ligand-dependent manner is contemplated within the scope of this disclosure. In some embodiments, the ligand-binding domain is an antigen-binding domain. Exemplary antigen-binding domains include, among others, scFv, SdAb, Vβ-only domains, and TCR antigen-binding domains derived from TCR α and β chain variable domains.

[0101] Any type of antigen binding domain is contemplated within the scope of this disclosure.

[0102] For example, the first extracellular ligand-binding domain can be part of a continuous polypeptide chain, including, for example, a Vβ-only domain, a single-domain antibody fragment (sdAb), or a heavy-chain antibody HCAb, a single-chain antibody (scFv) derived from a murine, humanized, or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the first extracellular ligand-binding domain comprises an antigen-binding domain, including an antibody fragment. In further embodiments, the first extracellular ligand-binding domain comprises an antibody fragment, including an scFv or sdAb.

[0103] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins or fragments thereof, of polyclonal or monoclonal origin, and can be derived from natural or recombinant sources.

[0104] The term "antibody fragment" or "antibody binding domain" refers to at least a portion of an antibody or recombinant variant thereof that contains the antigen-binding domain, i.e., the antigen-determining variable region of an intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments, single-chain (sc)Fv ("scFv") antibody fragments, linear antibodies, single-domain antibodies (abbreviated as "sdAb") (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.

[0105] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short, flexible polypeptide linker and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0106] "Heavy chain variable region" or "VH" (or "VHH" in the case of single domain antibodies, e.g., nanobodies) with respect to antibodies refers to the fragment of a heavy chain containing the three CDRs interposed between adjacent stretches known as framework regions, which are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0107] As used herein, unless otherwise specified, an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0108] In some embodiments, the antigen-binding domain of the activator and / or inhibitory receptor comprises an scFv. In some embodiments, the scFv comprises a VL and a VH region connected by a linker. In some embodiments, the linker comprises a glycine serine linker, e.g., GGGGSGGGGSGGGGSGG (SEQ ID NO: 146). In some embodiments, the scFv further comprises a signal sequence at the N-terminus of the scFv. An exemplary signal sequence includes MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 184), which is encoded by ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGT (SEQ ID NO: 185).

[0109] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules, in their naturally occurring conformations. Kappa ("K") and lambda ("λ") light chains refer to the two major antibody light chain isotypes.

[0110] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, e.g., an antibody expressed in a bacteriophage or yeast expression system. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses the antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology that is available and well known in the art.

[0111] The terms "Vβ domain," "Vβ-only domain," "β-chain variable domain," or "single variable domain TCR (svd-TCR)" refer to an antigen-binding domain consisting essentially of a single T cell receptor (TCR) beta variable domain that specifically binds to an antigen in the absence of a second TCR variable domain. Vβ-only domains engage antigen using complementarity-determining regions (CDRs). Each Vβ-only domain contains three complementarity-determining regions (CDR1, CDR2, and CDR3). Additional elements may be combined, provided that the Vβ domain is configured to bind to an epitope in the absence of a second TCR variable domain.

[0112] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises an antibody fragment, a single-chain Fv antibody fragment (scFv), or a β-chain variable domain (Vβ).

[0113] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a TCR alpha chain variable domain and a TCR beta chain variable domain.

[0114] In some embodiments, the first extracellular ligand-binding domain comprises a TCR ligand-binding domain that binds to the CEA antigen. In some embodiments, the CEA antigen is complexed with MHC-I, and the MHC-I comprises an HLA-A*02 allele. Exemplary TCR antigen-binding domains that bind to and recognize the CEA MHC-I HLA-A*02 antigen are described in Parkhurst et al. Molecular Therapy 2011 19(3):P620-626, the contents of which are incorporated herein by reference. Exemplary TCR extracellular ligand-binding domains that recognize amino acids 691-699 of SEQ ID NO: 1 (IMIGVLVGV) complexed with HLA-A*02 MHC-I include the TCR alpha domains of TRAV8-1*01 and TRAJ6*01, and the TCR beta domains of TRBV26*01, TRBD1*01, TRBJ2-7*01, and TRBC2.

[0115] Exemplary CDRs for recognizing the CEA MHC-I HLA-A*02 antigen, including IMIGVLVGV (SEQ ID NO: 2), are shown in Table 1 below. [Table 1]

[0116] In some embodiments, the first extracellular ligand-binding domain comprises a complementarity determining region (CDR) selected from SEQ ID NOs: 3-12, or a sequence having at least 85%, or at least 95%, identity thereto.

[0117] In some embodiments, the ligand-binding domain of the first receptor comprises a TCR ligand-binding domain. In some embodiments, the TCR α chain variable domain comprises CDR-1 of TSITA (SEQ ID NO: 3), CDR-2 of IRSNER (SEQ ID NO: 4), and CDR-3 comprising ATDLTSGGNYK (SEQ ID NO: 5), ATDFTSGGNYK (SEQ ID NO: 6), ATDLTTGGNYK (SEQ ID NO: 7), or ATDFTTGGNYK (SEQ ID NO: 8), and the TCR β chain variable domain comprises CDR-1 of KGHPV (SEQ ID NO: 9), CDR-2 of FQNQEV (SEQ ID NO: 10), and CDR-3 of ASSLGLGDYEQ (SEQ ID NO: 11) or ASSLGTGDYEQ (SEQ ID NO: 12), or a sequence having at least 85%, or at least 95%, identity thereto. In some embodiments, the TCR alpha chain variable domain comprises CDR-1 of SEQ ID NO: 9, CDR-2 of SEQ ID NO: 10, and CDR-3 of SEQ ID NO: 11 or SEQ ID NO: 12, and the TCR beta chain variable domain comprises CDR-1 of SEQ ID NO: 3, CDR-2 of SEQ ID NO: 4, and CDR-3 comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a sequence having at least 85%, or at least 95%, identity thereto.

[0118] Exemplary TCR alpha and beta chains comprising the CDRs from Table 1 are shown below in Table 2. The CDRs are underlined in the sequences in Table 2. In Table 2, the TCR alpha and TCR beta chains are separated by a P2A self-cleaving peptide (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 186)) and a GSG linker. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0119] In some embodiments, the first receptor comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to the sequence or subsequence of any one of SEQ ID NOs: 16-31 or 36-51. In some embodiments, the first receptor comprises the sequence or subsequence of any one of SEQ ID NOs: 16-31 or 36-51.

[0120] In some embodiments, the first receptor comprises a TCR alpha chain comprising or consisting essentially of amino acids 1-270 of any one of SEQ ID NOs: 16-31, or a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto. In some embodiments, the first receptor comprises a TCR alpha chain comprising or consisting essentially of amino acids 1-270 of any one of SEQ ID NOs: 16-31.

[0121] In some embodiments, the first receptor comprises a TCR beta chain comprising or consisting essentially of amino acids 293-598 of any one of SEQ ID NOs: 16-31, or a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto. In some embodiments, the first receptor comprises a TCR beta chain comprising or consisting essentially of amino acids 293-598 of any one of SEQ ID NOs: 16-31.

[0122] In some embodiments, the first receptor comprises a TCR alpha chain comprising amino acids 1-270 of any one of SEQ ID NOs: 16-31, and a TCR beta chain comprising amino acids 293-598 of any one of SEQ ID NOs: 16-31.

[0123] In some embodiments, the first receptor comprises a TCR alpha chain comprising or consisting essentially of amino acids 1-268 of any one of SEQ ID NOs: 36-51, or a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto. In some embodiments, the first receptor comprises a TCR alpha chain comprising or consisting essentially of amino acids 1-268 of any one of SEQ ID NOs: 36-51.

[0124] In some embodiments, the first receptor comprises a TCR beta chain comprising or consisting essentially of amino acids 291-596 of any one of SEQ ID NOs: 36-51, or a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical thereto. In some embodiments, the first receptor comprises a TCR beta chain comprising or consisting essentially of amino acids 291-596 of any one of SEQ ID NOs: 36-51.

[0125] In some embodiments, the first receptor comprises a TCR alpha chain comprising amino acids 1-268 of any one of SEQ ID NOs: 36-51, and a TCR beta chain comprising amino acids 291-596 of any one of SEQ ID NOs: 36-51.

[0126] 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 the CAR. Exemplary CAR sequences comprising a CEA-targeting scFv domain are shown in Table 3 below. In Table 3, the CDR sequences are underlined. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0127] In some embodiments, the CEA scFv comprises CDR-H1 of EFGMN (SEQ ID NO: 55), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 56), CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 57) or WDFAHYFQTMDY (SEQ ID NO: 58), CDR-L1 of KASQNVGTNVA (SEQ ID NO: 59) or KASAAVGTYVA (SEQ ID NO: 60), CDR-L2 of SASYRYS (SEQ ID NO: 61) or SASYRKR (SEQ ID NO: 62), and CDR-L3 of HQYYTYPLFT (SEQ ID NO: 63), or a sequence having at least 85% or at least 95% identity thereto. In some embodiments, the CEA scFv comprises CDR-H1 of EFGMN (SEQ ID NO: 55), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 56), CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 57) or WDFAHYFQTMDY (SEQ ID NO: 58), CDR-L1 of KASQNVGTNVA (SEQ ID NO: 59) or KASAAVGTYVA (SEQ ID NO: 60), CDR-L2 of SASYRYS (SEQ ID NO: 61) or SASYRKR (SEQ ID NO: 62), and CDR-L3 of HQYYTYPLFT (SEQ ID NO: 63). In some embodiments, the CEA scFv comprises CDR-H1 of EFGMN (SEQ ID NO:55), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO:56), CDR-H3 of WDFAYYVEAMDY (SEQ ID NO:57), CDR-L1 of KASQNVGTNVA (SEQ ID NO:59), CDR-L2 of SASYRYS (SEQ ID NO:61), and CDR-L3 of HQYYTYPLFT (SEQ ID NO:63). In some embodiments, the CEA scFv comprises CDR-H1 of EFGMN (SEQ ID NO:55), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO:56), CDR-H3 of WDFAYYVEAMDY (SEQ ID NO:57), CDR-L1 of KASAAVGTYVA (SEQ ID NO:60), CDR-L2 of SASYRKR (SEQ ID NO:62), and CDR-L3 of HQYYTYPLFT (SEQ ID NO:63).In some embodiments, the CEA scFv comprises CDR-H1 of EFGMN (SEQ ID NO: 56), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 56), CDR-H3 of WDFAHYFQTMDY (SEQ ID NO: 58), CDR-L1 of KASAAVGTYVA (SEQ ID NO: 60), CDR-L2 of SASYRKR (SEQ ID NO: 62), and CDR-L3 of HQYYTYPLFT (SEQ ID NO: 63).

[0128] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising a set of heavy chain complementarity-determining regions (HC-CDRs) selected from the group consisting of SEQ ID NOs: 55-58, and a light chain variable (VL) portion comprising a set of light chain complementarity-determining regions selected from the group consisting of SEQ ID NOs: 59-63, or CDR sequences having up to one, two, or three substitutions, deletions, or insertions compared to SEQ ID NOs: 55-58 or SEQ ID NOs: 59-63. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising a set of heavy chain complementarity-determining regions (HC-CDRs) selected from the group consisting of SEQ ID NOs: 55-57, and a light chain variable (VL) portion comprising a set of light chain complementarity-determining regions including SEQ ID NOs: 59, 61, and 63, or CDR sequences having up to one, two, or three substitutions, deletions, or insertions compared to SEQ ID NOs: 55-57 or SEQ ID NOs: 59, 61, and 63. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising a set of heavy chain complementarity-determining regions (HC-CDRs) comprising SEQ ID NOs: 55-57, and a light chain variable (VL) portion comprising a set of light chain complementarity-determining regions comprising SEQ ID NOs: 59, 61, and 63.

[0129] Exemplary scFv domains that recognize CEA are shown below in Table 4. The underlined CDR sequences are shown. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0130] In some embodiments, the CEA scFv comprises a sequence selected from the group consisting of SEQ ID NOs: 64-70, 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 CEA scFv comprises or consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 64-70. Further exemplary anti-CEA antibody sequences are provided in Stewart et al. Cancer Immunol. Immunother. 47:299-306 (1999), WO1999 / 043817A1, US2002 / 0018750A1, US2011 / 0104148A1, US2016 / 0108131A1, US2016 / 0075795A1, US2019 / 0185583A1, US2020 / 0123270A1, WO2020 / 259550A1, WO2021 / 053587A1, WO2021 / 110647A1, the contents of which are incorporated herein by reference for purposes of providing anti-CEA VH, VL, scFv, and / or ligand-binding domain sequences.

[0131] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising SEQ ID NO: 144, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a light chain variable (VL) portion comprising SEQ ID NO: 148, 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 heavy chain variable (VH) portion comprising SEQ ID NO: 144, and a light chain variable (VL) portion comprising SEQ ID NO: 148. In some embodiments, the extracellular ligand-binding domain of the first receptor further comprises a linker between the VH and VL portions.

[0132] In some embodiments, the extracellular ligand-binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOs: 66-70, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises the scFv sequence of SEQ ID NO: 68, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the extracellular ligand-binding domain of the first receptor comprises the scFv sequence of SEQ ID NO: 68.

[0133] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) amino acid residues in the CDR of the antigen-binding domain provided herein are substituted with another amino acid. The substitution may be "conservative," meaning that the substitution is within the same family of amino acids. Naturally occurring amino acids can be divided into four families, and conservative substitutions are made within these families: (1) amino acids with basic side chains: lysine, arginine, and histidine; (2) amino acids with acidic side chains: aspartic acid and glutamic acid; (3) amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, and tyrosine; and (4) amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine. Altering the amino acid sequence of an antibody CDR by adding, deleting, or substituting amino acids can produce various effects, such as increased binding affinity for a target antigen.

[0134] Chimeric antigen receptor (CAR) The present disclosure provides a first activator receptor and an immune cell comprising the same. In some embodiments, the first receptor is a chimeric antigen receptor.

[0135] As used herein, the term "chimeric antigen receptor (CAR)" can refer to an artificial receptor derived from a T cell receptor and encompasses engineered receptors that graft artificial specificity onto specific immune effector cells. CARs can be used to confer the specificity of a monoclonal antibody to T cells, thereby enabling the generation of large numbers of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, a CAR directs the specificity of a cell to, for example, a tumor-associated antigen. Exemplary CARs include an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen-binding region. In some embodiments, a CAR further comprises a hinge domain. In certain aspects, a CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3 transmembrane domain and endodomain. The specificity of other CAR designs can be derived from the receptor's ligand (e.g., a peptide). In certain cases, the CAR contains domains for additional costimulatory signaling, such as CD3, 4-1BB, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules can be co-expressed with the CAR, including costimulatory molecules, reporter genes for imaging, gene products that conditionally eliminate T cells upon addition of a prodrug, homing receptors, cytokines, and cytokine receptors.

[0136] In some embodiments, the extracellular ligand-binding domain of the first receptor is fused to the extracellular domain of the CAR.

[0137] In some embodiments, the CAR of the present disclosure comprises an extracellular hinge region. Incorporation of the hinge region can affect cytokine production from CAR-T cells and improve in vivo expansion of CAR-T cells. Exemplary hinges can be isolated or derived from IgD and CD8 domains, e.g., IgG1. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0138] In some embodiments, the hinge is isolated or derived from CD8α or CD28. In some embodiments, the CD8α hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 71). In some embodiments, the CD8α hinge comprises SEQ ID NO: 71. In some embodiments, the CD8α hinge consists essentially of SEQ ID NO: 71. In some embodiments, the CD8 alpha hinge is encoded by a nucleotide sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 72). In some embodiments, the CD8 alpha hinge is encoded by SEQ ID NO: 72.

[0139] In some embodiments, the CD8α hinge is encoded by a nucleotide sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of SEQ ID NO: 156. In some embodiments, CD8α is encoded by SEQ ID NO: 156.

[0140] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 73). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 73. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 74). In some embodiments, the CD28 hinge is encoded by SEQ ID NO:74.

[0141] The CAR of the present disclosure can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In some embodiments, a transmembrane domain that naturally associates with one of the domains in the CAR is used. For example, a CAR that includes a CD28 costimulatory domain can also use a CD28 transmembrane domain. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or different surface membrane proteins and minimize interaction with other members of the receptor complex.

[0142] The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region can be isolated from or derived from (i.e., comprising at least the transmembrane region 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, CD154, or an immunoglobulin such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0143] In some embodiments of the CAR of the present disclosure, the CAR comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 75). In some embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 75. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 76). In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 76. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of SEQ ID NO: 157. In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 157.

[0144] In some embodiments of the CAR of the present disclosure, the CAR comprises an IL-2R beta transmembrane domain. In some embodiments, the IL-2R beta transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 77). In some embodiments, the IL-2R beta transmembrane domain comprises or consists essentially of SEQ ID NO: 77. In some embodiments, the IL-2R beta transmembrane domain comprises [ka] (SEQ ID NO: 78). In some embodiments, the IL-2R beta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of (SEQ ID NO: 78).

[0145] The cytoplasmic domain, or otherwise, intracellular signaling domain, of a CAR of the present disclosure is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and causes the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion may be used in place of the intact chain, so long as it transduces an effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired function of the CAR-T cells of the present disclosure. Thus, the term intracellular signaling domain is meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce an effector function signal.

