Compositions and methods for treating cancers

Abstract

The disclosure provides engineered T cells comprising a first activator receptor specific to a target antigen, and a second inhibitory receptor specific to a non-target antigen, and methods of making and using same for the treatment of cancer.

Description

COMPOSITIONS AND METHODS FOR TREATING CANCERSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent applications 63 / 677,990, filed on July 31, 2024, 63 / 716,045, filed on November 4, 2024, 63 / 815,413, filed on May 30, 2025, and 63 / 815,409, filed on May 30, 2025 the contents of which are incorporated herein by reference in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 061250-565001WO, created on July 31, 2025 and is 1,460 kilobytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.TECHNICAL FIELD

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

[0004] Logic-gated chimeric antigen T cells are engineered for more selective tumor killing. However, the safety and efficacy of such engineered T cells may depend on correct dosing and other factors.

[0005] Provided herein are methods and composition for cancer treatment with logic-gated Logic-gated chimeric antigen T cells.SUMMARY

[0006] The present disclosure provides methods for treating solid tumors using engineered T cells. In certain aspects, the methods involve administering to a subject a therapeutically effective amount of engineered T cells that express a first receptor specific for a target antigen and a second receptor specific for a non-target antigen. The first receptor includes an intracellular signaling domain that activates and / or co-stimulates the T cell, while the second receptor includes an intracellular domain that inhibits T cell activity. In some embodiments,the non-target antigen is an allelic variant of a polymorphic cell surface protein or a major histocompatibility complex (MHC) protein that is absent from tumor cells but present on normal tissue.

[0007] The methods may further include administering the engineered T cells following a preconditioning lymphodepletion (PCLD) regimen. The PCLD regimen may include administration of cyclophosphamide and / or fludarabine, alone or in combination, at various dosages and schedules. Exemplary regimens include:Fludarabine at doses ranging from 25 mg / m2 / day to 30 mg / m2 / day, administered for 3 to 5 consecutive days;Cyclophosphamide at doses ranging from 250 mg / m2 / day to 750 mg / m2 / day, administered for 2 to 3 consecutive days, or at 60 mg / kg / day for 2 days;In some embodiments, fludarabine is administered for 4 or 5 days, and cyclophosphamide is administered for 2 or 3 days, either concurrently or sequentially.

[0008] In certain embodiments, the engineered T cells are administered approximately 2 days after completion of the lymphodepletion regimen. The methods may also include administration of recombinant interleukin-2 (IL-2) or a fragment thereof, optionally beginning 3 to 24 hours after T cell infusion and continuing every 12 hours over a 7-day period. The IL-2 dose may range from about 500,000 to about 2,000,000 IU, including a preferred dose of 1,000,000 IU.

[0009] The disclosure also encompasses a range of dosing regimens for the engineered T cells, including doses from about 0.25 x io8to about 36 x io8cells, with specific embodiments tailored to subject characteristics such as body weight (e.g., less than or greater than 50 kg).

[0010] In another aspect, the disclosure provides a method of treating a carcinoembryonic antigen-positive (CEA+) solid tumor in a subject comprising administering to the subject an effective amount of an engineered T cell, wherein the engineered T cell comprises: a first receptor, comprising an extracellular ligand binding domain specific to the CEA and an intracellular domain comprising at least one signal transduction element that activates and / or co-stimulates the engineered T cell; and a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen lost in a cancer cell and an intracellular domain comprising at least one signal transduction element that inhibit the engineered T cell.

[0011] In another aspect, the disclosure provides a method of treating a mesothelin antigenpositive (MSLN+) solid tumor in a subject, comprising administering to the subject an effective amount of an engineered T cell, wherein the engineered T cell comprises:a first receptor, comprising an extracellular ligand binding domain specific to the MSLN and an intracellular domain comprising at least one signal transduction element that activates and / or co-stimulates the engineered T cell; and a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen lost in a cancer cell and an intracellular domain comprising at least one signal transduction element that inhibit the engineered T cell.

[0012] In another aspect, the disclosure provides a method of treating a receptor tyrosineprotein kinase erbB-2 (HER2+) solid tumor in a subject comprising administering to the subject an effective amount of an engineered T cell, wherein the engineered T cell comprises: a first receptor, comprising an extracellular ligand binding domain specific to the HER2 and an intracellular domain comprising at least one signal transduction element that activates and / or co-stimulates the engineered T cell; and a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen lost in a cancer cell and an intracellular domain comprising at least one signal transduction element that inhibit the engineered T cell.