[0146] Examples of intracellular signaling domains for use in the CARs of the present disclosure include the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences that have the same functional capability.

[0147] Thus, the intracellular domain of the CAR of the present disclosure comprises at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures that the T cell receptor (TCR) signaling necessary to activate the effector function of the CAR T cell is present. In some embodiments, the at least one cytoplasmic activation domain is a CD247 molecule (CD3ζ) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX activation protein of 12 kDa (DAP12) activation domain.

[0148] In some embodiments, the CD3ζ activation domain comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 79).

[0149] In some embodiments, the CD3 zeta activation domain comprises or consists essentially of SEQ ID NO:79. In some embodiments, the CD3 zeta activation domain is encoded by a nucleotide sequence that has at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to, or is identical to, the sequence: AGATGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGCGTAGAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGACTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 80). In some embodiments, the CD3 zeta activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of SEQ ID NO: 163. In some embodiments, the CD3 zeta activation domain is encoded by SEQ ID NO: 163.

[0150] It is known that signals generated through the TCR alone are often insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0151] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. In some embodiments, the ITAM contains a tyrosine separated from a leucine or isoleucine by any two other amino acids (YxxL / I (SEQ ID NO: 983). In some embodiments, the cytoplasmic domain contains 1, 2, 3, 4, or 5 ITAMs. An exemplary ITAM-containing cytoplasmic domain is the CD3ζ activation domain. Further examples of ITAM-containing primary cytoplasmic signaling sequences that can be used in the CARs of the present disclosure include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d.

[0152] In some embodiments, a CD3 zeta activation domain comprising a single ITAM comprises an amino acid sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 81). In some embodiments, the CD3 zeta activation domain comprises SEQ ID NO: 81. In some embodiments, a CD3 zeta activation domain comprising a single ITAM consists essentially of the amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 81). In some embodiments, a CD3 zeta activation domain comprising a single ITAM comprises [ka] (SEQ ID NO: 82). In some embodiments, the CD3 zeta activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of (SEQ ID NO: 82).

[0153] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise a CD3ζ signaling domain by itself or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3ζ chain portion and a costimulatory domain. A costimulatory domain refers to a portion of the CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. Examples of such molecules include costimulatory domains, which are IL-2Rβ, Fc receptor gamma (FcRγ), Fc receptor beta (FcRβ), CD3g molecule gamma (CD3γ), CD3δ, CD3ε, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen-related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor The intracellular domain of the CAR of the present disclosure is selected from the group consisting of: oxidase superfamily member 4 (OX40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory domain (ICOS), lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin-like receptor C2 (NKG2C), and CD276 molecule (B7-H3) c-stimulatory domain, or a functional variant thereof. In some embodiments, the intracellular domain of the CAR of the present disclosure comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is isolated or derived from CD28.

[0154] In some embodiments, the intracellular domain of a CAR of the present disclosure comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 83). In some embodiments, the CD28 costimulatory domain comprises or consists essentially of SEQ ID NO: 83. In some embodiments, the CD28 costimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to, or identical to, the sequence of AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC (SEQ ID NO: 84). In some embodiments, the CD28 costimulatory domain is encoded by SEQ ID NO: 84. In some embodiments, the CD28 costimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to, or identical to, the sequence of SEQ ID NO: 160. In some embodiments, the CD28 costimulatory domain is encoded by SEQ ID NO: 160.

[0155] In some embodiments, the costimulatory domain is isolated or derived from 4-1BB. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 161). In some embodiments, the 4-1BB costimulatory domain comprises or consists essentially of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 161). In some embodiments, the 4-1BB costimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGGCCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 162).

[0156] In some embodiments, the intracellular domain of the CAR comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain. In some embodiments, the intracellular domain of the CAR comprises the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 158), or a sequence with at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity thereto. In some embodiments, the intracellular domain of the CAR is encoded by SEQ ID NO: 159, or a sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity thereto. In some embodiments, the intracellular domain of the CAR is encoded by SEQ ID NO: 159.

[0157] The cytoplasmic domains within the cytoplasmic signaling portion of a CAR of the present disclosure can be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides an example of a suitable linker. An exemplary linker comprises the sequence GGGGSGGGGSGGGGSGG (SEQ ID NO: 146).

[0158] The cytoplasmic domains within the cytoplasmic signaling portion of a CAR of the present disclosure can be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides an example of a suitable linker.

[0159] T cell receptor (TCR) The present disclosure provides a first activator receptor and an immune cell comprising the same. In some embodiments, the first receptor is a T cell receptor (TCR).

[0160] Exemplary TCRs comprising an intracellular domain for use in the present disclosure are described in PCT / US2020 / 045250, filed September 6, 2020, the contents of which are incorporated herein by reference.

[0161] As used herein, "TCR," sometimes referred to as "TCR complex" or "TCR / CD3 complex," refers to a protein complex comprising one or more of the TCR alpha chain, TCR beta chain, and invariant CD3 chains (zeta, gamma, delta, and epsilon), sometimes referred to as subunits. The TCR alpha and beta chains may be disulfide-linked to function as a heterodimer that binds to peptide-MHC complexes. Engagement of the TCR alpha / beta heterodimer induces a conformational change in the TCR complex within the associated invariant CD3 subunit, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal. In an exemplary TCR complex, TCR alpha and TCR beta polypeptides form a heterodimer, CD3 epsilon and CD3 delta form a heterodimer, and CD3 epsilon and CD3 gamma for the heterodimer and two CD3 zetas form homodimers.

[0162] Any suitable ligand binding domain may be fused to the extracellular domain, hinge domain or transmembrane domain of a TCR as described herein. For example, the ligand binding domain may be an antibody or antigen binding domain of a TCR, or may comprise an antibody fragment, a Vβ-only domain, a linear antibody, a single chain variable fragment (scFv), or a single domain antibody (sdAb).

[0163] In some embodiments, the ligand-binding domain is fused to one or more extracellular or transmembrane domains of one or more TCR subunits. The TCR subunits may be TCR alpha, TCR beta, CD3 delta, CD3 epsilon, CD3 gamma, or CD3 zeta. For example, the ligand-binding domain may be fused to TCR alpha or TCR beta, or the ligand-binding portion may be fused to two subunits, e.g., the ligand-binding domain may be fused to both TCR alpha and TCR beta.

[0164] TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma, and CD3 epsilon. Any one or more of the TCR alpha, TCR beta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta, or fragments or derivatives thereof, can be fused to one or more domains capable of providing a stimulatory signal of the present disclosure, thereby enhancing TCR function and activity.

[0165] TCR transmembrane domains isolated or derived from any source are contemplated within the scope of this disclosure. The transmembrane domains may be derived from either natural or recombinant sources. If the source is natural, the domains may be derived from any membrane-bound or transmembrane protein.

[0166] In some embodiments, the transmembrane domain can transmit a signal to the intracellular domain whenever the TCR complex is bound to a target. A transmembrane domain of particular use can include, for example, at least the transmembrane region of the alpha, beta, or zeta chain of the TCR, CD3 delta, CD3 epsilon, or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0167] In some embodiments, the transmembrane domain can be connected to the extracellular region of a polypeptide of a TCR, e.g., the antigen-binding domain of a TCR alpha or beta chain, via a hinge, e.g., a hinge derived from a human protein. For example, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge. In some embodiments, the hinge is isolated from or derived from CD8α or CD28.

[0168] In some embodiments, the extracellular ligand-binding domain is linked to one or more transmembrane domains of a TCR. In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain, a TCR beta transmembrane domain, or both. In some embodiments, the transmembrane comprises a CD3 zeta transmembrane domain.

[0169] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 15 amino acids of the extracellular region), and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 15 amino acids of the intracellular region).

[0170] In some embodiments, transmembrane domains may be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0171] If present, the transmembrane domain may be a native TCR transmembrane domain, a native transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain may be a membrane anchor domain. Without limitation, a native or artificial transmembrane domain may often comprise a hydrophobic a-helix of about 20 amino acids, with a positive charge adjacent to the transmembrane segment. The transmembrane domain may have one transmembrane segment or more than one transmembrane segment. Predictions of transmembrane domains / segments may be made using published prediction tools (e.g., TMHMM, Krogh et al. Journal of Molecular Biology 2001;305(3):567-580, or TMpred, Hofmann & Stoffel Biol. Chem. Hoppe-Seyler 1993;347:166). Non-limiting examples of membrane anchor systems include the platelet-derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (post-translationally added to a signal sequence), and the like.

[0172] In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain. In some embodiments, the TCR alpha transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or is identical to the sequence VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 85). In some embodiments, the TCR alpha transmembrane domain comprises or consists essentially of SEQ ID NO: 85. In some embodiments, the TCR alpha transmembrane domain is encoded by the sequence GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG (SEQ ID NO: 86).

[0173] In some embodiments, the transmembrane domain comprises a TCR beta transmembrane domain. In some embodiments, the TCR beta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or is identical to the sequence of TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 87). In some embodiments, the TCR beta transmembrane domain comprises or consists essentially of SEQ ID NO: 87. In some embodiments, the TCR beta transmembrane domain is encoded by the sequence ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO: 88).

[0174] The TCRs of the present disclosure can comprise one or more intracellular domains. In some embodiments, the intracellular domain comprises one or more domains capable of providing a stimulatory signal to the transmembrane domain. In some embodiments, the intracellular domain comprises a first intracellular domain capable of providing a stimulatory signal and a second intracellular domain capable of providing a stimulatory signal. In other embodiments, the intracellular domain comprises first, second, and third intracellular domains capable of providing a stimulatory signal. The intracellular domain capable of providing a stimulatory signal is selected from the group consisting of a CD28 molecule (CD28) domain, an LCK proto-oncogene, a Src family tyrosine kinase (Lck) domain, a TNF receptor superfamily member 9 (4-1BB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, a Src family tyrosine kinase (Fyn) domain, a zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, a lymphocyte cytoplasmic protein 2 (SLP76) domain, (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, and a CD3 epsilon intracellular domain.

[0175] In some embodiments, the intracellular domain comprises at least one intracellular signaling domain. The intracellular signaling domain generates a signal that promotes immune effector function of a functional cell, e.g., a TCR-containing cell, e.g., a TCR-expressing T cell. In some embodiments, the intracellular domain of a first receptor of the present disclosure comprises at least one intracellular signaling domain. For example, the intracellular domain of CD3 gamma, delta, or epsilon comprises a signaling domain.

[0176] In some embodiments, the extracellular domain, transmembrane domain, and intracellular domain are isolated from or derived from the same protein, e.g., T cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, CD3 epsilon, or CD3 zeta.

[0177] Examples of intracellular domains for use in the activator receptors of the present disclosure include the cytoplasmic sequences of TCR alpha, TCR beta, CD3 zeta, and 4-1BB, and intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences having the same functional capabilities.

[0178] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from proteins responsible for primary, or antigen-dependent, stimulation.

[0179] 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.

[0180] In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain. In some embodiments, the TCR alpha intracellular domain comprises Ser-Ser. In some embodiments, the TCR alpha intracellular domain is encoded by the sequence TCCAGC.

[0181] In some embodiments, the intracellular domain comprises a TCR beta intracellular domain. In some embodiments, the TCR beta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, or identical to the sequence of MAMVKRKDSR (SEQ ID NO: 89). In some embodiments, the TCR beta intracellular domain comprises or consists essentially of SEQ ID NO: 89. In some embodiments, the TCR beta intracellular domain is encoded by the sequence ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 90).

[0182] In some embodiments, the intracellular signaling domain comprises at least one stimulatory intracellular domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma, or CD3 epsilon intracellular domain, and one additional stimulatory intracellular domain, e.g., a costimulatory domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma, or CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.

[0183] Exemplary costimulatory intracellular signaling domains include those derived from proteins responsible for costimulatory signals or antigen-independent stimulation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll ligand receptors, and DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137, TNF receptor superfamily member 9), and CD28 molecules (CD28). Costimulatory proteins may be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and NK cell activating receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83, a ligand that specifically binds to CD4, etc. A costimulatory domain can comprise the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional variant thereof.

[0184] In some embodiments, the stimulatory domain comprises a costimulatory domain. In some embodiments, the costimulatory domain comprises a CD28 or 4-1BB costimulatory domain. CD28 and 4-1BB are well-characterized costimulatory molecules required for full T cell activation and are known to enhance T cell effector function. For example, CD28 and 4-1BB have been utilized in chimeric antigen receptors (CARs) to enhance cytokine release, cytolytic function, and persistence over first-generation CARs containing only the CD3 zeta signaling domain. Similarly, including a costimulatory domain, e.g., a CD28 and 4-1BB domain, in a TCR can increase T cell effector function and specifically enable costimulation in the absence of costimulatory ligands that are typically downregulated on the surface of tumor cells. In some embodiments, the stimulatory domain comprises a CD28 intracellular domain or a 4-1BB intracellular domain.

[0185] inhibitory receptors The present disclosure provides a second receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is lost in cancer cells, e.g., an allelic variant of a gene. The non-target allelic variant may be lost in cancer cells through any mechanism, including, but not limited to, epigenetic changes affecting the expression of the non-target allelic variant, mutations in the gene encoding the non-target allelic variant, disruption of cell signaling regulating the expression of the non-target allelic variant, chromosomal loss, partial or complete deletion of a genomic locus, gene silencing due to nucleic acid or heterochromatin modifications, or loss of expression due to other mechanisms. In variations of the compositions and methods disclosed herein, treated cells or subjects may exhibit loss of expression of the non-target allelic variant due to non-genetic changes. Thus, the present disclosure provides compositions and methods for killing cells and / or treating subjects lacking expression of the non-target antigen due to any cause, including, but not limited to, loss of heterozygosity.

[0186] The non-target antigen may be a protein in complex with major histocompatibility complex class I (MHC-I) or an antigenic peptide thereof, and the non-target antigen may include polymorphisms. Because the non-target antigen is polymorphic, loss of a single copy of the gene encoding the non-target antigen, which can occur through loss of heterozygosity in cancer cells, results in cancer cells that retain other polymorphic variants of the gene but have lost the non-target antigen. For example, a subject with HLA-A*02 and HLA-A*01 alleles at the HLA locus may have a cancer in which only the HLA-A*02 allele is lost. In such a subject, the HLA-A*01 protein is still present, but is not recognized by the inhibitory receptors of immune cells that encounter cancer cells because the inhibitory receptor is engineered to be specific for HLA-A*02 (or other non-target antigen). In normal, non-malignant cells, HLA-A*02 (or other non-target antigen) is present and inhibits the activation of the engineered immune cells. In cancer cells with loss of heterozygosity, the HLA-A*02 allele variant (or other non-target antigen) is lost. Because inhibitory receptors respond only to HLA-A*02 (or other non-target antigens) that are not present in cancer cells, immune cells engineered to express inhibitory receptors do not receive inhibitory signals from the inhibitory receptors. Through this mechanism, immune cells are selectively activated and selectively kill cancer cells that express CEA but have lost HLA-A*02 (or another non-target antigen) due to loss of heterozygosity. Herein, HLA-A is used as an example. Similar polymorphic variations occur in other MHC gene groups and other non-MHC genes. Thus, the present disclosure provides a second receptor comprising an extracellular ligand-binding domain specific for a non-target antigen selected from TNFRSF11A, ACHRB, ITGAE, TRPV1, and SREC, or antigenic peptides thereof, in a complex with major histocompatibility complex class I (MHC-I), wherein the non-target antigen comprises a polymorphism, and an immune cell comprising the second receptor.

[0187] In some embodiments, the second receptor is an inhibitory chimeric antigen receptor (inhibitory receptor).

[0188] In some embodiments, the second receptor is an inhibitory receptor. In some embodiments, the second receptor is humanized.

[0189] In some embodiments, the second receptor comprises SEQ ID NO: 164, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, 174, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0190] The present disclosure provides a second receptor, which is an inhibitory receptor, comprising extracellular ligand binding and capable of distinguishing between single amino acid variant alleles of non-target antigens.This ability to distinguish between allelic variants of non-target antigens allows the second receptor to suppress the activation of immune cells comprising the second receptor in the presence of non-target cells expressing the allele recognized by the ligand binding domain.However, the activation of immune cells is not suppressed in the presence of target cells that have lost the allele, for example, cancer cells that have lost one allele of a gene due to loss of heterozygosity.

[0191] The present disclosure provides a second receptor that is an inhibitory receptor, comprising extracellular ligand binding that can distinguish between different levels of expression of a non-target antigen, thereby allowing the second receptor to suppress activation of an immune cell that contains the second receptor in the presence of non-target cells that express a ligand for the second receptor, but allows activation of the immune cell in the presence of cancer cells that express low levels or no ligand for the second receptor.

[0192] Repressor Ligand In some embodiments, the non-target antigen is not expressed by target cells but is expressed by non-target cells. In some embodiments, the non-target antigen is expressed by healthy cells, i.e., cells that are not cancer cells. In some embodiments, the target cells are a plurality of cancer cells that have lost expression of the non-target antigen through loss of heterozygosity (LOH). In some embodiments, the non-target cells are a plurality of healthy cells (i.e., non-cancer cells, normal cells, or healthy cells) that express both the target and non-target antigens.

[0193] Any cell surface molecule expressed by a non-target cell that is not expressed by a target cell can be a suitable non-target antigen for the extracellular ligand-binding domain of a second receptor. For example, a cell adhesion molecule, an intercellular signaling molecule, an extracellular domain, a molecule involved in chemotaxis, a glycoprotein, a G protein-coupled receptor, a transmembrane receptor, a receptor for a neurotransmitter, or a voltage-gated ion channel can be used as a non-target antigen.

[0194] In some embodiments, the non-target antigen is selected from the group consisting of polymorphic variants of TNFRSF11A, ACHRB, ITGAE, TRPV1, and SREC. In some embodiments, the non-target antigen is an antigenic peptide that includes a polymorphic residue of TNFRSF11A, ACHRB, ITGAE, TRPV1, or SREC in complex with major histocompatibility complex class I (MHC-I).

[0195] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in complex with major histocompatibility complex class I (MHC-I).

[0196] Non-target MHC-1 (pMHC) antigens, including any of HLA-A, HLA-B, HLA-C, or HLA-E, are contemplated within the scope of this disclosure.

[0197] In some embodiments, the non-target antigen comprises a major histocompatibility complex (MHC) protein. In some embodiments, the MHC is MHC class I. In some embodiments, the MHC class I protein comprises a human leukocyte antigen (HLA) protein. In some embodiments, the non-target antigen comprises an allele of an HLA class I protein selected from the group consisting of HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the HLA-A allele comprises HLA-A*01, HLA-A*02, HLA-A*03, or HLA-A*11. In some embodiments, the HLA-B allele comprises HLA-B*07. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0198] In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the non-target antigen comprises an allele of HLA-A. In some embodiments, the allele of HLA-A comprises HLA-A*01, HLA-A*02, HLA-A*03, or HLA-A*11. In some embodiments, the non-target antigen comprises HLA-A*69.

[0199] In some embodiments, the non-target antigen comprises the allele HLA-B. In some embodiments, the allele of HLA-B comprises HLA-B*11.

[0200] In some embodiments, the non-target antigen comprises an allele of HLA-C. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0201] In some embodiments, the non-target antigen is selected from the group consisting of TNFRSF11A, ACHRB, ITGAE, TRPV1, and SREC. CEA and TNFRSF11A (RANK) are low / absent in T cells, thus avoiding in cis challenge with other ligands. The LOH frequency for the TNFRSF11A locus is very high (approximately 90% in rectal cancer).

[0202] In some embodiments, the non-target antigen comprises TNFRSF11A or an antigenic peptide thereof in a complex with MHC-I. Human TNFRSF11A is located at Chr18q:35,237,593-37,208,541 and is frequently lost via loss of heterozygosity in colorectal cancer cells.

[0203] Wild-type human TNFRSF11A isoform 1 is described in NCBI accession number NP_003830.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, TNFRSF11A is [ka] It contains the amino acid sequence of In some embodiments, TNFRSF11A comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 13. Polymorphic residues of TNFRSF11A are marked as bold and underlined in SEQ ID NO: 13.

[0204] In some embodiments, the non-target antigen comprises a polymorphism of TNFRSF11A. For example, the non-target antigen comprises a peptide derived from TNFRSF11A that includes a polymorphic residue of TNFRSF11A. The polymorphic residue of TNFRSF11A includes amino acid residues 141 and 192 of SEQ ID NO: 13. In some embodiments, the non-target antigen comprises a peptide of TNFRSF11A that includes amino acid 141 (rs35211496, H141Y) or 192 (rs1805034, V192A) of SEQ ID NO: 13.

[0205] In some embodiments, the polymorphism of TNFRSF11A comprises the H141 / A192V allele of TNFRSF11A. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 229).

[0206] In some embodiments, the polymorphism in TNFRSF11A comprises the H141Y / A192 allele of TNFRSF11A. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 230).

[0207] In some embodiments, the polymorphism in TNFRSF11A comprises the H141Y / A192V allele of TNFRSF11A. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 231).

[0208] In some embodiments, the non-target antigen comprises a TNFRSF11A polymorphism having an A at position 192 of SEQ ID NO: 13, and the second receptor comprises a ligand-binding domain with higher affinity for a TNFRSF11A ligand having an A at position 192 of SEQ ID NO: 13 than for a TNFRSF11A ligand having a V at position 192 of SEQ ID NO: 13. In some embodiments, the non-target antigen comprises a TNFRSF11A polymorphism having a V at position 192 of SEQ ID NO: 13, and the second receptor comprises a ligand-binding domain with higher affinity for a TNFRSF11A ligand having a V at position 192 of SEQ ID NO: 13 than for a TNFRSF11A ligand having an A at position 192 of SEQ ID NO: 13. In some embodiments, the non-target antigen comprises a TNFRSF11A polymorphism having an H at position 141 of SEQ ID NO: 13, and the second receptor comprises a ligand-binding domain with higher affinity for a TNFRSF11A ligand having an H at position 141 of SEQ ID NO: 13 than for a TNFRSF11A ligand having a Y at position 141 of SEQ ID NO: 13. In some embodiments, the non-target antigen comprises a TNFRSF11A polymorphism having a Y at position 141 of SEQ ID NO: 13, and the second receptor comprises a ligand-binding domain with higher affinity for a TNFRSF11A ligand having a Y at position 141 of SEQ ID NO: 13 than for a TNFRSF11A ligand having an H at position 141 of SEQ ID NO: 13.

[0209] Mouse TNFRSF11A isoform 1 is described in NCBI accession number AH19185.1, the contents of which are incorporated by reference in their entirety. In some embodiments, TNFRSF11A is [ka] It contains the amino acid sequence of In some embodiments, TNFRSF11A comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 32. Polymorphic residues of TNFRSF11A are marked as bold and underlined in SEQ ID NO: 32.

[0210] In some embodiments, the non-target antigen comprises a polymorphism of TNFRSF11A. The polymorphic residues of TNFRSF11A include positions 142 and 193 of SEQ ID NO: 32. In some embodiments, the non-target antigen comprises a peptide of TNFRSF11A comprising amino acid 142 or 193 of SEQ ID NO: 32.

[0211] In some embodiments, the non-target antigen comprises integrin alpha-E (ITGAE) or an antigenic peptide thereof in complex with MHC-I. ITGAE contains two polymorphisms in the extracellular domain: R950W (rs1716) with a minor allele frequency (MAF) of 0.2654 and V1019A / V1019G (rs2976230) with a MAF of 0.282.

[0212] Human ITGAE (R950 / V10109) is described in NCBI accession number NP_002199.3, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the ITGAE comprises the following amino acid sequence: [ka] In some embodiments, ITGAE comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 14. Polymorphic residues of ITGAE are marked as bold and underlined in SEQ ID NO: 14.

[0213] In some embodiments, the ITGAE polymorphism comprises the R950W / V1019 allele of ITGAE. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 232).

[0214] In some embodiments, the ITGAE polymorphism comprises the R950 / V1019A allele of ITGAE. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 233).

[0215] In some embodiments, the ITGAE polymorphism comprises the R950 / V1019G allele of ITGAE. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 234).

[0216] In some embodiments, the ITGAE polymorphism comprises the R950W / V1019 allele of ITGAE. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 235).

[0217] In some embodiments, the polymorphism in ITGAE comprises the R950W / V1019A allele of ITGAE. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 236).

[0218] In some embodiments, the ITGAE polymorphism comprises the R950W / V1019G allele of ITGAE. [ka] (Polymorphic amino acids are bold and underlined) (SEQ ID NO: 237).

[0219] In some embodiments, the non-target antigen comprises a polymorphism of ITGAE. For example, the non-target antigen comprises a peptide derived from ITGAE that includes a polymorphic residue of ITGAE. The polymorphic residue of ITGAE includes amino acids 950 and 1019 of SEQ ID NO: 14. In some embodiments, the non-target antigen comprises a peptide of ITGAE that includes amino acid 950 or 1019 of SEQ ID NO: 14.

[0220] In some embodiments, the non-target antigen comprises an ITGAE polymorphism having an R at position 950 of SEQ ID NO: 14, and the second receptor comprises a ligand binding domain with higher affinity for an ITGAE ligand having an R at position 950 of SEQ ID NO: 14 than for an ITGAE ligand having a W at position 950 of SEQ ID NO: 14. In some embodiments, the non-target antigen comprises an ITGAE polymorphism having a W at position 950 of SEQ ID NO: 14, and the second receptor comprises a ligand binding domain with higher affinity for an ITGAE ligand having a W at position 950 of SEQ ID NO: 14 than for an ITGAE ligand having an R at position 950 of SEQ ID NO: 14. In some embodiments, the non-target antigen comprises an ITGAE polymorphism having a V at position 1019 of SEQ ID NO: 14, and the second receptor comprises a ligand binding domain with higher affinity for an ITGAE ligand having a V at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand having an A or G at position 1019 of SEQ ID NO: 14. In some embodiments, the non-target antigen comprises an ITGAE polymorphism having an A at position 1019 of SEQ ID NO: 14, and the second receptor comprises a ligand-binding domain with higher affinity for an ITGAE ligand having an A at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand having a V or G at position 1019 of SEQ ID NO: 14. In some embodiments, the non-target antigen comprises an ITGAE polymorphism having a G at position 1019 of SEQ ID NO: 14, and the second receptor comprises a ligand-binding domain with higher affinity for an ITGAE ligand having a G at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand having a V or A at position 1019 of SEQ ID NO: 14.

[0221] In some embodiments, the non-target antigen comprises ACHRB (also called CHRNB or CHRNB1) in complex with MHC-I, or an antigenic peptide thereof. Human ACHRB is described in NCBI accession number NP_000738.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, ACHRB comprises the following amino acid sequence: [ka] In some embodiments, ACHRB comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 33. Polymorphic residues of ACHRB are marked as bold and underlined in SEQ ID NO: 33.

[0222] In some embodiments, the non-target antigen comprises a polymorphism of ACHRB. For example, the non-target antigen comprises a peptide derived from ACHRB that comprises a polymorphic residue of ACHRB. The polymorphic residue of ACHRB comprises 32 of SEQ ID NO: 33. In some embodiments, the non-target antigen comprises a peptide of ACHRB that comprises amino acid 32 of SEQ ID NO: 33. In some embodiments, the non-target antigen comprises a peptide of ACHRB that comprises E at amino acid 32 of SEQ ID NO: 33. In some embodiments, the non-target antigen comprises a peptide of ACHRB that comprises G at amino acid 32 of SEQ ID NO: 33.

[0223] In some embodiments, the non-target antigen comprises TRPV1 or an antigenic peptide thereof in complex with MHC-I. Human TRPV1 is described in NCBI accession number NP_542435.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, TRPV1 is [ka] (SEQ ID NO: 34) In some embodiments, TRPV1 comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 94%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 34. Polymorphic residues of TRPV1 are marked as bold and underlined in SEQ ID NO: 34.

[0224] In some embodiments, the non-target antigen comprises a polymorphism of TRPV1. For example, the non-target antigen comprises a peptide derived from TRPV1 that comprises a polymorphic residue of TRPV1. The polymorphic residues of TRPV1 include positions 585, 459, and 469 of SEQ ID NO: 34. In some embodiments, the non-target antigen comprises a peptide of TRPV1 that comprises amino acid 585, 459, or 469 of SEQ ID NO: 34. In some embodiments, the non-target antigen comprises a peptide of TRPV1 that comprises an I at amino acid 585 of SEQ ID NO: 34. In some embodiments, the non-target antigen comprises a peptide of TRPV1 that comprises a V at amino acid 585 of SEQ ID NO: 34.

[0225] In some embodiments, the non-target antigen comprises an SREC or an antigenic peptide thereof in a complex with MHC-I. Human SREC isoform 1 is described in NCBI accession number NP_003684.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the SREC comprises the following amino acid sequence: [ka] In some embodiments, the SREC comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 94%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 35. Polymorphic residues of SREC are marked as bold and underlined in SEQ ID NO: 35.

[0226] In some embodiments, the non-target antigen comprises a polymorphism of an SREC. For example, the non-target antigen comprises a peptide derived from an SREC that comprises a polymorphic residue of an SREC. The polymorphic residue of an SREC comprises positions 339 and 425 of SEQ ID NO: 35. In some embodiments, the non-target antigen comprises a peptide of an SREC that comprises amino acid 339 or 425 of SEQ ID NO: 35. In some embodiments, the non-target antigen comprises a peptide of an SREC that comprises an A at amino acid 425 of SEQ ID NO: 35. In some embodiments, the non-target antigen comprises a peptide of an SREC that comprises a V at amino acid 425 of SEQ ID NO: 35.

[0227] In some embodiments, the non-target antigen comprises a C-X-C motif chemokine ligand 16 (CXCL16) or an antigenic peptide thereof in complex with MHC-I. Human CXCL16 precursor is described in NCBI accession number NP_001094282.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, CXCL16 is [ka] It contains the amino acid sequence of

[0228] In some embodiments, the non-target antigen comprises a polymorphism of CXCL16. For example, the non-target antigen comprises a peptide derived from CXCL16 that comprises a polymorphic residue of CXCL16. The polymorphic residue of CXCL16 comprises positions 142 and 200 of SEQ ID NO: 136. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises amino acid 142 or 200 of SEQ ID NO: 136. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises an A at amino acid 200 of SEQ ID NO: 136. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises a V at amino acid 200 of SEQ ID NO: 136. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises an I at amino acid 142 of SEQ ID NO: 136. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises a T at amino acid 142 of SEQ ID NO: 136.

[0229] In some embodiments, the non-target antigen comprises collectin subfamily member 12 (COLEC12) or an antigenic peptide thereof in complex with MHC-I. Human COLEC12 is described in NCBI accession number NP_569057.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, COLEC12 is [ka] It contains the amino acid sequence of

[0230] In some embodiments, COLEC12 comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 94%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 137. Polymorphic residues in COLEC12 are marked as bold and underlined in SEQ ID NO: 137.

[0231] In some embodiments, the non-target antigen comprises a polymorphism of COLEC12. For example, the non-target antigen comprises a peptide derived from COLEC12 that comprises a polymorphic residue of COLEC12. The polymorphic residue of COLEC12 comprises position 522 of SEQ ID NO: 137. In some embodiments, the non-target antigen comprises a peptide of COLEC12 that comprises amino acid 522 of SEQ ID NO: 137. In some embodiments, the non-target antigen comprises a peptide of COLEC12 that comprises an S at amino acid 522 of SEQ ID NO: 137. In some embodiments, the non-target antigen comprises a peptide of COLEC12 that comprises a P at amino acid 522 of SEQ ID NO: 137.

[0232] In some embodiments, the non-target antigen comprises APC downregulation 1 (APCDD1) or an antigenic peptide thereof in complex with MHC-I. An exemplary human APCDD1 is described in UniProtKB accession number Q8J025, the contents of which are incorporated herein by reference in their entirety. In some embodiments, APCDD1 is [ka] It contains the amino acid sequence of

[0233] In some embodiments, the non-target antigen comprises a polymorphism of APCDD1. Exemplary polymorphisms of APCDD1 include rs3748415, which can be V, I, or L at position 150 of SEQ ID NO: 138. In some embodiments, the non-target antigen comprises a peptide of APCDD1 comprising amino acid 150 of SEQ ID NO: 138. In some embodiments, the non-target antigen comprises a peptide of APCDD1 comprising V at amino acid 150 of SEQ ID NO: 138. In some embodiments, the non-target antigen comprises a peptide of APCDD1 comprising I at amino acid 150 of SEQ ID NO: 138. In some embodiments, the non-target antigen comprises a peptide of APCDD1 comprising L at amino acid 150 of SEQ ID NO: 138.

[0234] Further exemplary human APCDD1 is described in UniProtKB Accession No. V9GY82, the contents of which are incorporated herein by reference in their entirety. In some embodiments, APCDD1 is [ka] It contains the amino acid sequence of

[0235] Exemplary polymorphisms of APCDD1 include rs1786683, which can be Y or S at position 165 of SEQ ID NO: 139. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes amino acid 165 of SEQ ID NO: 139. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes a Y at amino acid 165 of SEQ ID NO: 139. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes an S at amino acid 165 of SEQ ID NO: 139.

[0236] Further exemplary human APCDD1 is described in UniProt accession number J3QSE3, the contents of which are incorporated herein by reference in their entirety. In some embodiments, APCDD1 is [ka] It contains the amino acid sequence of

[0237] Exemplary polymorphisms of APCDD1 include rs9952598, which can be Q or R at position 28 of SEQ ID NO: 140. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes amino acid 28 of SEQ ID NO: 140. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes Q at amino acid 28 of SEQ ID NO: 140. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes R at amino acid 28 of SEQ ID NO: 140.

[0238] In some embodiments, APCDD1 comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 94%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 138-140. Polymorphic residues of APCDD1 are marked as bold and underlined in SEQ ID NOs: 138-140.