[0013] The data supporting the disclosed methods and dosing regimens are derived from ongoing clinical trials.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a pair of diagrams showing discrimination between tumor and normal tissue using loss of heterozygosity (LOH). Engineered immune cells kill tumors but spare normal cells. In the case of an illustrative embodiment, immune cells express CEA CAR, the activator antigen is CEA, and the blocker antigen is HLA-A*02. Patients with germline heterozygosity of HLA-A*02 and clonal LOH of HLA-A*02 in tumors are selected.

[0015] FIG. 2A is a diagram showing the molecular composition of an illustrative dual receptor system of the disclosure, comprising a CEA CAR and an HLA-A*02 scFv LILRB1 inhibitory receptor. The Tmod format shown in this figure is also consistent with herein disclosed MSLH-directed Tmods and Her2-directed Tmods.

[0016] FIG. 2B is a diagram showing the molecular composition of an illustrative dual receptor system of the disclosure, comprising a MSLN CAR and an HLA-A*02 scFv LILRB1 inhibitory receptor. The Tmod format shown in this figure is also consistent with herein disclosed MSLN-directed Tmods and Her2-directed Tmods.

[0017] FIG. 3 shows that the CEA activator and HL A- A* 02 LILRB1 inhibitory receptor function in Jurkat cells using engineered HeLa cells as targets for cytotoxicity. A*02: HLA- A*02; Tmod: the cells express the CEA CAR and the HLA-A*02 inhibitory receptor; CAR: cells express the CEA CAR only.

[0018] FIG. 4 shows that the CEA activator and HL A- A* 02 LILRB1 inhibitory receptor function in donor T cells from a single donor on HeLa cells. Tmod: the cells express the CEA CAR and the HLA-A*02 inhibitory receptor; CAR: cells express the CEA CAR only.

[0019] FIG. 5 shows that the CEA activator and HL A- A* 02 LILRB1 inhibitory receptor function in T cells from four donors on HeLa cells. Tmod: the cells express the CEA CAR and the HL A- A* 02 inhibitory receptor; CAR: cells express the CEA CAR only. Target cells are HeLa cells expressing CEA only or CEA and HL A- A* 02.

[0020] FIG. 6 shows CEA CAR activator and HLA-A*02 LILRB1 blocker sensitivity measured as a function of the number of CEA surface molecules in HeLa cells using Jurkat effector cells with stably expressed CEA activator and HLA-A*02 blocker receptors.

[0021] FIG. 7 shows sensitivity of activator and blocker of primary T cells expressing CEA CAR Tmod (both the CEA CAR and HLA-A*02 and LILRB1 inhibitory receptors), CAR- only, and CEA TCR. The dose response curve for the activator (right) is shown for the CEA CAR, CEA CAR with the HLA-A*02 blocker (Tmod), and the CEA TCR, while the dose response curve for the inhibitory receptor (blocker) is only for the CEA CAR and the CEA CAR with the HLA-A*02 blocker (Tmod). A*02: HLA-A*02.

[0022] FIG. 8 shows images of five mice from each group (a subset of those in FIG. 40) which were used to measure bioluminescence (luciferase) over time. One Tmod mouse (2ndfrom the left, day64) did not receive BLI substrate by mistake.

[0023] FIG. 9 shows that CRISPR using a guide RNA (gRNA) to B2M and a B2M shRNA reduce HLA expression on cell surface and increase blocker receptor availability in HLA- A*02(+) T cells.

[0024] FIG. 10 shows the effect of a B2M shRNA construct on cis binding for the 1 st generation autologous T cells expressing the CEA CAR and HLA-A*02 scFvLILRBl inhibitory receptor (Tmod).

[0025] FIG. 11 shows 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.

[0026] FIG. 12 shows a graph of a cytotoxicity assay of tumor cells in mixed cultures with Normal Cells and Tmod cells. Abbreviations: AB=MSLN(+)HLA-A*02(+); B-only=MSLN(- )HLA-A*02(+); CAR=chimeric antigen receptor; E:T=effector-to-target ratio; FLuc=firefly luciferase; hrs=hours; RLuc=renilla luciferase; % Kill=percent specific killing. Panel A: Baseline specific killing 48 hours after co-culture between unmixed target cells and MSLN Tmod at E:T=0.5: l measured by dual luciferase-endpoint readouts. Panel B: Specific killing measurement after 48 hours of MSLN Tmod in mixed culture with tumor cells and ABnormal cells at E:T ratio of 0.5: 1 (based on total target cells) measured by dual luciferaseendpoint readouts. Panel C: Specific killing measurement after 48 hours of MSLN Tmod in mixed culture with tumor cells and B-only normal cells at E:T ratio of 0.5: 1 (based on total target cells) measured by dual luciferase-endpoint readouts. Tumor cells stably express FLuc, while AB normal and B-only normal cells both stably express RLuc. Each symbol represents a unique donor (data shown is mean ± standard deviation; n=3 donors) with each donor’s symbol representing an average of 3 technical replicates.