[0239] In some embodiments, the non-target antigen comprises HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07. Various single variable domains that bind to or recognize specific HLA alleles for use in the embodiments described herein are set forth in Table 5. Such scFvs include, but are not limited to, the following murine and humanized scFv antibodies that bind to HLA alleles in a peptide-independent manner as shown in Table 5 below (complementarity-determining regions are underlined): [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9]

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

Table 5-26

Table 5-27

Table 5-28

Table 5-29

Table 5-30

Table 5-31

Table 5-32

Table 5-33

Table 5-34

Table 5-35

Table 5-36

Table 5-37

Table 5-38

[0240] In some embodiments, the ligand-binding domain of the second inhibitory receptor comprises an scFv. In some embodiments, the scFv binds to HLA-A*01, HLA-A*02, HLA-A*3, HLA-A*11, HLA-B*07, or HLA-C*07 and comprises a sequence selected from the group of sequences set forth in SEQ ID NOs: 91-102, 250-260, 262, 264, 266, 268, 270, 272, 274, 276, and 278-345, or Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the scFv binds to HLA-A*01, HLA-A*02, HLA-A*3, HLA-A*11, HLA-B*07, or HLA-C*07 and comprises a sequence selected from the group of sequences set forth in Table 5. In some embodiments, the non-target antigen comprises HLA-A*01, and the non-target extracellular ligand-binding domain of the second receptor comprises an HLA-A*01 scFv sequence comprising SEQ ID NOs: 337-345 set forth in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-A*02, and the non-target extracellular ligand-binding domain of the second receptor comprises an HLA-A*02 scFv sequence comprising SEQ ID NOs: 91-102 in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-A*03, and the non-target extracellular ligand-binding domain of the second receptor comprises an HLA-A*03 scFv sequence comprising SEQ ID NOs: 323-336 in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.In some embodiments, the non-target antigen comprises HLA-A*11, and the non-target extracellular ligand-binding domain of the second receptor comprises an HLA-A*11 scFv sequence comprising SEQ ID NO: 260, 262, 264, 266, 268, 270, 272, 274, or 276 in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-B*07, and the non-target extracellular ligand-binding domain of the second receptor comprises an HLA-B*07 scFv sequence comprising SEQ ID NO: 250-259 in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. In some embodiments, the non-target antigen comprises HLA-C*07, and the non-target extracellular ligand-binding domain of the second receptor comprises an HLA-C*07 scFv sequence comprising SEQ ID NOs: 278-322 set forth in Table 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0241] Exemplary heavy and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3, respectively) for the HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, and HLA-C*07 ligand binding domains are shown in Table 6 below. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

[0242] In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the ligand-binding domain of the second inhibitory receptor comprises an HLA-A*01, HLA-A*02, HLA-A*03, or HLA-A*11 ligand-binding domain comprising the CDR sequences set forth in Table 6.

[0243] In some embodiments, the non-target antigen comprises HLA-B. In some embodiments, the ligand-binding domain of the second inhibitory receptor comprises an HLA-B*07 ligand-binding domain comprising the CDR sequences set forth in Table 6.

[0244] In some embodiments, the non-target antigen comprises HLA-C. In some embodiments, the ligand-binding domain of the second inhibitory receptor comprises an HLA-C*07 ligand-binding domain comprising the CDR sequences set forth in Table 6.

[0245] In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to an allelic variant of an HLA-A, HLA-B, or HLA-C protein, hi some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07.

[0246] In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A*01. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises HLA-A*01 complementarity-determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the HLA-A*01 CDRs of Table 6.

[0247] In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A*02. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises HLA-A*02 complementarity-determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the HLA-A*02 CDRs of Table 6.

[0248] In some embodiments, the extracellular ligand-binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 103-108 or 109-114, or CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the CDRs of SEQ ID NOs: 103-108 or 109-114.

[0249] In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A*03. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises HLA-A*03 complementarity-determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the HLA-A*03 CDRs of Table 6.

[0250] In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-A*11. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises HLA-A*11 complementarity-determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the HLA-A*11 CDRs of Table 6.

[0251] In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-B*07. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises HLA-B*07 complementarity-determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the HLA-B*07 CDRs of Table 6.

[0252] In some embodiments, the extracellular ligand-binding domain of the second receptor specifically binds to HLA-C*07. In some embodiments, the extracellular ligand-binding domain of the second receptor comprises HLA-C*07 complementarity-determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed in Table 6, or CDR sequences having up to 1, 2, or 3 substitutions, deletions, or insertions compared to the HLA-C*07 CDRs of Table 6.

[0253] In further embodiments of any of the ligand-binding domains, each CDR sequence may have one, two, three or more substitutions, insertions, or deletions. The CDR sequences may tolerate substitutions, deletions, or insertions. Using sequence alignment tools, routine experimentation, and known assays, one skilled in the art can generate and test variant sequences with one, two, three or more substitutions, insertions, or deletions in the CDR sequences without undue experimentation.

[0254] In some embodiments, the non-target antigen comprising HLA-A*02 and the ligand-binding domain of the second receptor comprise an HLA-A*02 ligand-binding domain. In some embodiments, the ligand-binding domain binds to HLA-A*02 independently of the peptide in a pMHC complex comprising HLA-A*02. In some embodiments, the HLA-A*02 ligand-binding domain comprises an scFv domain. In some embodiments, the HLA-A*02 ligand-binding domain comprises the sequence of any one of SEQ ID NOs: 91-102. In some embodiments, the HLA-A*02 ligand-binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of SEQ ID NOs: 91-102.

[0255] In some embodiments, the HLA-A*02 scFv comprises the complementarity determining regions (CDRs) of any one of SEQ ID NOs: 103 to 114. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOs: 103 to 114. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOs: 103 to 114. In some embodiments, the heavy chain of the antigen-binding domain comprises the heavy chain CDRs of any one of SEQ ID NOs: 103 to 114, and the light chain of the antigen-binding domain comprises the light chain CDRs of any one of SEQ ID NOs: 103 to 114. In some embodiments, the HLA-A*02 antigen-binding domain comprises a heavy chain and a light chain, wherein the heavy chain comprises a CDR selected from SEQ ID NOs: 106 to 108 and 112 to 14, and the light chain comprises a CDR selected from SEQ ID NOs: 103 to 15 and 109 to 111.

[0256] In some embodiments, the HLA-A*02 antigen-binding domain comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 91-102, and the light chain comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOs: 91-102.

[0257] In some embodiments, the heavy chain comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 91-102, and the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 91-102.

[0258] In some embodiments, the HLA-A*02 scFv comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to any one of SEQ ID NOs: 91-102. In some embodiments, the HLA-A*02 scFv comprises a sequence that is identical to any one of SEQ ID NOs: 91-102.

[0259] In some embodiments, the non-target antigen comprises HLA-A*01 and the extracellular ligand-binding domain of the second receptor comprises an HLA-A*01 ligand-binding domain. In some embodiments, the HLA-A*1 ligand-binding domain comprises an scFv domain comprising a sequence selected from the group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-A*01 scFv comprises an HLA-A*1 CDR sequence set forth in Table 6.

[0260] In some embodiments, the non-target antigen comprises HLA-A*03 and the extracellular ligand-binding domain of the second receptor comprises an HLA-A*03 ligand-binding domain. In some embodiments, the HLA-A*03 ligand-binding domain comprises an scFv domain comprising a sequence selected from the group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-A*03 scFv comprises an HLA-A*03 CDR sequence set forth in Table 6.

[0261] In some embodiments, the non-target antigen comprises HLA-A*111 and the extracellular ligand-binding domain of the second receptor comprises an HLA-A*11 ligand-binding domain. In some embodiments, the HLA-A*11 ligand-binding domain comprises an scFv domain comprising a sequence selected from the group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-A*11 scFv comprises an HLA-A*11 CDR sequence set forth in Table 6.

[0262] In some embodiments, the non-target antigen comprises HLA-B*07 and the extracellular ligand-binding domain of the second receptor comprises an HLA-B*07 ligand-binding domain. In some embodiments, the HLA-B*07 ligand-binding domain comprises an scFv domain comprising a sequence selected from the group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-B*07 scFv comprises an HLA-B*07 CDR sequence set forth in Table 6.

[0263] In some embodiments, the non-target antigen comprises HLA-C*07 and the extracellular ligand-binding domain of the second receptor comprises an HLA-C*07 ligand-binding domain. In some embodiments, the HLA-C*07 ligand-binding domain comprises an scFv domain comprising a sequence selected from the group of sequences set forth in Table 5, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the HLA-C*07 scFv comprises an HLA-C*07 CDR sequence set forth in Table 6.

[0264] inhibitory receptors The present disclosure provides a second receptor that is an inhibitory chimeric antigen receptor. The inhibitory receptor may comprise an extracellular ligand-binding domain that binds to and recognizes a non-target antigen or a peptide derivative thereof in the MHC-I complex.

[0265] Exemplary inhibitory receptors are described in PCT / US2020 / 045228, filed September 6, 2020, PCT / US2020 / 064607, filed December 11, 2020, PCT / US2021 / 029907, filed April 29, 2021, and PCT / US2020 / 059856, filed November 10, 2020, the contents of each of which are incorporated herein by reference.

[0266] As used herein, the term "inhibitory receptor" refers to a ligand-binding domain fused to an intracellular signaling domain that can transduce an inhibitory signal that inhibits or suppresses the immune activity of an immune cell. Inhibitory receptors have immune cell inhibitory potential and are distinct from and distinguishable from CARs, which are receptors that have immune cell activation potential. For example, CARs contain an intracellular stimulatory domain and / or a costimulatory domain, making them activating receptors. Inhibitory receptors are inhibitory receptors that contain an intracellular inhibitory domain.

[0267] As used herein, an "inhibitory signal" refers to a signal transduction or change in protein expression in an immune cell that results in the suppression of an immune response (e.g., decreased cytokine production or decreased immune cell activation). The inhibition or suppression of an immune cell can be selective and / or reversible, or it is not selective and / or reversible. An inhibitory receptor responds to a non-target antigen (e.g., HLA-A*02). For example, when a non-target antigen (e.g., HLA-A*02) binds to or contacts the inhibitory receptor, the inhibitory receptor is responsive and activates an inhibitory signal in an immune cell expressing the inhibitory receptor upon binding of the non-target antigen by the extracellular ligand-binding domain of the inhibitory receptor.

[0268] Inhibitory receptors of the present disclosure can include an extracellular ligand-binding domain. Any type of ligand-binding domain that can regulate the activity of the receptor in a ligand-dependent manner is contemplated within the scope of the present disclosure.

[0269] In some embodiments, the ligand-binding domain is an antigen-binding domain. Exemplary antigen-binding domains include, inter alia, scFvs, SdAbs, Vβ-only domains, and TCR antigen-binding domains derived from TCR α and β chain variable domains.

[0270] Any type of antigen binding domain is contemplated within the scope of this disclosure.

[0271] In some embodiments, the extracellular ligand-binding domain of the second receptor is an scFv.

[0272] In some embodiments, the extracellular ligand-binding domain of the second receptor is fused to the extracellular domain of the inhibitory CAR.

[0273] In some embodiments, an inhibitory receptor of the present disclosure comprises an extracellular hinge region. Exemplary hinges can be isolated or derived from IgD and CD8 domains, e.g., IgG1. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0274] Inhibitory receptors of the present disclosure can be designed to include a transmembrane domain fused to the extracellular domain of the inhibitory receptor. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to transmembrane domains of the same or different surface membrane proteins and to minimize interactions with other members of the receptor complex.

[0275] The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region can be isolated from or derived from (i.e., comprising at least the transmembrane region 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, CD154, or an immunoglobulin such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular domain of the inhibitory receptor. A glycine-serine doublet provides a particularly suitable linker.

[0276] The present disclosure provides inhibitory receptors comprising an intracellular domain. The intracellular domain of the inhibitory receptor of the present disclosure is responsible for suppressing activation of immune cells comprising the inhibitory receptor that would otherwise be activated in response to an activating signal from a first receptor. In some embodiments, the inhibitory intracellular domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory intracellular domain comprising an ITIM can be isolated or derived from immune checkpoint inhibitors such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immunoinhibitory receptors expressed on the surface of T cells and play important roles in dampening or terminating T cell responses.

[0277] In some embodiments, the inhibitory intracellular domain is isolated from the human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and CD200 receptor 1. In some embodiments, the TRAIL receptor comprises TR10A, TR10B, or TR10D.

[0278] In some embodiments, the inhibitory intracellular domain is isolated from a phosphoprotein membrane anchor having glycosphingolipid microdomain 1 (PAG1). In some embodiments, the inhibitory intracellular domain is isolated from leukocyte immunoglobulin-like receptor B1 (LILRB1).

[0279] In some embodiments, the inhibitory domain is isolated from or derived from a human protein, for example, a human TRAIL receptor, CTLA-4, PD-1, PAG1, or LILRB1 protein.

[0280] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region, or a combination thereof.

[0281] In some embodiments, the inhibitory domain is isolated from or derived from killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin-like receptor B1 (LIR1, also known as LIR-1 and LILRB1), programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin-like receptor K1 (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N- and C-terminal SH2 domains), or ZAP70 KI_K369A (kinase-inactive ZAP70).

[0282] In some embodiments, the inhibitory domain is isolated or derived from a human protein.

[0283] In some embodiments, the second inhibitory receptor comprises an inhibitory domain. In some embodiments, the second inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of the inhibitory receptor. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of the inhibitory receptor.

[0284] In some embodiments, the second inhibitory receptor comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, or portions thereof, isolated or derived from the same protein, e.g., an ITIM-containing protein. In some embodiments, the second inhibitory receptor comprises an intracellular domain and / or a transmembrane domain and a hinge region, isolated or derived from the same protein, e.g., an ITIM-containing protein.

[0285] In some embodiments, the second receptor is a TCR comprising an inhibitory domain (inhibitory TCR). In some embodiments, the inhibitory TCR comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma, or CD3 epsilon, or a portion thereof. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma, or CD3 epsilon.

[0286] In some embodiments, the second receptor is a TCR that includes an inhibitory domain (an inhibitory TCR). In some embodiments, the inhibitory domain is isolated from or derived from LILRB1.

[0287] LILRB1 inhibitory receptor The present disclosure provides a second inhibitory receptor comprising a LILRB1 inhibitory domain and, optionally, a LILRB1 transmembrane domain and / or hinge domain, or a functional variant thereof. By including the LILRB1 transmembrane domain and / or LILRB1 hinge domain in an inhibitory receptor, the inhibitory signal generated by the inhibitory receptor can be increased compared to a reference inhibitory receptor having a different transmembrane domain or a different hinge domain. The second inhibitory receptor comprising the LILRB1 inhibitory domain may be a CAR or TCR as described herein. Any suitable ligand-binding domain as described herein can be fused to the LILRB1-based second inhibitory receptor.

[0288] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), also known as leukocyte immunoglobulin-like receptor B1, as well as ILT2, LIR1, MIR7, PIRB, CD85J, ILT-2, LIR-1, MIR-7, and PIR-B, are members of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1 protein belongs to the subfamily B class of LIR receptors. These receptors contain two to four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces negative signals that inhibit stimulation of the immune response. LILRB1 is thought to play a role in regulating inflammatory responses and cytotoxicity and limiting autoreactivity. Multiple transcriptional variants encoding different isoforms of LILRB1 exist, all of which are contemplated within the scope of the present disclosure.

[0289] 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 receptors having one or more domains isolated or derived from LILRB1, one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or identical to the sequence or subsequence of SEQ ID NO: 115. In some embodiments, one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or identical to the sequence or subsequence of SEQ ID NO: 115. In some embodiments, one or more domains of LILRB1 consist of an amino acid sequence identical to the sequence or subsequence of SEQ ID NO:115.

[0290] In some embodiments of receptors having one or more domains isolated or derived from LILRB1, one or more domains of LILRB1 are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or identical to, the sequence or subsequence of SEQ ID NO:116.

[0291] In some embodiments of a receptor having one or more domains of LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is identical to the sequence or subsequence of SEQ ID NO:116.

[0292] In various embodiments, an inhibitory receptor comprising a polypeptide is provided, wherein the polypeptide comprises one or more of a LILRB1 hinge domain or a functional variant thereof, a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain or an intracellular domain comprising at least one, or at least two immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0293] As used herein, "immunoreceptor tyrosine-based inhibitory motif" or "ITIM" refers to a conserved sequence of amino acids with a consensus sequence, such as S / I / V / LxYxxI / V / L (SEQ ID NO: 984), found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-bearing inhibitory receptors interact with their ligands, the ITIM motif becomes phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or an inositol-phosphatase called SHIP.

[0294] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), at least two ITIMs, at least three ITIMs, at least four ITIMs, at least five ITIMs, or at least six ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0295] 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: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0296] In further specific embodiments, the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0297] In some embodiments, the intracellular domain comprises both the ITIMs NLYAAV (SEQ ID NO: 117) and VTYAEV (SEQ ID NO: 118). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 121. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 121.

[0298] In some embodiments, the intracellular domain comprises both the ITIMs VTYAEV (SEQ ID NO: 118) and VTYAQL (SEQ ID NO: 119). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 122. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 122.

[0299] In some embodiments, the intracellular domain comprises both the ITIMs VTYAQL (SEQ ID NO: 119) and SIYATL (SEQ ID NO: 120). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 123. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 123.

[0300] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), and VTYAQL (SEQ ID NO: 119). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 124. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 124.

[0301] In some embodiments, the intracellular domain comprises the ITIM VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 125. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 125.

[0302] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120). In embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 126. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 126.

[0303] In some embodiments, the intracellular domain comprises a sequence at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 131). In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to the LILRB1 intracellular domain (SEQ ID NO: 131).

[0304] The LILRB1 intracellular domain or functional variant thereof of the present disclosure can have at least 1, at least 2, at least 4, at least 4, at least 5, at least 6, at least 7, or at least 8 ITIMs. In some embodiments, the LILRB1 intracellular domain or functional variant thereof has 2, 3, 4, 5, or 6 ITIMs.

[0305] In certain embodiments, the intracellular domain comprises two, three, four, five, or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0306] In certain embodiments, the intracellular domain comprises at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0307] In certain embodiments, the intracellular domain comprises three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0308] In certain embodiments, the intracellular domain comprises four immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0309] In certain embodiments, the intracellular domain comprises five immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0310] In certain embodiments, the intracellular domain comprises six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0311] In certain embodiments, the intracellular domain comprises at least seven immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0312] The LILRB1 protein has four immunoglobulin (Ig)-like domains designated D1, D2, D3, and D4. In some embodiments, the LILRB1 hinge domain comprises a LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1 D3D4 domain comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or identical to SEQ ID NO: 127. In some embodiments, the LILRB1 D3D4 domain comprises or consists essentially of SEQ ID NO: 127.

[0313] In some embodiments, the polypeptide comprises a LILRB1 hinge domain or a functional variant thereof. In embodiments, the LILRB1 hinge domain, or a functional variant thereof, comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or identical to, SEQ ID NO: 134, SEQ ID NO: 127, or SEQ ID NO: 128. In embodiments, the LILRB1 hinge domain, or a functional variant thereof, comprises a sequence that is at least 95% identical to, SEQ ID NO: 134, SEQ ID NO: 127, or SEQ ID NO: 128.

[0314] In some embodiments, the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO:134, SEQ ID NO:127, or SEQ ID NO:128.

[0315] In some embodiments, the LILRB1 hinge domain consists essentially of a sequence identical to SEQ ID NO:134, SEQ ID NO:127, or SEQ ID NO:128.

[0316] In some embodiments, the transmembrane domain is a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 135. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 135. In some embodiments, the LILRB1 transmembrane domain comprises a sequence identical to SEQ ID NO: 135. In embodiments, the LILRB1 transmembrane domain consists essentially of a sequence identical to SEQ ID NO: 135.

[0317] In some embodiments, the transmembrane domain can be attached to the extracellular region of a second inhibitory receptor, e.g., an antigen-binding domain or a ligand-binding domain, via a hinge, e.g., a hinge derived from a human protein. For example, in some embodiments, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, a CD8a hinge, or a LILRB1 hinge.

[0318] In some embodiments, the second inhibitory receptor comprises an inhibitory domain. In some embodiments, the second inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory domain is isolated from or derived from LILR1B.

[0319] Inhibitory receptors containing combinations of LILRB1 domains In some embodiments, the LILRB1-based inhibitory receptors of the present disclosure comprise more than one LILRB1 domain or functional equivalents thereof, for example, in some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and an intracellular domain, or a LILRB1 hinge domain, a transmembrane domain, and an intracellular domain.

[0320] In certain embodiments, the inhibitory receptor comprises a LILRB1 hinge domain, or a functional fragment thereof, and a LILRB1 transmembrane domain, or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 129. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 129. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 129.

[0321] In further embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibition motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120). In some embodiments, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least two ITIMs, wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0322] In some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and an intracellular domain. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 130. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 130. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 130.

[0323] In a preferred embodiment, the inhibitory receptor comprises a LILRB1 hinge domain, or a functional variant thereof, a LILRB1 transmembrane domain, or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from LYAAV (SEQ ID NO: 117), VTYAE (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 11).

[0324] In some embodiments, the inhibitory receptor comprises a sequence at least 95% identical to SEQ ID NO:132 or SEQ ID NO:133, or at least 99% identical to SEQ ID NO:132 or SEQ ID NO:133, or identical to SEQ ID NO:132 or SEQ ID NO:133.

[0325] In some embodiments, the polypeptide comprises a sequence at least 99% identical to SEQ ID NO:129, or at least 99% identical to SEQ ID NO:129, or identical to SEQ ID NO:129.

[0326] In some embodiments, the polypeptide comprises a sequence at least 99% identical to SEQ ID NO:130, or at least 99% identical to SEQ ID NO:130, or identical to SEQ ID NO:130. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0327] Polynucleotides and Vectors The present disclosure provides polynucleotides encoding the sequences of the first and second receptors of the present disclosure.The present disclosure provides immune cells comprising the polynucleotides and vectors described herein.

[0328] In some embodiments, the sequences of the first and / or second receptors are operably linked to promoters, ie, the sequence encoding the first receptor is operably linked to a first promoter and the sequence encoding the second receptor is operably linked to a second promoter.

[0329] The present disclosure provides vectors comprising the polynucleotides described herein.

[0330] In some embodiments, the first receptor is encoded by a first vector and the second receptor is encoded by a second vector. In some embodiments, both receptors are encoded by a single vector. In some embodiments, the first and / or second vector comprises an shRNA, e.g., a B2M shRNA.

[0331] In some embodiments, both receptors are encoded by a single vector. In some embodiments, the vector comprises an shRNA, e.g., a B2M shRNA.

[0332] In some embodiments, the first and second receptors are encoded by a single vector. Methods for encoding multiple polypeptides using a single vector are known to those of skill in the art and may include, among other things, encoding the multiple polypeptides under the control of different promoters, or, when using a single promoter to control transcription of multiple polypeptides, the use of an internal ribosome entry site (IRES) and / or a sequence encoding a self-cleaving peptide. Exemplary self-cleaving peptides include the T2A, P2A, E2A, and F2A self-cleaving peptides. In some embodiments, the T2A self-cleaving peptide comprises the sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 489). In some embodiments, the P2A self-cleaving peptide comprises the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 186). In some embodiments, the E2A self-cleaving peptide comprises the sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 490). In some embodiments, the F2A self-cleaving peptide comprises the sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 491). In some embodiments, the T2A self-cleaving peptide comprises the sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 489). Any of the foregoing can also include an N-terminal GSG linker. For example, the T2A self-cleaving peptide can also comprise the sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 181), which can be encoded by the sequence GGATCCGGAGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCC (SEQ ID NO: 492).

[0333] In some embodiments, the vector is an expression vector, ie, for expression of the first and / or second receptor in a suitable cell.

[0334] Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, because they allow long-term and stable integration of transgenes and their proliferation in daughter cells.Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of low immunogenicity.

[0335] Expression of natural or synthetic nucleic acids encoding receptors is typically achieved by operably linking the nucleic acid encoding the receptor or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0336] The polynucleotide encoding the receptor can be cloned into several types of vectors. For example, the polynucleotide can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0337] Furthermore, expression vectors can be provided to cells, such as immune cells, in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0338] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0339] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 base pairs (bp) upstream of the start site, although many promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription.

[0340] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, U6 promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0341] To assess receptor expression, the expression vector introduced into cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells to be transfected or infected via the viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA or used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0342] Reporter genes are used to identify potentially transfected or transduced cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with the minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions may be linked to reporter genes and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0343] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0344] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0345] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0346] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0347] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to inhibitors of the present disclosure, various assays can be performed to confirm the presence of recombinant DNA sequences within the host cells. Such assays include "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present disclosure.

[0348] immune cells The present disclosure provides immune cells comprising the receptors, vectors, and polynucleotides described herein.

[0349] In some embodiments, the immune cells comprise: (a) a first receptor comprising a first extracellular ligand-binding domain specific for a target antigen selected from (i) a cancer cell-specific antigen in complex with major histocompatibility complex class I (MHC-I) or a peptide antigen thereof, or (ii) CEA cell adhesion molecule 5 (CEA) in complex with major histocompatibility complex class I (MHC-I) or a peptide antigen thereof; and (b) a second receptor comprising a second extracellular ligand-binding domain specific for a non-target antigen selected from TNFRSF11, ACHRB, ITGAE, TRPV1, and SREC, or antigenic peptides thereof, in complex with major histocompatibility complex class I (MHC-I), wherein the non-target antigen comprises a polymorphism. 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.

[0350] As used herein, the term "immune cell" refers to a cell involved in the innate or adaptive (acquired) immune system. Exemplary innate immune cells include phagocytes such as neutrophils, monocytes, and macrophages, natural killer (NK) cells, polymorphonuclear leukocytes such as neutrophils, eosinophils, and basophils, and mononuclear cells such as monocytes, macrophages, and mast cells. Immune cells that play a role in acquired immunity include lymphocytes such as T cells and B cells.

[0351] The present disclosure provides immune cells comprising a first receptor comprising the sequence of SEQ ID NO: 52 and a second receptor comprising the 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. In some embodiments, the immune cells comprise an shRNA encoded by a sequence comprising GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 179), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the immune cells comprise a first receptor comprising the sequence of SEQ ID NO: 52, a second receptor comprising the sequence of SEQ ID NO: 164, and a sequence encoding an shRNA comprising the sequence of SEQ ID NO: 179. In some embodiments, the first receptor and the second receptor are encoded by a single polynucleotide, and the sequences encoding the first and second receptors are separated by a sequence encoding a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising the sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 181).

[0352] The present disclosure provides immune cells comprising a polypeptide comprising the sequence of SEQ ID NO: 141, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the polypeptide comprises SEQ ID NO: 141.

[0353] The present disclosure provides an immune cell comprising a polynucleotide comprising the sequence of SEQ ID NO: 142, or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the polynucleotide comprises SEQ ID NO: 142.

[0354] As used herein, "T cells" refers to a type of lymphocyte derived from myeloid precursors that develop in the thymus. There are several different types of T cells that develop upon migration to the thymus, including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells, and stem memory T cells. Different types of T cells can be distinguished by those skilled in the art based on the expression of their markers. Methods for distinguishing between types of T cells will be readily apparent to those skilled in the art.

[0355] In some embodiments, the first receptor and the second receptor together specifically activate an immune cell in the presence of a target cell.

[0356] In some embodiments, the immune cell is selected from the group consisting of a T cell, a B cell, and a natural killer (NK) cell. In some embodiments, the immune cell is a gamma delta (γδ) 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, an NK cell, or a macrophage. 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.

[0357] In some embodiments, the immune cells are non-naturally occurring. In some embodiments, the immune cells are isolated.

[0358] Methods for transducing populations of immune cells, such as T cells, using the vectors of the present disclosure will be readily apparent to those skilled in the art. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi) according to the manufacturer's instructions. T cells can be cultured at a density of 1 x 10^6 cells / mL in X-Vivo15 medium supplemented with 5% human A / B serum and 1% Pen / strep in the presence of CD3 / 28 Dynabeads (1:1 cell-to-bead ratio) and 300 units / mL of IL-2 (Miltenyi). After two days, T cells can be transduced with a viral vector, such as a lentiviral vector, using methods known in the art. In some embodiments, the viral vector is transduced at a multiplicity of infection (MOI) of 5. Cells can then be cultured in IL-2 or other cytokines, such as an IL-7 / 15 / 21 combination, for an additional five days before enrichment. Methods for isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells, will be readily apparent to those skilled in the art. While this method outlines potential approaches, it should be noted that these methodologies are rapidly evolving. For example, successful viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of expansion, generating a population of cells in which >99% of CD3+ are highly transduced.

[0359] Methods for activating and culturing populations of T cells containing TCR, CAR, inhibitory receptor or vectors encoding them will be readily apparent to one of skill in the art.

[0360] Whether before or after genetic modification of the T cells to express a TCR, the T cells may be engineered to express TCRs as described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, Activation and propagation can be achieved using methods generally described in U.S. Patent Nos. 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, 10040846, and U.S. Patent Application Publication No. 2006 / 0121005.

[0361] In some embodiments, T cells of the present disclosure are expanded and activated in vitro. Generally, T cells of the present disclosure are expanded in vitro by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, a population of T cells can be stimulated as described herein, for example, by contact with an anti-CD3 antibody. Costimulation of an accessory molecule on the surface of the T cells uses a ligand that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate proliferation of either CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), and can be used as well as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0362] In some embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be bound to a surface and the other agent can be in solution. In some embodiments, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor, or an antibody or other binding agent that binds to the agent. In this regard, see, for example, U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810 for artificial antigen presenting cells (aAPCs) contemplated for use in the activation and expansion of T cells in the present disclosure.

[0363] In some embodiments, the two agents are immobilized on beads either on the same bead, i.e., "cis," or on separate beads, i.e., "trans." For example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, with both agents co-immobilized on the same bead with comparable molecular weights. In one embodiment, a 1:1 ratio of each antibody bound to beads for CD4+ T cell expansion and T cell proliferation is used. In some embodiments, the ratio of CD3:CD28 antibody bound to beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one aspect of the present disclosure, more anti-CD28 antibody than anti-CD3 antibody is bound to the particles, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the present disclosure, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to beads is greater than 2:1.

[0364] To stimulate T cells or other target cells, particle-to-cell ratios ranging from 1:500 to 500:1 and any integer value therebetween can be used. As one of ordinary skill in the art would readily appreciate, the particle-to-cell ratio can depend on the particle size relative to the target cells. For example, small beads can bind only a few cells, while larger beads can bind many cells. In certain embodiments, the cell-to-particle ratio ranges from 1:100 to 100:1 and any integer value therebetween. In further embodiments, the ratio includes 1:9 to 9:1 and any integer value therebetween, and can also be used to stimulate T cells. In some embodiments, a 1:1 cell-to-bead ratio is used. One of ordinary skill in the art will appreciate that various other ratios may be suitable for use with the present disclosure. In particular, the ratio will vary depending on particle size and the size and type of cells.

[0365] In further embodiments of the present disclosure, cells, such as T cells, are combined with agent-coated beads, followed by separation of the beads and cells, and then culturing the cells. In alternative embodiments, the agent-coated beads and cells are not separated but are cultured together prior to culturing. In further embodiments, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0366] As an example, cell surface proteins can be ligated by binding anti-CD3 and anti-CD28 to paramagnetic beads, allowing them to contact T cells. In one embodiment, cells (e.g., CD4+ T cells) and beads (e.g., DYNABEADS CD3 / CD28T paramagnetic beads in a 1:1 ratio) are combined in a buffer. Again, one skilled in the art can readily appreciate that any cell concentration can be used. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and particles. For example, in one embodiment, a concentration of approximately 2 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In further embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml are used. In yet other embodiments, concentrations of cells from 75, 80, 85, 90, 95, or 100 million cells / ml are used. In further embodiments, concentrations of 125 or 150 million cells / ml may be used. In some embodiments, concentrations of 1 x 10 6 Cells cultured at a density of 10 cells / mL are used.

[0367] In some embodiments, the mixture may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value in between. In another embodiment, the beads and T cells are cultured together for 2 to 3 days. Suitable conditions for T cell culture include an appropriate medium (e.g., minimal essential medium or RPMI medium 1640 or X-vivo15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additive for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetylcysteine ​​and 2-mercaptoethanol. Media may include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo15, and X-Vivo20, Optimizer, and are supplemented with amino acids, sodium pyruvate, and vitamins, and are serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in amounts sufficient for T cell growth and expansion. In some embodiments, the media comprises X-VIVO-15 medium supplemented with 5% human A / B serum, 1% penicillin / streptomycin (pen / strep), and 300 units / ml of IL-2 (Miltenyi).

[0368] The T cells are maintained under conditions necessary to support proliferation, eg, at an appropriate temperature (eg, 37° C.) and atmosphere (eg, air plus 5% CO 2 ).

[0369] In some embodiments, T cells comprising the TCR, CAR, and inhibitory receptor of the present disclosure are autologous. Prior to expansion and genetic modification, a source of T cells is obtained from the subject. Immune cells such as T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T cell lines available in the art can be used. In certain embodiments of the present disclosure, T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation.

[0370] In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, may lack magnesium, or may lack many, but not all, divalent cations. As one of ordinary skill in the art would readily appreciate, wash steps can be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca2+-free PBS, Mg2+-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0371] In some embodiments, immune cells such as T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, e.g., by centrifugation through a PERCOLL™ gradient or by counterflow centrifugation. Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD4 conjugated beads for a period sufficient for positive selection of the desired T cells.

[0372] Enrichment of immune cell populations, such as T cell populations, by negative selection can be achieved by a combination of antibodies directed against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0373] For isolation of a desired population of immune cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and beads.

[0374] In some embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at either 2-10° C. or room temperature.

[0375] PBMCs from which immune cells such as stimulatory T cells or T cells are isolated can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogenous product by removing granulocytes, and to some extent monocytes, in the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40, 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40, 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled immediate freezing at -20°C or in liquid nitrogen, may also be used.

[0376] The present disclosure provides immune cells that express an activator and / or blocker receptor described herein, wherein the immune cells have reduced expression and / or function of major histocompatibility (MHC) class I complexes.

[0377] In some embodiments, the immune cells are autologous. For example, the immune cells are isolated or derived from the same subject who will receive the cells as part of a therapeutic regimen. Modifying autoimmune cells to reduce MHC class I expression and / or function via blocker receptors can be advantageously specific to MHC class I antigens. Without wishing to be bound by theory, modifying autoimmune cells to reduce MHC class I expression and / or function reduces blocker receptor binding by MHC class I expressed by the immune cells, either in cis or trans.