[0027] FIG. 13 shows bar graphs of activity and selectivity cytotoxicity assays of MSLN Tmod for Tumor vs Normal Cells in the Presence of Soluble MSLN. Endpoint luciferase readout after 48 hours of coculture with MSLN Tmod and HeLa target cells at a 0.5: 1 effective E:T ratio in the presence of 0, 20, 200 or 2000 ng / mL of sMSLN. Each symbol represents a unique donor (data shown is mean ± standard deviation; n=3 donors) with each donor’s symbol representing an average of 3 technical replicates. Abbreviations: AB=MSLN(+)HLA-A*02(+); B-only=MSLN(-)HLA-A*02(+); E:T=effector-to-target ratio; hrs=hours; sMSLN=soluble MSLN; % Kill=percent specific killing.

[0028] FIG. 14 shows graphs of in vivo efficacy and selectivity of MSLN Tmod and M5 CAR T cells across a 40-fold dose range in a xenograft mouse model. NSG mice with dualflank xenografts representing HLA-A*02(-) tumor and HLA-A*02(+) normal cells were treated with saline or MSLN Tmod, M5 CAR T cells, or UTD T cells from Donor 204382. Mice were treated with 1 of 5 different doses of transduced cells or an equivalent total number of UTD T cells (dose groups are listed from high to low, top to bottom; vertical black line indicates time of T-cell injection). Xenograft volumes were measured at each time point by caliper. Shaded areas indicate the onset of allo- and / or xeno-reactivity as indicated by the reduction of graft volume in the UTD T cell-treated mice in the top 2 doses. All mice, except those which reached maximum-permitted xenograft burden earlier were euthanized on day 27. N=7 mice per group, except for saline group where n=5 mice (data shown are mean ± SEM).. Abbreviations: CAR=chimeric antigen receptor; HLA-A*02=human leukocyte antigen serotype A*02; sub-Q=subcutaneous; UTD=untransduced.

[0029] FIG. 15 is a graph depicting the number of copies of the blocker receptor DNA per pg of gDNA. The number of copies of blocker receptor DNA was measured by digital drop PCR (ddPCR) at the indicated time points before and after infusion with engineered T cells. Plotted values represent mean and standard deviation. (SCR=screening and LD= lymphodepletion chemotherapy). Each line represents an individual patient treated with the engineered T cells. The a indicates the patient received half dose due to body weight. DL,Dose level; MSLN, mesothelin; NSLC, non-small cell lung cancer; OVC, ovarian cancer; PANC, pancreatic cancer; PD, progressive disease; PR, partial response SD, stable disease.

[0030] FIG. 16A is a graph depicting the level of IFN-y in the serum of patients treated with engineered T cells.

[0031] FIG. 16B is a graph depicting the level of Interleukin- 15 in the serum of patients treated with engineered T cells.

[0032] FIG. 16C is a graph depicting the level of Interleukin-6 in the serum of patients treated with engineered T cells.

[0033] FIG. 17 is a series of CT scans from participant five treated with CAR-T cells expressing the MSLN activator and HLA-A*02 inhibitor receptor of the present disclosure. Participant five has KRAS G12V / STK111 commutated NSLC adenocarcinoma that progress on carboplatin, pemetrexed and pembrolizumab treatment. Participant five achieved partial response at day 90 post infusion. Imaging was performed before cell infusion and 30 days and 90 days after infusion.

[0034] FIG. 18A is a series of CT scans from a patient treated with CEA Tmod CAR-T cells of the present disclosure. Imaging was performed before cell infusion and 30 days and 90 days after infusion.

[0035] FIG. 18B is a graph quantifying the number of copies of blocker DNA per μg of genomic DNA (gDNA) from peripheral blood of the patient treated with CEA Tmod CAR-T cells in FIG. 18A. Peripheral blood samples were harvested at the indicated time points. Digital drop PCR (ddPCR) was done to detect blocker DNA. The level of IFN-γ, granzyme B and TNFα was also determined from the same peripheral blood samples.

[0036] FIG. 19 is a graph depicting the number of copies of the blocker receptor DNA per pg of gDNA. The number of copies of blocker receptor DNA was measured by digital drop PCR (ddPCR) at the indicated time points before and after infusion with engineered T cells. Plotted values represent mean and standard deviation. (SCR=screening and PCLD=preconditioning lymphodepletion). Each line represents an individual patient treated with the engineered T cells. Patient 14 from DL5 had the highest number of copies of Blocker receptor DNA per μg of gDNA on day three. Patient 12 from DL4 had the second highest number of copies of Blocker receptor DNA per μg of gDNA on day three. Patient 11 from DL4 and Patient 13 from DL5 had the third highest number of copies of Blocker receptor DNA per μg of gDNA on day three.

[0037] FIG. 20 is a graph depicting the level of IFN-γ in the serum of patients treated with engineered T cells. Patient 14 from DL5 had the highest concentration of interferon-y on day nine. Patient 5 from DL2 had the second highest concentration of interferon-y on day nine.

[0038] FIG. 21 is a graph depicting the level of interleukin-6 in the serum of patients treated with engineered T cells. Patient 14 from DL5 had the highest concentration of interleukin-6 on day nine.

[0039] FIG. 22 is a graph depicting the level of interleukin- 15 in the serum of patients treated with engineered T cells. Patient 14 from DL5 had the highest concentration of interleukin-15 on day seven. The order of samples from highest to lowest concentration of interleukin-15 on day seven is: Patient 14, Patient 12, Patient 2, Patient 11, Patient 3, Patient 13, Patient 7, Patient 6, Patient 8, Patient 4, Patient 1, Patient 5, and Patient 9.

[0040] FIG. 23A is a graph depicting the number and severity of gastrointestinal adverse events over time. Each grade 1 adverse event is noted by a circle, each grade 2 adverse event is marked by a triangle, and each grade 3 adverse event is marked by square. PCLD treatment began on day zero. Infusion of engineered T cell began on day six. Each bar represents a single patient, and their dose level is indicated (e.g. DL1, DL2, DL3, DL4, or DL5 as identified in FIG. 19 to FIG. 22).

[0041] FIG. 23B is a graph depicting the number and severity of gastrointestinal adverse events over time. Each grade 1 adverse event is noted by a circle, each grade 2 adverse event is marked by a triangle, and each grade 3 adverse event is marked by square. PCLD treatment began on day zero. Infusion of engineered T cell began on day six. Each bar represents a single patient, and their dose level is indicated (e.g. DL1, DL2, DL3, DL4, or DL5 as identified in FIG. 19 to FIG. 22).

[0042] FIG. 24 shows the CT scan of a patient at Baseline and Day 90 after MSLN Tmod CAR-T cell dosing in Example 25.

[0043] FIG. 25 shows the CT scan of a patient at Baseline and Day 90 after MSLN Tmod CAR-T cell dosing in Example 25.

[0044] FIG. 26 shows the PET-CT scan of a patient at pre-baseline (Day -115) and Day 90 after MSLN Tmod CAR-T cell dosing in Example 25.

[0045] FIG. 27 is a graph depicting the number of copies of the blocker receptor DNA per pg of gDNA. The number of copies of blocker receptor DNA was measured by digital drop PCR (ddPCR) at the indicated time points before and after infusion with engineered T cells. Plotted values represent mean and standard deviation. (SCR=screening and LD= lymphodepletion chemotherapy). Each line represents an individual patient treated with theengineered T cells. The a indicates the patient received half dose due to body weight. DL, Dose level; MSLN, mesothelin; NSLC, non-small cell lung cancer; OVC, ovarian cancer; PANC, pancreatic cancer; PD, progressive disease; PR, partial response SD, stable disease.