[0378] In some embodiments, the immune cells are all allogeneic. Allogeneic immune cells may be derived from a donor other than the subject to whom the immune cells are administered. Because allogeneic cells can be prepared and stored for use in subjects of various genotypes, allogeneic immune cells are commonly referred to as "off-the-shelf" or "universal" in cell therapy.

[0379] Any suitable method of reducing the expression and / or function of an MHC class I complex is contemplated within the scope of the present disclosure, including, inter alia, expression of an interfering RNA that knocks down one or more RNAs encoding an MHC class I component, or modification of a gene encoding an MHC class I component. The methods of reducing the expression and / or function of an MHC class I complex described herein are suitable for use with both allogeneic and autoimmune cells.

[0380] The major histocompatibility complex (MHC) is a genetic locus on the vertebrate genome that encodes a set of polypeptides required for the adaptive immune system. Among these are MHC class I polypeptides, including HLA-A, HLA-B, and HLA-C, and their alleles. MHC class I alleles are highly polymorphic and expressed in all nucleated cells. MHC class I polypeptides encoded by HLA-A, HLA-B, and HLA-C and their alleles form heterodimers with β2-microglobulin (B2M) and are present in complexes with antigens on the surface of cells. As referred to herein, an MHC class I gene or polypeptide may refer to any polypeptide found in the MHC or the corresponding gene encoding the polypeptide. In some embodiments, the immune cells of the present disclosure are inactivated by inhibitory ligands, including MHC class I polypeptides, e.g., HLA-A, HLA-B, and HLA-C, and their alleles. HLA-A alleles can be, for example, but not limited to, HLA-A*02, HLA-A*02:01, HLA-A*02:01:01, HLA-A*02:01:01:01, and / or any gene encoding a protein identical or similar to an HLA-A*02 protein. Thus, to prevent the autocrine signaling / binding described herein, it is desirable to remove or reduce expression of polypeptides encoded by HLA-A, HLA-B, and HLA-C, and their alleles, in immune cells.

[0381] Immune cells with reduced MHC class I polypeptide expression In some embodiments, the immune cells described herein are modified to inactivate, reduce, or eliminate expression or function of endogenous genes encoding alleles of endogenous MHC class I polypeptides. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A, HLA-B, and / or HLA-C. HLA-A, HLA-B, and HLA-C are encoded by the HLA-A, HLA-B, and HLA-C loci. Each of HLA-A, HLA-B, and HLA-C includes many variant alleles, all of which are contemplated within the scope of the present disclosure. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A*02. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A*02:01. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A*02:01:01. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A*02:01:01:01.

[0382] In some embodiments, the genetically engineered immune cells described herein are modified to reduce or eliminate expression of the B2M gene product. The beta-2 microglobulin (B2M) gene encodes a protein that associates with the major histocompatibility complex (MHC) class I, i.e., the MHC-I complex. The MHC-I complex is required for antigen presentation on the cell surface. When B2M is deleted, the MHC-I complex is disrupted and nonfunctional (Wang D et al. Stem Cells Transl Med. 4:1234-1245 (2015)). Furthermore, the B2M gene can be disrupted with high efficiency using gene editing techniques known in the art (Ren et al. Clin. Cancer Res. 23:2255-2266 (2017)). Reducing or eliminating B2M can reduce or eliminate functional MHC I on the surface of immune cells.

[0383] The present disclosure provides a gene editing system for editing endogenous target genes in immune cells. The present disclosure provides interfering RNA specific to the sequence of the target gene. Gene editing systems such as CRISPR / Cas systems, TALENs, and zinc fingers can be used to generate double-strand breaks, which can be used to introduce mutations through gene repair mechanisms such as homology-directed repair or non-homologous end joining (NHEJ). NHEJ after resection of the broken end, or improper end joining, can be used to introduce deletions. In some embodiments, the target gene includes a gene encoding a subunit of the MHC-I complex.

[0384] Target gene sequences include, but are not limited to, promoters, enhancers, introns, exons, intron / exon junctions, transcripts (pre-mRNA, mRNA, and splice variants), and / or 3' and 5' untranslated regions (UTRs). Any genetic element or combination of genetic elements can be targeted for the purposes of gene editing in immune cells described herein. Modifications to the target gene can be achieved by editing the target gene, resulting in altered or disrupted expression or function of the target gene or gene product, using any method known in the art.

[0385] In some embodiments, modifying a gene encoding an MHC class I polypeptide comprises deleting all or a portion of the gene. In some embodiments, modifying a gene encoding an MHC class I polypeptide comprises introducing a mutation into the gene. In some embodiments, the mutation comprises a deletion, insertion, substitution, or frameshift mutation. In some embodiments, modifying the gene comprises using a nucleic acid-guided endonuclease.

[0386] Gene sequences for the target genes described herein are known in the art. The sequences can be found in public databases such as NCBI GenBank or the NCBI nucleotide database. The sequences can be found using gene identifiers, for example, the HLA-A gene has NCBI gene ID: 3105, the HLA-B gene has NCBI gene ID: 3106, the HLA-C gene has NCBI gene ID: 3107, and the B2M gene has NCBI gene ID: 567 and NCBI reference sequence: NC_000015.10. Gene sequences can also be found by searching public databases using keywords. For example, HLA-A alleles can be found in the NCBI nucleotide database by searching the keywords "HLA-A*02," "HLA-A*02:01," "HLA-A*02:01:01," or "HLA-A*02:01:01:01." These sequences can be used for targeting in various gene editing techniques known in the art. Table 8 provides non-limiting exemplary sequences of HLA-A alleles and B2M gene sequences targeted for modification as described herein. [Table 8]

[0387] One of skill in the art will understand that T can be substituted for U to convert an RNA sequence to a DNA sequence, and vice versa, and both are contemplated as target gene sequences in the present disclosure.

[0388] In some embodiments, the target gene is edited in the immune cells described herein using a nucleic acid-guided endonuclease. Exemplary nucleic acid-guided endonucleases include Class II endonucleases such as CRISPR / Cas9.

[0389] As used herein, "CRISPR" or "CRISPR gene editing" refers to a set of clustered regularly interspaced short palindromic repeats or a system containing such a set of repeats. "Cas" as used herein refers to a CRISPR-associated protein. The "CRISPR / Cas" system refers to a system derived from CRISPR and Cas that can be used to silence, knock out, or mutate target genes. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. The CRISPR / Cas system has been modified for use in gene editing. This is achieved by introducing into a eukaryotic cell one or more specifically designed guide nucleic acids (gNAs), typically guide RNAs (gRNAs), and an appropriate Cas endonuclease that forms a ribonucleoprotein complex with the gNA. The gNA guides the gNA-endonuclease protein complex to the target genomic location, and the endonuclease introduces a strand break at the target genomic location. This strand break can be repaired by cellular mechanisms such as non-homologous end joining (leading to a deletion) or homologous repair (which can generate an insertion), thereby introducing the genetic modification into the host cell genome.

[0390] CRISPR / Cas systems are classified by class and type. Class 2 systems represent a single interfering protein that is currently classified into three different types (Type II, Type V, and Type VI). Any Class 2 CRISPR / Cas system suitable for gene editing, such as a Type II, Type V, or Type VI system, is contemplated within the scope of the present disclosure. Exemplary Class 2 Type II CRISPR systems include Cas9, Csn2, and Cas4. Exemplary Class 2 Type V CRISPR systems include Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, and Cas12k (C2c5). Exemplary Class 2 Type VI systems include Cas13, Cas13a (C2c2), Cas13b, Cas13c, and Cas13d.

[0391] CRISPR sequence, sometimes called CRISPR locus, comprises alternating repeats and spacers. In naturally occurring CRISPR, the spacer usually comprises a sequence foreign to bacteria, such as a plasmid or phage sequence. As described herein, the spacer sequence may also be referred to as "targeting sequence". In the CRISPR / Cas system for genetic manipulation, the spacer is derived from the target gene sequence (gNA).

[0392] An exemplary Class 2 type II CRISPR system relies on the protein Cas9, which is a nuclease with two active cleavage sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi (2013) Science 341:833-836. In some embodiments, the Cas protein used to modify immune cells is Cas9.

[0393] Therefore, the CRISPR / Cas system can be used to edit target genes, such as those targeted for editing in immune cells described herein, by adding or deleting base pairs or by introducing premature stops, thereby reducing target expression. Alternatively, the CRISPR / Cas system can be used like RNA interference to reversibly turn off target genes. In mammalian cells, for example, RNA can guide Cas proteins to target gene promoters and sterically block RNA polymerase.

[0394] The Cas protein can be derived from any bacterial or archaeal Cas protein. Any suitable CRISPR / Cas system is contemplated within the scope of this disclosure. In other embodiments, the Cas protein is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a (Cpf1), Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, In some embodiments, the Cas protein comprises one or more of Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, CasY, homologs thereof, or modified forms thereof. In some embodiments, the Cas protein is a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, or a combination or complex thereof. In some embodiments, the Cas protein is a Cas9 protein.

[0395] Artificial CRISPR / Cas systems that inhibit target genes can be generated using techniques known in the art, such as those described in U.S. Publication No. 2014 / 0068797 and Cong (2013) Science 339:819-823. For example, those described in Tsai (2014) Nature Biotechnol., 32:6 569-576, U.S. Patent Nos. 8,871,445, 8,865,406, 8,795,965, 8,771,945, and 8,697,359, but other artificial CRISPR / Cas systems known in the art that inhibit target genes can also be generated. Those skilled in the art will know how to design suitable gNAs for specific Cas proteins.

[0396] The present disclosure provides gene-targeting guide nucleic acids (gRNAs) that can direct the activity of an associated polypeptide (e.g., a nucleic acid-guided endonuclease) to a specific target gene sequence within a target nucleic acid genome. The genome-targeting nucleic acid can be RNA. The genome-targeting RNA is referred to herein as a "guide RNA" or "gRNA." The guide RNA can include at least a targeting sequence that hybridizes to a target nucleic acid sequence of interest and a CRISPR repeat sequence. In some type II systems, the gRNA also includes a second RNA called a tracrRNA sequence, also referred to herein as a "scaffold" sequence. In type II guide RNAs (gRNAs), the CRISPR repeat sequence and the scaffold sequence hybridize to each other to form a duplex. In type V guide RNAs (gRNAs), the crRNA forms a duplex. In both systems, the duplex can bind to a site-directed polypeptide such that the guide RNA and the site-directed polypeptide form a complex. The gene-targeting nucleic acid can provide target specificity to the complex through its association with the site-directed polypeptide. Thus, the gene-targeting nucleic acid can direct the activity of the site-specific polypeptide.

[0397] In some embodiments, the present disclosure provides a guide RNA comprising a targeting sequence and a guide RNA scaffold sequence, wherein the targeting sequence is complementary to a sequence of a target gene.

[0398] Exemplary guide RNAs include a targeting sequence of approximately 15-20 bases. As will be appreciated by those skilled in the art, each gRNA can be designed to include a targeting sequence complementary to its genomic target sequence. For example, each of the targeting sequences, e.g., the RNA versions of the DNA sequences shown in Table 9, minus the three 3' nucleotides representing their PAM sites, can be incorporated into a single RNA chimera or crRNA.

[0399] The gene targeting nucleic acid can be a bimolecular guide RNA.The gene targeting nucleic acid can be a single-molecule guide RNA.The gene targeting nucleic acid can be any known configuration of guide RNA known in the art, including, for example, paired gRNA, or multiple gRNAs used in a single step.The coding sequence and splice junctions are clear from the genome sequence, but other features required for gene expression can be specific and unclear.

[0400] A bimolecular guide RNA can comprise two strands of RNA. The first strand comprises a 5' to 3' sequence, an optional spacer extension sequence, a targeting sequence, and a minimal CRISPR repeat sequence. The second strand comprises a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0401] In a Type II system, a single-molecule guide RNA (sgRNA) can include, from 5' to 3', an optional spacer extension sequence, a targeting sequence, a minimal CRISPR repeat sequence, a single-molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension can include elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can include one or more hairpins.

[0402] In some embodiments, the guide RNA or single-molecule guide RNA (sgRNA) can comprise a targeting sequence and a scaffold sequence. In some embodiments, the scaffold sequence is a Cas9 gRNA sequence. In some embodiments, the scaffold sequence is encoded by a DNA sequence comprising a sequence sharing at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 497). In some embodiments, the scaffold sequence is encoded by a DNA sequence comprising GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 497).

[0403] In some embodiments, for example, in those embodiments in which the CRISPR / Cas system is a Cas9 system, the sgRNA can comprise a 20-nucleotide targeting sequence at the 5' end of the sgRNA sequence. The sgRNA can comprise a targeting sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a targeting sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a variable-length targeting sequence having 17 to 30 nucleotides at the 5' end of the sgRNA sequence.

[0404] Suitable scaffold sequences and the placement of scaffold target sequences will depend on the choice of endonuclease and will be known to those skilled in the art.

[0405] The single molecule guide RNA (sgRNA) in the Type II system, e.g., Cas9, can comprise, from 5' to 3', a minimal CRISPR repeat sequence and a targeting sequence.

[0406] By way of example, guide RNAs or other smaller RNAs used in the CRISPR / Cas9 or CRISPR / Cpf1 systems can be easily synthesized by chemical means, as exemplified below and described in the art. While chemical synthesis procedures continue to expand, as polynucleotide lengths increase significantly beyond approximately 100 nucleotides, purification of such RNAs by procedures such as high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult. One approach used to generate longer RNAs is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding Cas9 or Cpf1 endonucleases, are more easily produced enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic production of the RNA, for example, modifications that improve stability, reduce the likelihood or severity of innate immune responses, and / or improve other attributes can be introduced, as described in the art.

[0407] The targeting sequence of gRNA hybridizes with the sequence in the target nucleic acid of interest.The targeting sequence of genome targeting nucleic acid can interact with target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing).The nucleotide sequence of targeting sequence can vary according to the sequence of the target nucleic acid of interest.

[0408] In the Cas9 system described herein, the targeting sequence can be designed to hybridize to the target nucleic acid located 5' of the reverse complement of the PAM of the Cas9 enzyme used in the system. The targeting sequence can perfectly match the target sequence or can have a mismatch. Each CRISPR / Cas system protein may have a specific PAM sequence recognized in a specific orientation and position in the target DNA. For example, S. pyogenes Cas9 recognizes a PAM containing the sequence 5'-NRG-3' in the target nucleic acid, where R contains either A or G, and N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the targeting sequence. The selection of an appropriate PAM sequence will be apparent to one of skill in the art.

[0409] The target sequence is complementary to and hybridizes with the targeting sequence of the gRNA. The target nucleic acid sequence can comprise 20 nucleotides. The target nucleic acid can comprise fewer than 20 nucleotides. The target nucleic acid can comprise more than 20 nucleotides. The target nucleic acid can comprise at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. In some embodiments, for example, in those embodiments where the CRISPR / Cas system is a Cas9 system, the target nucleic acid sequence can comprise the 20 nucleotides immediately 5' to the first nucleotide of the reverse complement of the PAM sequence. This target nucleic acid sequence is often referred to as the PAM strand or target strand, and the complementary nucleic acid sequence is often referred to as the non-PAM strand or non-target strand. Those skilled in the art will recognize that the targeting sequence hybridizes to the non-PAM strand of the target nucleic acid. See, e.g., US2019 / 0185849A1.

[0410] In some cases, the percent complementarity between the targeting sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some cases, the percent complementarity between the targeting sequence and the target nucleic acid is up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%. In some instances, the percent complementarity between the targeting sequence and the target nucleic acid is 100% over the six contiguous 5'-most nucleotides of the target sequence on the complementary strand of the target nucleic acid. The percent complementarity between the targeting sequence and the target nucleic acid may be at least 60% over about 20 contiguous nucleotides. The lengths of the targeting sequence and the target nucleic acid may vary by 1 to 6 nucleotides, which may be considered a bulge or multiple bulges.

[0411] Targeting sequences can be designed or selected using computer programs known to those skilled in the art. Computer programs can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic frequency (e.g., of sequences that are identical or similar but vary in one or more spots as a result of mismatches, insertions, or deletions), methylation status, and the presence of SNPs. Available computer programs can take as input an NCBI gene ID, official gene symbol, Ensembl Gene ID, genomic coordinates, or DNA sequence and create an output file containing sgRNAs targeting the appropriate genomic region specified as input. Computer programs can also provide summary statistics and scores indicating on-target and off-target binding of the sgRNA for the target gene (Doench et al. Nat Biotechnol. 34:184-191 (2016)). The present disclosure provides guide RNAs comprising a targeting sequence. In some embodiments, the guide RNA further comprises a guide RNA scaffold sequence. In some embodiments, the targeting sequence is complementary to a sequence of a target gene selected from the group consisting of HLA-A, HLA-B, HLA-C, B2M, or an allele thereof. In some embodiments, the target gene is an HLA-A gene. In some embodiments, the target gene is an HLA-B gene. In some embodiments, the target gene is an HLA-C gene. In some embodiments, the target gene is HLA-A, HLA-B, HLA-C, or a combination thereof. In some embodiments, the targeting sequence comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity with, or is identical to, a sequence disclosed in Table 8.

[0412] In some embodiments, a gNA specifically targets a sequence at the endogenous HLA-A locus. In some embodiments, a gNA that specifically targets a sequence at the HLA-A locus comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with a sequence selected from the sequences disclosed in Table 9. In some embodiments, a gNA that specifically targets a sequence at the HLA-A locus comprises a sequence selected from the sequences disclosed in Table 9.

[0413] In some embodiments, the gNA specifically targets a sequence of the HLA-A*02 allele. For example, the gRNA specifically targets and hybridizes to a sequence shared by all HLA-A*02 alleles, but not shared by HLA-A*02 and HLA-A*03 alleles. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele.

[0414] In some embodiments, the gNA specifically targets the coding DNA sequence of HLA-A*02.

[0415] In some embodiments, gNAs specifically target coding DNA sequences shared by more than 1000 HLA-A*02 alleles. In some embodiments, gNAs that specifically target coding DNA sequences in more than 1000 HLA-A*02 alleles comprise a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity with, or is identical to, a sequence selected from SEQ ID NOs: 400-465.

[0416] The sequences in Tables 9-12 are presented as DNA sequences. One skilled in the art will understand that thymine (T) can be substituted for uracil (U) in any DNA sequence, including those set forth in Tables 9-12, to arrive at the corresponding RNA sequence. [Table 9-1] [Table 9-2]

[0417] The sequences disclosed in Table 9 include the corresponding genomic sequences, including the PAM sequence. One skilled in the art will understand that the targeting sequence of a gRNA does not include the three 3'-most nucleotides of the sequences in Table 9, which represent the corresponding PAM site for the gRNA.

[0418] The present disclosure provides a gNA comprising a targeting sequence specific to the B2M gene. In some embodiments, the gNA specifically targets the coding sequence (CDS) sequence of the B2M gene. In some embodiments, the gNA comprises a sequence that targets the B2M gene promoter sequence.

[0419] In some embodiments, the gNA comprises a targeting sequence and a gNA scaffold sequence. In some embodiments, the targeting sequence comprises a sequence set forth in Table 10, or a sequence sharing about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto.

[0420] In some embodiments, the targeting sequence is complementary to a sequence of the B2M gene. In some embodiments, the B2M gene comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with a B2M sequence shown in Table 8. [Table 10-1] [Table 10-2]

[0421] In some embodiments, the immune cells described herein are edited using TALEN gene editing.

[0422] "TALEN" or "TALEN gene editing" refers to Transcription Activator-Like Effector Nuclease, an artificial nuclease used to edit target genes.

[0423] TALENs are artificially produced by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) derived from Xanthomonas bacteria can be engineered to bind to any desired DNA sequence, including portions of target genes such as TCR subunits, MHC class I complex components, or CD52. By combining the engineered TALE with a DNA cleavage domain, restriction enzymes specific for any desired DNA sequence, including target gene sequences, can be produced. These can then be introduced into cells and used for genome editing.

[0424] To produce a TALEN, a TALE protein is fused to a nuclease (N), which is a wild-type or mutant Fold endonuclease. Several mutations to FokI have been made for use in TALENs, which improve, for example, cleavage specificity or activity.

[0425] The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains to be properly oriented and spaced apart to target sites in the genome. Both the number of amino acid residues between the TALE DNA-binding domain and the FokI cleavage domain, and the number of bases between the two individual TALEN binding sites, appear to be important parameters for achieving high levels of activity.

[0426] TALENs specific to sequences within target genes can be constructed using any method known in the art, including various schemes that use modular components.

[0427] In some embodiments, target genes are edited in immune cells described herein using ZFN gene editing.

[0428] "ZFN" or "zinc finger nuclease" or "ZFN gene editing" refers to zinc finger nucleases, which are artificial nucleases that can be used to edit target genes.

[0429] Similar to TALENs, ZFNs contain a Fold nuclease domain (or a derivative thereof) fused to a DNA-binding domain, which in the case of ZFNs contains one or more zinc fingers.

[0430] Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. A zinc finger can contain, for example, Cys2His2 and recognize approximately 3-bp sequences. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize approximately 6, 9, 12, 15, or 18-bp sequences. A variety of selection and modular assembly techniques are available for generating zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0431] Like TALENs, ZFNs must dimerize to cleave DNA. Therefore, a pair of ZFNs is required to target non-palindromic DNA sites. Two individual ZFNs must bind to opposite strands of DNA with their nucleases appropriately spaced apart.

[0432] Also, like TALENs, ZFNs can create double-strand breaks in DNA, which, if improperly repaired, can create frameshift mutations, resulting in reduced expression and amount of target genes or gene products in cells. ZFNs can also be used in conjunction with homologous recombination to create mutations in target genes.

[0433] ZFNs specific for a sequence within a target gene can be constructed using any method known in the art.

[0434] In some embodiments, the expression and function of one or more MCH-I components are reduced using RNA interference. "RNAi" or "RNA interference" refers to the process of sequence-specific post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA). Double-stranded RNAs, such as siRNA (small interfering RNA), miRNA (microRNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeat-associated siRNA), and modified forms thereof, can all mediate RNA interference. These dsRNA molecules may be commercially available or may be designed and prepared based on known sequence information. The antisense strand of these molecules may comprise RNA, DNA, PNA, or a combination thereof. DNA / RNA chimeric polynucleotides include, but are not limited to, double-stranded polynucleotides consisting of DNA and RNA that inhibit the expression of a target gene. dsRNA molecules may also contain one or more modified nucleotides, as described herein, which may be incorporated into either or both strands.

[0435] In RNAi gene silencing or knockdown, dsRNA is introduced into an organism, which comprises a first (antisense) strand that is complementary to a portion of the target gene, and a second (sense) strand that is completely or partially complementary to the first antisense strand.After being introduced into an organism, the target gene-specific dsRNA is processed into relatively small fragments (siRNAs), which are then distributed throughout the organism, reducing the messenger RNA of the target gene and causing a phenotype that can be very similar to the phenotype resulting from the complete or partial deletion of the target gene.

[0436] Certain dsRNA in cells can be acted upon by the Dicer enzyme, which is a ribonuclease III enzyme.Dicer can process dsRNA into shorter parts of dsRNA, namely, siRNA.RNAi also involves an endonuclease complex known as the RNA-induced silencing complex (RISC).After cleavage by Dicer, siRNA enters the RISC complex and directly cleaves the single-stranded RNA target that has a sequence complementary to the antisense strand of the siRNA duplex.The other strand of the siRNA is the passenger strand.Cutting of target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex.Therefore, siRNA can downregulate or knock down gene expression by mediating RNA interference in a sequence-specific manner.

[0437] As used herein with respect to RNA interference, a "target gene" or "target sequence" refers to a gene or gene sequence whose corresponding RNA is targeted for degradation via the RNAi pathway using dsRNA or siRNA, as described herein. Exemplary target gene sequences are shown in Table 8. For example, to target a gene using siRNA, the siRNA comprises an antisense region that is complementary or substantially complementary to at least a portion of the target gene or sequence, and a sense strand that is complementary to the antisense strand. Once introduced into a cell, the siRNA directs the RISC complex to cleave the RNA containing the target sequence, thereby degrading the RNA. The present disclosure provides interfering RNA. The double-stranded RNA molecule of the present disclosure may be in the form of any type of RNA interference molecule known in the art. In some embodiments, the double-stranded RNA molecule is a small interfering RNA (siRNA). In other embodiments, the double-stranded RNA molecule is a short hairpin RNA (shRNA) molecule. In other embodiments, the double-stranded RNA molecule is a Dicer substrate that is processed in the cell to produce siRNA. In other embodiments, the double-stranded RNA molecule is part of a microRNA precursor molecule.

[0438] In some embodiments, the shRNA is of a length suitable as a Dicer substrate and can be processed to produce a RISC-active siRNA molecule. See, e.g., Rossi et al., US2005 / 0244858.

[0439] Dicer substrate double-stranded RNAs (e.g., shRNAs) can be of sufficient length to be processed by Dicer to produce active siRNAs and can further comprise one or more of the following properties: (i) Dicer substrate shRNAs can be asymmetric, e.g., with a 3' overhang on the antisense strand, (ii) Dicer substrate shRNAs can have a modified 3' end on the sense strand to direct the orientation of Dicer binding and processing of the dsRNA into active siRNA, e.g., the incorporation of one or more DNA nucleotides, and (iii) the first and second strands of the Dicer substrate dsRNA can be 21-30 bp in length.

[0440] In some embodiments, the interfering RNA comprises a sequence complementary to the sequence of B2M mRNA. In some embodiments, the interfering RNA can induce RNAi-mediated degradation of B2M mRNA. In some embodiments, the B2M mRNA sequence comprises a coding sequence. In some embodiments, the B2M mRNA sequence comprises an untranslated region.

[0441] In some embodiments, the interfering RNA comprises a sequence complementary to the sequence of HLA-A*02 mRNA. In some embodiments, the interfering RNA can induce RNAi-mediated degradation of HLA-A*02 mRNA. In some embodiments, the HLA-A*02 mRNA sequence comprises a coding sequence. In some embodiments, the HLA-A*02 mRNA sequence comprises an untranslated region.

[0442] In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises a first sequence having a sequence complementary to B2M mRNA at its 5' to 3' end and a second sequence having a sequence complementary to the first sequence at its 5' to 3' end, wherein the first sequence and the second sequence form the shRNA.

[0443] In some embodiments, the first sequence is 18, 19, 20, 21, or 22 nucleotides. In some embodiments, the first sequence is complementary to a sequence selected from the sequences shown in Tables 11 and 12. In some embodiments, the first sequence has a GC content of 25% or more and less than 60%. In some embodiments, the first sequence is complementary to a sequence selected from the sequences shown in Tables 11 and 12. In some embodiments, the first sequence does not contain four nucleotides of the same base or a run of seven C or G nucleotide bases. In some embodiments, the first sequence is 21 nucleotides.

[0444] Exemplary target B2M sequences complementary to the first sequence are shown in Table 11.

[0445] In some cases, the first sequence may have 100% identity, i.e., complete identity, homology, or complementarity, with the target nucleic acid sequence. In other cases, there may be one or more mismatches between the first sequence and the target nucleic acid sequence. For example, there may be 1, 2, 3, 4, 5, 6, or 7 mismatches between the sense region and the target nucleic acid sequence.

[0446] The sequences shown in Table 11 are presented as DNA sequences. In all sequences shown in Table 11, thymine (T) can be replaced by uracil (U) to arrive at the sequence of the target mRNA sequence. [Table 11-1] [Table 11-2]

[0447] Exemplary sequences encoding B2M shRNAs include the sequence GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 179), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. Further exemplary sequences encoding B2M shRNAs include the sequence GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 180), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0448] In some embodiments, the interfering RNA comprises a sequence complementary to the sequence of HLA-A*02 mRNA. In some embodiments, the interfering RNA can induce RNAi-mediated degradation of HLA-A*02 mRNA. In some embodiments, the HLA-A*02 mRNA sequence comprises a coding sequence. In some embodiments, the HLA-A*02 mRNA sequence comprises an untranslated region.

[0449] In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises a first sequence having a sequence complementary to HLA-A*02 mRNA from its 5' to 3' end, and a second sequence having a sequence complementary to the first sequence from its 5' to 3' end, wherein the first sequence and the second sequence form the shRNA.

[0450] Exemplary target HLA sequences complementary to the first sequence are shown in Table 12. [Table 12-1] [Table 12-2]

[0451] In some embodiments, the first and second sequences are separated by a linker, sometimes referred to as a loop. In some embodiments, both the first and second sequences are encoded by a single-stranded RNA or DNA vector. In some embodiments, a loop is located between the first and second sequences. In these embodiments, the first and second sequences hybridize to form a double-stranded region. The first and second sequences are connected by a linker sequence to form a "hairpin" or "stem-loop" structure. An shRNA can have complementary first and second sequences at opposite ends of a single-stranded molecule, such that the molecule can form a double-stranded region with the complementary sequence portion, and the strands are connected at one end of the double-stranded region by a linker (i.e., loop sequence). The linker, or loop sequence, can be either a nucleotide or a non-nucleotide linker. The linker can interact with the first sequence, and optionally the second sequence, through covalent or non-covalent interactions.

[0452] Any suitable nucleotide loop sequence is contemplated within the scope of the present disclosure. The shRNAs of the present disclosure may include a nucleotide, non-nucleotide, or mixed nucleotide / non-nucleotide linker that connects the first sequence of the shRNA to the second sequence of the shRNA. The nucleotide loop sequence can be two or more nucleotides in length, for example, about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. Exemplary loop sequences are disclosed in Table 14.

[0453] In some embodiments, the shRNA further comprises a 5' flanking sequence and a 3' flanking sequence, in which the 5' flanking sequence is linked to the 5' end of the first sequence and the 3' flanking sequence is linked to the 3' end of the second sequence.

[0454] Without wishing to be bound by theory, it is believed that flanking shRNA stem-loop sequences with 5' and 3' sequences similar to those found in microRNAs can target the shRNA for processing by the endogenous microRNA processing machinery and increase the efficiency of shRNA processing. Alternatively, or in addition, the flanking sequences may increase shRNA compatibility with polymerase II or polymerase III promoters, resulting in more effective regulation of shRNA expression.

[0455] In some embodiments, the 5' flanking sequence is selected from the sequences shown in Table 13. Exemplary flanking sequences are shown in Table 13. [Table 13]

[0456] In some embodiments, the first and second sequences are present on a single-stranded polynucleotide, and the first and second sequences are separated by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, and the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides form a loop region within the shRNA. In some embodiments, the loop region comprises a sequence selected from the sequences set forth in Table 14. [Table 14]

[0457] The shRNAs of the present disclosure can be produced exogenously by chemical synthesis, in vitro transcription, or by cleavage of longer double-stranded RNAs using Dicer or another suitable nuclease with similar activity.Chemically synthesized siRNAs produced from protected ribonucleoside phosphoramidites using conventional DNA / RNA synthesizers can be obtained from commercial sources such as Millipore Sigma (Houston, Tex.), Ambion Inc. (Austin, Tex.), Invitrogen (Carlsbad, Calif.), or Dharmacon (Lafayette, Colo.). siRNAs can be purified, for example, by extraction with solvents or resins, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, siRNAs can be used with little or no purification to avoid losses due to sample processing.

[0458] In some embodiments, shRNAs of the present disclosure can be produced using an expression vector into which a nucleic acid encoding the double-stranded RNA has been cloned, for example, under the control of a suitable promoter.

[0459] Pharmaceutical Composition The present disclosure provides a pharmaceutical composition comprising an immune cell comprising a first and second receptor of the present disclosure and a pharmaceutically acceptable diluent, carrier, or excipient.

[0460] Such compositions may include a buffer such as neutral buffered saline, phosphate buffered saline, a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol, a protein, polypeptide or amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, and a preservative.

[0461] In some embodiments, immune cells express both the first receptor and the second receptor. In some embodiments, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of immune cells express both the first receptor and the second receptor. In some embodiments, at least 90% of immune cells express both the first receptor and the second receptor.

[0462] Cancer treatment Provided herein are methods of killing multiple cancer cells or treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising an immune cell comprising a first and second receptor of the present disclosure, wherein the immune cell expresses both receptors within the same cell.

[0463] Cancer is a disease in which abnormal cells divide uncontrollably and spread to nearby tissues. In some embodiments, cancer comprises a liquid tumor or a solid tumor. Exemplary liquid tumors include leukemia and lymphoma. Cancer can arise in virtually any organ in the body, including epithelial tissue. Any cancer in which multiple cancer cells express a first activator ligand but do not express a second inhibitory ligand is contemplated within the scope of the present disclosure. For example, CEA-positive cancers that can be treated using the methods described herein include colorectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung adenocarcinoma, head and neck cancer, gallbladder cancer, diffuse large B-cell carcinoma, or acute myeloid leukemia.

[0464] In some embodiments, a plurality of cancer cells express the target antigen. In some embodiments, a plurality of cancer cells in a subject express CEA. Any cancer in which cells express CEA, i.e., are CEA-positive, is contemplated within the scope of the present disclosure. Exemplary CEA-positive cancers include, but are not limited to, prostate cancer, ovarian cancer, lung cancer, thyroid cancer, gastrointestinal cancer, breast cancer, and liver cancer. Additional CEA-positive cancers include colorectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung cancer, head and neck cancer, gallbladder cancer, diffuse large B-cell carcinoma, or acute myeloid leukemia cancer. In some embodiments, the cancer comprises colon cancer, lung cancer, or pancreatic cancer. In some embodiments, the CEA-positive cancer comprises lung cancer or colorectal cancer. In some embodiments, the lung cancer comprises lung adenocarcinoma, small cell lung cancer (SCLC), or non-small cell lung cancer (NSCLC). In some embodiments, the lung cancer comprises lung adenocarcinoma. The compositions and methods disclosed herein can be used to treat CEA-positive cancers that are recurrent, refractory, and / or metastatic.