[0046] FIG. 28 is a set of graphs measuring engineered immune cells, serum cytokine levels, and other biomarkers through day 180 in peripheral blood samples for a patient 5 with aggressive, metastatic NSCLC.DETAILED DESCRIPTION

[0047] Provided herein are compositions and methods for treating cancers using engineered T cells comprising a two-receptor system responsive to differences in gene expression of a ligand between cancer and normal, wild type cells. These differences in expression can be due to loss of heterozygosity in the cancer cells. Alternatively, the differences in expression can be because the gene expression is not expressed in cancer cells, or is expressed in cancer cells at a lower level than normal cells. The two-receptor system is expressed in engineered T cells, for example T cells used in adoptive cell therapy, and targets activity of these T cells to cancer cells exhibiting loss of heterozygosity or expression differences. In this two-receptor system, the first receptor (an activator receptor, sometimes referred to herein as an A module) activates, or promotes activation of the T cell, while the second receptor (an inhibitory receptor, sometimes referred to herein as a blocker, or inhibitor receptor, or a B module) acts to inhibit activation of the T cell by the first receptor. Each receptor contains a ligand-binding domain (LBD) that binds a specific ligand. Signals from the two receptors upon ligand binding are integrated by the T cell. Differential expression of ligands for the first and second receptors in cancer and normal cells, for example through loss of heterozygosity of the locus encoding the inhibitory ligand in cancer cells, or differences in transcription levels, mediates activation of T cells by target cancer cells that express the first activator ligand but not the second inhibitory ligand.

[0048] In variations, the compositions and methods described herein can be used to kill target cells and / or treat subjects in which expression of the non-target antigen is partially or completely decreased by causes other than loss of heterozygosity, including but not limited to partial gene deletion, epigenetic silencing, and point mutations or truncating mutations in the sequence encoding the non-target antigen.Definitions

[0049] Prior to setting forth this disclosure in more detail, it can be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0051] As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0052] As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. As also used herein, in any instance or embodiment described herein, “comprising” may be replaced with “consisting essentially of’ and / or “consisting of’ used herein, in any instance or embodiment described.

[0053] As used herein, the phrases “at least one”, “one or more”, and “and / or” are open- ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0054] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0055] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered tohave specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96- 99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

[0056] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0057] As used herein, the term “isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated.

[0058] In embodiments, the terms “obtained” or “derived” mean selecting a known amino acid sequence and including the known amino acid sequence into an antigen binding domain, ligand binding domain, construct, receptor, polynucleotide, or polypeptide of the present disclosure. As an example, the expression a “ligand binding domain may be derived from commercially available antibodies” may be interpreted as incorporating the amino acid sequence for one or more complement determining regions (CDRs) of the commercially available antibodies into an antigen binding domain, ligand binding domain, construct, receptor, or polypeptide of the present disclosure. Additionally, a VH or VL of one or more commercially available antibodies may be incorporated into an antigen binding domain, ligand binding domain, construct, receptor, or polypeptide of the present disclosure. A known amino acid sequence comprises a non-antigen binding domain of a commercially available antibody; here the non-antigen binding domain is incorporated into a construct, receptor, or polypeptide of the present disclosure. In some embodiments, “incorporating” comprises including the nucleic acid sequence encoding the known amino acid sequence into a polynucleotide that encodes an antigen binding domain, ligand binding domain, construct, receptor, or polypeptide of the present disclosure.

[0059] The terms “subject,” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0060] As used herein “treatment” or “treating,” includes any beneficial or desirable effect, and can include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0061] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of disease prior to onset or recurrence.

[0062] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a virus to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0063] A “therapeutically effective amount” of a virus or cell can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).

[0064] An “increased” or “enhanced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a “statistically significant” amount, and can include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0065] A “decreased” or “reduced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a “statistically significant”amount, and can include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0066] By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule / composition. A comparable response is one that is not significantly different or measurable different from the reference response.

[0067] As used herein, a “polynucleotide system” refers to one or more polynucleotides. The one or more polynucleotides can be designed to work in concert for a particular application, or to produce a desired transformed cell.

[0068] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0069] The term “MOI” is used herein to refer to multiplicity of infection, which is the ratio of agents (e.g. viral particles) to infection targets (e.g. cells).

[0070] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.

[0071] As used herein, a “target cell” refers to cell that is targeted by an adoptive cell therapy. For example, a target cell can be cancer cell, which can be killed by the transplanted T cells of the adoptive cell therapy. Target cells of the disclosure express a target antigen, as described herein, and do not express a non-target antigen.

[0072] As used herein, a “non-target cell” refers to cell that is not targeted by an adoptive cell therapy. For example, in an adoptive cell targeting cancer cells, normal, healthy, non- cancerous cells are non-target cells. Some, or all, non-target cells in a subject can express both the target antigen and the non-target antigen. Non-target cells in a subject can express the non-target antigen irrespective of whether or not these cells also express the target antigen.

[0073] As used herein, a “non-target allelic variant” refers to an allele of a gene whose product is expressed by non-target cells, but is not expressed by target cells. For example, anon-target allelic variant is an allele of a gene that is expressed by normal, non-cancer cells of subject, but not expressed by cancer cells of the subject. The expression of the non-target allelic variant can be lost in the cancer cells by any mechanism, including, but not limited to, loss of heterozygosity, mutation, or epigenetic modification of the gene encoding the non- target allelic variant.

[0074] As used herein, “specific to” or “specifically binds to” when used with respect to a ligand binding domain, such as an antigen binding domain, refers to a ligand binding domain that has a high specificity for a named target. Antibody specificity can be viewed as a measure of the goodness of fit between the ligand binding domain and the corresponding ligand, or the ability of the ligand binding domain to discriminate between similar or even dissimilar ligands. In comparison with specificity, affinity is a measure of the strength of the binding between the ligand binding domain and ligand, such that a low-affinity ligand binding domain binds weakly and high-affinity ligand binding domain binds firmly. A ligand binding domain that is specific to a target allele is one that can discriminate between different alleles of a gene. For example, a ligand binding domain that is specific to HLA-A*03 will not bind, or bind only weakly to, other HLA-A alleles such as HLA-A*01 or HLA-A*02. The person of skill in the art will appreciate that a ligand binding domain can be said to be specific to a particular target, and yet still have low levels of binding to one or more additional targets that do not affect its function in the receptor systems described herein.

[0075] As used herein, a “target antigen,” whether referred to using the term antigen or the name of a specific antigen, refers to an antigen expressed by a target cell, such as a cancer cell. Expression of target antigen is not limited to target cells. Target antigens can be expressed by both cancer cells and normal, non-cancer cells in a subject.

[0076] As used herein, a “non-target antigen” (or “blocker antigen”) whether referred to using the term antigen or the name of a specific antigen, refers to an antigen that is expressed by normal, non-cancer cells and is not expressed in cancer cells. This difference in expression allows the inhibitory receptor to inhibit immune cell activation in the presence of non-target cells, but not in the presence of target cells.

[0077] Polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population. A polymorphism can comprise one or more base changes, an insertion, a repeat, or a deletion. A polymorphism includes e.g., a simple sequence repeat (SSR) and a single nucleotide polymorphism (SNP), which is a variation, occurring when a single nucleotide of adenine (A), thymine (T), cytosine (C) or guanine (G) is altered.

[0078] As used herein, “affinity” refers to strength of binding of a ligand to a single ligand binding site on a receptor, for example an antigen for the antigen binding domain of any of the receptors described herein. Ligand binding domains can have a weaker interaction (low affinity) with their ligand, or a stronger interaction (high affinity).

[0079] As used herein, “HLA-A*02 antibody” and “anti -HL A- A* 02 antibody” are used interchangeably and refer to an antibody that specifically binds to an HLA-A*02 polypeptide. Similarly, the term “HLA-A*02”, as mentioned in “HLA-A*02 binding”, “HLA-A*02 targeting”, “HLA-A*02 specific”, “HLA-A*02 expression” or other similar terms here, refers to HLA-A*02 polypeptide. The term “A*02” is understood to refer to HLA-A*02 in any instance. HLA-A*02 antibodies of the disclosure may bind to proteins falling within the HLA-A*02 allele group (for example HLA-A*02:01, HLA-A*02:02 and the like), and may not bind, or bind with lower affinity, to proteins falling within other HL A- A allele groups (for example, HLA-A*01, HLA- A* 11 , and the like). The person of ordinary skill in the art will recognize that some cross-reactivity with other HLA antigens may exist, but that the HLA-A*02 antibodies of the disclosure will still be considered to be specific to HLA-A*02. In some cases, specificity is considered in the context of the subject to be treated with an HLA-A*02 antibody or receptor of the disclosure. When the subject has both an HLA-A*02 allele and a second HLA-A allele not recognized, or only poorly recognized, by the HLA-A*02 antibody or receptor comprising an equivalent antigen binding domain, the HLA-A*02 antibody is specific to the HLA-A*02 allele of the subject.