[0465] Provided herein are methods for treating CEA+ cancer in a subject with a CEA+ tumor, wherein the tumor has loss of heterozygosity at the MHC class I locus. In some embodiments, the method comprises administering to the subject an effective amount of immune cells or pharmaceutical compositions described herein. In some embodiments, the method comprises (a) determining the HLA-A, HLA-B, or HLA-C genotype or expression of normal cells and a plurality of cancer cells of the subject, (b) determining the expression of CEA in a plurality of cancer cells of the subject, and (c) if the normal cells express HLA-A, HLA-B, or HLA-C non-target antigen 2, the plurality of cancer cells do not express HLA-A, HLA-B, or HLA-C non-target antigen, and the plurality of cancer cells are CEA-positive, administering to the subject an effective amount of the immune cells or pharmaceutical composition of the present disclosure. In some embodiments, for example, in those embodiments in which the cancer is known to be CEA+, the method includes (a) determining the HLA-A, HLA-B, or HLA-C genotype or expression of normal cells and a plurality of cancer cells of the subject, and (b) administering to the subject an effective amount of an immune cell or pharmaceutical composition of the present disclosure if the normal cells express an HLA-A, HLA-B, or HLA-C non-target antigen and the plurality of cancer cells do not express the non-target antigen. In some embodiments, the non-target antigen comprises HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07.

[0466] Administration of the immune cells or pharmaceutical compositions described herein can reduce the size of a tumor in a subject. In some embodiments, the size of 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% compared to the size of the tumor before administration of the immune cells or pharmaceutical composition. In some embodiments, the tumor is eliminated.

[0467] Administration of the immune cells or pharmaceutical compositions described herein can inhibit tumor growth in a subject. For example, the immune cells or pharmaceutical compositions can kill tumor cells, causing the tumor to stop growing or shrink in size. In some cases, the immune cells or pharmaceutical compositions can prevent further tumors from forming in a subject or reduce the total number of tumors.

[0468] Administration of the immune cells or pharmaceutical compositions described herein can result in selective killing of cancer cells, but not wild-type cells, in a subject. In some embodiments, about 60% of the dead cells are cancer cells, about 65% of the dead cells are cancer cells, about 70% of the dead cells are cancer cells, about 75% of the dead cells are cancer cells, about 80% of the dead cells are cancer cells, about 85% of the dead cells are cancer cells, about 90% of the dead cells are cancer cells, about 95% of the dead cells are cancer cells, or about 100% of the dead cells are cancer cells.

[0469] Administration of the immune cells or pharmaceutical compositions described herein may result in the killing of about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or all of the subject's cancer cells.

[0470] Administration of the immune cells or pharmaceutical compositions described herein can result in fewer side effects in a subject than administration of other equivalent immune cells that include a first activator receptor but lack a second inhibitory receptor. For example, administration of the immune cells or pharmaceutical compositions described herein can result in reduced dose-limiting toxicity compared to CEA CARs or CEA TCRs administered without a second inhibitory receptor.

[0471] In some embodiments, the plurality of cancer cells do not express a polymorphic allele of TNFRSF11, ACHRB, ITGAE, TRPV1, or SREC, e.g., the cancer cells have lost an allele of TNFRSF11, ACHRB, ITGAE, TRPV1, or SREC through loss of heterozygosity at that locus.

[0472] The present disclosure provides a method of treating cancer in a subject, comprising: (a) determining the genotype of normal cells and a plurality of cancer cells of the subject at a polymorphic locus selected from the group consisting of rs1716 (ITGAE R950W), rs2976230 (ITGAE V1019A / V1019G), rs1805034 (TNFRSF11A V192A), and rs35211496 (TNFRSF11A H141Y); (b) determining the expression of CEA in the plurality of cancer cells; and (c) if the wild-type cells are heterozygous for the polymorphic locus and the plurality of cancer cells are hemizygous for the polymorphic locus, and the plurality of cancer cells are CEA-positive, administering a plurality of immune cells to the subject, wherein the plurality of immune cells (i) express the CEA-cell adhesion molecule in complex with major histocompatibility complex class I (MHC-I). and (ii) a second receptor, optionally an inhibitory receptor, comprising an extracellular ligand binding domain specific for a non-target antigen selected from TNFRSF11, ACHRB, ITGAE, TRPV1, and SREC, or antigenic peptides thereof, in complex with major histocompatibility complex class I (MHC-I), wherein the non-target antigen comprises a polymorphism.

[0473] Methods for genotyping cancer and normal cells from a subject for the presence or absence of SNPs will be readily apparent to those skilled in the art. SNP genotyping methods include PCR-based methods such as dual-probe TaqMan assays, array-based hybridization methods, and sequencing, among others.

[0474] Methods for measuring the expression of target antigens in cancer or wild-type cells derived from a subject will be readily apparent to those skilled in the art. These include, inter alia, methods for measuring RNA expression, such as RNA sequencing and reverse transcription polymerase chain reaction (RT-PCR), and methods for measuring protein expression, such as immunohistochemistry-based methods. Methods for measuring loss of heterozygosity in multiple cancer cells include, inter alia, high-throughput sequencing of genomic DNA extracted from cancer cells using methods known in the art.

[0475] In some embodiments, the first ligand comprises IMIGVLVGV (SEQ ID NO: 2). In some embodiments, the first ligand is complexed with a major histocompatibility complex comprising the human leukocyte antigen A*02 allele (HLA-A*02).

[0476] In some embodiments, the plurality of cancer cells comprises the TNFRSF11A 192A allele at rs1805034, and the ligand binding domain of the second receptor has a higher affinity for a TNFRSF11A ligand having a V at position 192 of SEQ ID NO: 13 than for a TNFRSF11A ligand having an A at position 192 of SEQ ID NO: 13.

[0477] In some embodiments, the plurality of cancer cells comprises the TNFRSF11A 192V allele at rs1805034, and the ligand binding domain of the second receptor has a higher affinity for a TNFRSF11A ligand having an A at position 192 of SEQ ID NO: 13 than for a TNFRSF11A ligand having a V at position 192 of SEQ ID NO: 13.

[0478] In some embodiments, the plurality of cancer cells comprises the TNFRSF11A 141H allele at rs35211496, and the ligand binding domain of the second receptor has a higher affinity for a TNFRSF11A ligand having a Y at position 141 of SEQ ID NO: 13 than for a TNFRSF11A ligand having an H at position 141 of SEQ ID NO: 13.

[0479] In some embodiments, the plurality of cancer cells comprises the TNFRSF11A 141Y allele at rs35211496, and the ligand binding domain of the second receptor has a higher affinity for a TNFRSF11A ligand having an H at position 141 of SEQ ID NO: 13 than for a TNFRSF11A ligand having a Y at position 141 of SEQ ID NO: 13.

[0480] In some embodiments, the plurality of cancer cells comprises an ITGAE 950R allele at rs1716, and the ligand-binding domain of the second receptor has a higher affinity for an ITGAE ligand having a W at position 950 of SEQ ID NO: 14 than for an ITGAE ligand having an R at position 950 of SEQ ID NO: 14.

[0481] In some embodiments, the cancer cells comprise ITGAE 950W at rs1716, and the ligand-binding domain of the second receptor has a higher affinity for an ITGAE ligand having an R at position 950 of SEQ ID NO: 14 than for an ITGAE ligand having a W at position 950 of SEQ ID NO: 14.

[0482] In some embodiments, the plurality of cancer cells comprises the ITGAE 1019V allele at rs2976230, and the ligand-binding domain of the second receptor has a higher affinity for an ITGAE ligand having an A or G at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand having a W at position 1019 of SEQ ID NO: 14.

[0483] In some embodiments, the plurality of cancer cells comprises the ITGAE 1019A allele at rs2976230, and the ligand-binding domain of the second receptor has a higher affinity for an ITGAE ligand having a V or G at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand having an A at position 1019 of SEQ ID NO: 14.

[0484] In some embodiments, the plurality of cancer cells comprises the ITGAE 1019G allele at rs2976230, and the ligand-binding domain of the second receptor has a higher affinity for an ITGAE ligand having a V or A at position 1019 of SEQ ID NO: 14 than for an ITGAE ligand having a G at position 1019 of SEQ ID NO: 14.

[0485] In some embodiments, the immune cell is a T cell.

[0486] In some embodiments, the immune cells are allogeneic or autologous.

[0487] In some embodiments, the second receptor increases the specificity of the immune cell for CEA-positive cancer cells compared to immune cells that express the first receptor but not the second receptor. In some embodiments, the immune cell has reduced side effects compared to immune cells that express the first receptor but not the second receptor.

[0488] Treatment of cancer can result in a reduction in tumor size. A reduction in tumor size is sometimes referred to as "tumor regression." Preferably, after treatment, the tumor size is reduced by 5% or more compared to the size before treatment, more preferably, the tumor size is reduced by 10% or more, more preferably, by 20% or more, more preferably, by 30% or more, more preferably, by 40% or more, even more preferably, by 50% or more, and most preferably, by 75% or more. Tumor size can be measured by any reproducible means of measurement. Tumor size can be measured as the diameter of the tumor.

[0489] Treatment of cancer can result in a reduction in tumor volume. Preferably, after treatment, tumor volume is reduced by 5% or more compared to pre-treatment size, more preferably by 10% or more, more preferably by 20% or more, more preferably by 30% or more, more preferably by 40% or more, even more preferably by 50% or more, and most preferably by 75% or more. Tumor volume can be measured by any reproducible means of measurement.

[0490] Treating cancer results in a reduction in the number of tumors. Preferably, after treatment, the number of tumors is reduced by 5% or more compared to the number before treatment, more preferably, the number of tumors is reduced by 10% or more, more preferably, by 20% or more, more preferably, by 30% or more, more preferably, by 40% or more, even more preferably, by 50% or more, and most preferably, by 75% or more. The number of tumors can be measured by any reproducible measurement means. The number of tumors can be measured by counting tumors visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0491] Treatment of cancer can result in a reduction in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or more compared to the number before treatment, more preferably by 10% or more, more preferably by 20% or more, more preferably by 30% or more, more preferably by 40% or more, even more preferably by 50% or more, and most preferably by 75% or more. The number of metastatic lesions can be measured by any reproducible measurement means. The number of metastatic lesions can be measured by counting metastatic lesions visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0492] Cancer treatment can result in an increase in the average survival time of a treated population compared to a population receiving only a carrier. Preferably, the average survival time increases by more than 30 days, more preferably by more than 60 days, more preferably by more than 90 days, and most preferably by more than 120 days. The increase in the average survival time of a population can be measured by any reproducible means. For example, the increase in the average survival time of a population can be measured by calculating the average survival time of a population after the start of treatment with an active compound. The increase in the average survival time of a population can also be measured by, for example, calculating the average survival time of a population after the completion of the first round of treatment with an active compound.

[0493] Treatment of cancer can result in an increase in the average survival time of a population of treated subjects compared to a population of untreated subjects.Preferably, the average survival time increases by more than 30 days, more preferably by more than 60 days, more preferably by more than 90 days, and most preferably by more than 120 days.The increase in the average survival time of a population can be measured by any reproducible means.The increase in the average survival time of a population can be measured, for example, by calculating the average survival time of a population after the start of treatment with an active compound.The increase in the average survival time of a population can also be measured, for example, by calculating the average survival time of a population after the completion of the first round of treatment with an active compound.

[0494] Treatment of cancer can result in an increase in the average survival time of a treated population compared to a population receiving monotherapy with a drug other than the compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, analog, or derivative thereof. Preferably, the average survival time increases by more than 30 days, more preferably by more than 60 days, more preferably by more than 90 days, and most preferably by more than 120 days. The increase in average survival time of a population can be measured by any reproducible means. For example, the increase in average survival time of a population can be measured by calculating the average survival time of a population after the start of treatment with an active compound. The increase in average survival time of a population can also be measured, for example, by calculating the average survival time of a population after the completion of the first round of treatment with an active compound.

[0495] Treating cancer may result in a reduction in mortality in a treated population compared to a population receiving carrier alone. Treating cancer may result in a reduction in mortality in a treated population compared to an untreated population. Treating cancer may result in a reduction in mortality in a treated population compared to a population receiving monotherapy with a drug other than a compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, analog, or derivative thereof. Preferably, the mortality rate is reduced by more than 2%, more preferably more than 5%, more preferably more than 10%, and most preferably more than 25%. The reduction in mortality in a treated population can be measured by any reproducible means. The reduction in mortality in a population can be measured, for example, by calculating the average number of disease-related deaths per unit time for the population after initiation of treatment with the active compound. The reduction in mortality in a population can also be measured, for example, ...

Claims

1. An immune cell, a. a first receptor comprising an extracellular ligand-binding domain specific for CEA cell adhesion molecule 5 (CEA), the first receptor being a chimeric antigen receptor (CAR) comprising a hinge, a transmembrane domain and an activator intracellular domain; b. a second receptor comprising an extracellular ligand-binding domain specific for HLA-A*02, the second receptor being a chimeric antigen receptor (CAR) comprising a hinge, a transmembrane domain and an inhibitory intracellular domain; The first receptor is an activator receptor that responds to CEA, and the second receptor is an inhibitory receptor that responds to HLA-A*02; the extracellular ligand-binding domain of the first receptor comprises a heavy chain variable (VH) portion comprising complementarity determining regions (CDRs) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NOs: 55, 56 and 57, respectively, and a light chain variable (VL) portion comprising CDRs CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NOs: 59, 61 and 63, respectively; 1. An immune cell, wherein the extracellular ligand binding domain of the second receptor comprises a VH portion comprising CDRs CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NOs: 106, 107 and 108, respectively, and a VL portion comprising CDRs CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NOs: 103, 104 and 105, respectively, wherein the immune cell is a T cell.

2. 2. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the first receptor comprises a VH portion comprising SEQ ID NO: 144, or a sequence having at least 85% identity thereto, and a VL portion comprising SEQ ID NO: 148, or a sequence having at least 85% identity thereto.

3. 2. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the second receptor comprises a VH portion comprising SEQ ID NO: 981, or a sequence having at least 85% identity thereto, and a VL portion comprising SEQ ID NO: 166, or a sequence having at least 85% identity thereto.

4. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the first receptor comprises an scFv sequence of SEQ ID NO: 68 or a sequence having at least 85% identity thereto.

5. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the second receptor comprises an scFv sequence of SEQ ID NO: 91 or a sequence having at least 85% identity thereto.

6. The immune cell of claim 1 , wherein the first receptor comprises a CD8α hinge domain.

7. The immune cell of claim 6, wherein the CD8α hinge domain of the first receptor comprises a sequence of SEQ ID NO: 71, or a sequence having at least 85% identity thereto.

8. The immune cell of claim 1 , wherein the transmembrane domain of the first receptor comprises a CD28 transmembrane domain.

9. The immune cell of claim 8, wherein the CD28 transmembrane domain of the first receptor comprises a sequence of SEQ ID NO: 75, or a sequence having at least 85% identity thereto.

10. The immune cell of claim 1, wherein the activator intracellular domain of the first receptor comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain.

11. The immune cell of claim 10, wherein the activator intracellular domain of the first receptor comprises a sequence of SEQ ID NO: 158, or a sequence having at least 85% identity thereto.

12. The immune cell of claim 1, wherein the first receptor comprises a sequence of SEQ ID NO: 52, or a sequence having at least 90% identity thereto.

13. The immune cell of claim 1 , wherein the inhibitory factor intracellular domain of the second receptor comprises a LILRB1 intracellular domain.

14. The immune cell of claim 13, wherein the LILRB1 intracellular domain comprises a sequence that is at least 90% identical to or identical to SEQ ID NO:

131.

15. The immune cell of claim 1 , wherein the second receptor comprises a LILRB1 transmembrane domain.

16. The immune cell of claim 15, wherein the LILRB1 transmembrane domain comprises a sequence that is at least 90% identical to or identical to SEQ ID NO:

135.

17. The immune cell of claim 1 , wherein the second receptor comprises a LILRB1 hinge domain.

18. The immune cell of claim 17, wherein the LILRB1 hinge domain comprises a sequence at least 90% identical to or identical to SEQ ID NO:

134.

19. The immune cell of claim 1, wherein the second receptor comprises a sequence of SEQ ID NO: 164, or a sequence having at least 90% identity thereto.

20. A pharmaceutical composition comprising a therapeutically effective amount of the immune cells of claim 1 and a pharma- ceutically acceptable carrier, diluent, or excipient.

21. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the second receptor comprises a VH portion comprising SEQ ID NO: 981, or a sequence having at least 95% identity thereto, and a VL portion comprising SEQ ID NO: 166, or a sequence having at least 95% identity thereto.

22. 22. The immune cell of claim 21 , wherein the extracellular ligand-binding domain of the first receptor comprises a VH portion comprising SEQ ID NO: 144, or a sequence having at least 95% identity thereto, and a VL portion comprising SEQ ID NO: 148, or a sequence having at least 95% identity thereto.

23. The immune cell of claim 21 , wherein the extracellular ligand-binding domain of the first receptor comprises an scFv sequence of SEQ ID NO: 68 or at least 95% identical thereto.

24. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the second receptor comprises an scFv sequence of SEQ ID NO: 91 or a sequence having at least 95% identity thereto.

25. The immune cell of claim 1, wherein the extracellular ligand-binding domain of the second receptor comprises a VH portion comprising SEQ ID NO: 981 and a VL portion comprising SEQ ID NO:

166.

26. The immune cell of claim 25, wherein the extracellular ligand-binding domain of the first receptor comprises a VH portion comprising SEQ ID NO: 144 and a VL portion comprising SEQ ID NO:

148.

27. The immune cell of claim 1, wherein the extracellular ligand binding domain of the first receptor comprises an scFv sequence of SEQ ID NO: 68 and the extracellular ligand binding domain of the second receptor comprises an scFv sequence of SEQ ID NO: 91.

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