[0080] As used herein, a receptor that is “responsive” or “responsive to” refers to a receptor comprising an intracellular domain, that when bound by a ligand (i.e. antigen) generates a signal corresponding to the known function of the intracellular domain. An activator receptor bound to a target antigen can generate a signal that causes activation of an immune cell expressing the activator receptor. An inhibitory receptor bound to a non-target antigen can generate an inhibitory signal that prevents or reduces an activation of an immune cell expressing the activator receptor. Responsiveness of receptors, and their ability to activate or inhibit immune cells expressing the receptors, can be assayed by any means known in the art and described herein, including, but not limited to, reporter assays and cytotoxicity assays.

[0081] As used herein, “activation” of an immune cell or an immune cell that is “activated” refers to an immune cell that can carry out one or more functions characteristic of an immune response. These functions include proliferation, release of cytokines, and cytotoxicity, i.e. killing of a target cell. Activated immune cells express markers that will be apparent to persons of skill in the art. For example, activated T cells can express one or more of CD69, CD71, CD25 and HLA-DR. An immune cell expressing an activator receptor (e.g. a MSLNCAR) can be activated by the activator receptor when it becomes responsive to the binding of the receptor to a target antigen (e.g. MSLN) expressed by the target cell. A “target antigen” can also be referred to an “activator antigen” and can be isolated or expressed by a target cell. Activation of an immune cell expressing an inhibitory receptor can be prevented when the inhibitory receptor becomes responsive to a non-target antigen (e.g. HLA-A*03), even when the activator receptor is bound to the target activator ligand. A “non-target antigen” can also be referred to as an “inhibitory ligand” or a “blocker”, and can be isolated or expressed by a target cell.

[0082] As used herein, the term “immune cell” refers to a cell involved in the innate or adaptive (acquired) immune systems. Exemplary innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, Natural Killer (NK) cells, polymorphonuclear leukocytes such as neutrophils eosinophils and basophils and mononuclear cells such as monocytes, macrophages and mast cells. Immune cells with roles in acquired immunity include lymphocytes such as T-cells and B-cells.

[0083] A responsive receptor expressed by the immune cells described herein can be verified by assays that measure the generation of a signal expected to be generated by the intracellular domain of the receptor.

[0084] As used herein, the term “functional variant” refers to a protein that has one or more amino-acid substitutions, insertions, or deletions as compared to a parental protein, and which retains one or more desired activities of the parental protein. A functional variant can be a fragment of the protein (i.e. a variant having N- and / or C-terminal deletions) that retain the one or more desired activities of the parental protein.

[0085] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.Activator Receptors

[0086] The disclosure provides a first receptor, comprising a first extracellular ligand binding domain specific to a target antigen comprising a cancer cell-specific antigen, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I). Thefirst receptor is an activator receptor, and mediates activation of an immune cell expressing the first receptor upon binding of the target antigen by the extracellular ligand binding domain of the first receptor. The first receptor is responsive to a target antigen (i.e. activator ligand). For example, when a target antigen binds to or contacts the first receptor, the first receptor is responsive and activates an immune cell expressing the first receptor upon binding of the target antigen by the extracellular ligand binding domain of the first receptor. In some embodiments, the first receptor is a chimeric antigen receptor (CAR). In some embodiments, the first receptor is a T cell receptor (TCR). In some embodiments, the first receptor comprises a signal transduction element that activates the immune cell.

[0087] In some embodiments, the first receptor is humanized. As used herein, “humanized” refers to the replacement of a sequence or a subsequence in a transgene that has been isolated or derived from a non-human species with a homologous, or functionally equivalent, human sequence. For example, a humanized antibody can be created by grafting mouse CDRs into human framework sequences, followed by back substitution of certain human framework residues for the corresponding mouse residues from the source antibody.Activator Targets

[0088] In some embodiments, the target antigen for the first receptor is a cancer cell specific antigen. Any cell surface molecule expressed by the target cancer cells can be a suitable target antigen for the first receptor ligand binding domain. For example, a cell adhesion molecule, a cell-cell signaling molecule, an extracellular domain, a molecule involved in chemotaxis, a glycoprotein, a G protein-coupled receptor, a transmembrane, a receptor for a neurotransmitter or a voltage gated ion channel can be used as a target antigen.

[0089] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in a complex with a major histocompatibility complex class I (MHC-I). Any molecule expressed by the target cancer cells and presented by the major histocompatibility complex class I (MHC-I) on the cancer cell surface as a peptide antigen (pMHC) can be a suitable target antigen for the first receptor extracellular ligand binding domain.

[0090] The major histocompatibility complex class I (MHC-I) is a protein complex that displays antigens to cells of the immune system, triggering an immune response. The Human Leukocyte Antigens (HLAs) corresponding to MHC-I are HLA-A, HLA-B and HLA-C.

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

[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 particular number (such as HLA-B27).

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

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

[0095] In some embodiments, the cancer cell-specific antigen is CEA cell adhesion molecule 5 (CEA), or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).

[0096] CEA is a 180-kDa glycoprotein tumor-associated protein expressed by a variety of cancer cells. CEA is a GPI-anchored adhesion molecule composed of repeated immunoglobulin domains. It is used as a biomarker in colon cancer, both as a diagnostic and as a surrogate for treatment response. Cancers that express CEA include adenocarcinomas, colorectal cancers and selected other epithelial cancers, including colorectal adenocarcinomas. However, CEA is also expressed in a variety of normal epithelial cells throughout the gastrointestinal tract, for example in the highly differentiated epithelial cells in the upper third of colonic crypts.

[0097] All isoforms of CEA are envisaged as cancer cell-specific antigens of the disclosure. CEA isoform 1 is described in NCBI record number NP 001278413.1, the contents of which are incorporated by reference herein. In some embodiments, CEA comprises an amino acid sequence of:( SEQ ID NO : 1000 ) .

[0098] 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 to SEQ ID NO: 1000. CEA isoform 2 is described in NCBI record number NP 001295327.1, the contents of which are incorporated by reference herein. In some embodiments, CEA comprises an amino acid sequence of: ( SEQ ID NO : 1001 ) .

[0099] 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 to SEQ ID NO: 1001.

[0100] In some embodiments, the cancer cell-specific antigen is a peptide antigen derived from CEA. In some embodiments, the peptide antigen is 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: 1000. In some embodiments, the peptide antigen comprises a sequence identical to a subsequence of SEQ ID NO: 1000. Exemplary CEA peptide antigens include amino acids 691-699 of SEQ ID NO: 1000 (IMIGVLVGV), amino acids 605-613 of SEQ ID NO: 1000 (YLSGANLNL), and amino acids 694-702 of SEQ ID NO: 1000 (GVLVGVALI). In some embodiments the CEA peptide antigen comprises, or consists essentially of, amino acids 691-699 of SEQ ID NO: 1000 (IMIGVLVGV).

[0101] 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: 1001. In some embodiments, the peptide antigen comprises a sequence identical to a subsequence of SEQ ID NO: 1001. In some embodiments, the CEA peptide antigen is complexed with MHC-I. In some embodiments, the MHC-I comprises a human leukocyte antigen A*02 allele (HLA-A*02).Mesothelin

[0102] In some embodiments, the cancer cell-specific antigen is MSLN, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).

[0103] All isoforms of MSLN are envisaged as cancer cell-specific antigens of the disclosure. MSLN isoform 1 preprotein is described in NCBI record number NP_005814.2, the contents of which are incorporated by reference herein. In some embodiments, MSLN comprises an amino acid sequence of:(SEQ ID NO: 1003).

[0104] In some embodiments, MSLN comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1003.

[0105] MSLN isoform 2 preprotein is described in NCBI record number NP_037536.2, the contents of which are incorporated by reference herein. In some embodiments, MSLN comprises an amino acid sequence of:(SEQ ID NO: 1004).

[0106] In some embodiments, MSLN comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1004.

[0107] In some embodiments, the cancer cell-specific antigen is a peptide antigen derived from MSLN. In some embodiments, the peptide antigen is 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: 1003 and / or SEQ ID NO: 1004. In some embodiments, the peptide antigen comprises a sequence identical to a subsequence of SEQ ID NO: 1003 and / or SEQ ID NO: 1004.HER.2

[0108] In some embodiments, the cancer cell-specific antigen is HER2, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).

[0109] The term "Her2 protein" or "Her2" as used herein includes any of the recombinant or naturally-occurring forms of Receptor tyrosine-protein kinase erbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human), or variants or homologs thereof that maintain Her2 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Her2). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Her2 protein. In embodiments, the Her2 protein is substantially identical to the protein identified by the UniProt reference number P04626 or a variant or homolog having substantial identity thereto.Illustrative HER2 sequence (SEQ ID NO: 1005)Additional Antigens

[0110] In some embodiments, the cancer cell-specific antigen is an antigen listed in Table 1, or a peptide antigen thereof in a complex with a major histocompatibility complex class I (MHC-I).Table 1: Exemplary Target Antigens, Antibodies, and diseases

[0111] In some embodiments, the activator antigen is selected from the group consisting of CEA cell adhesion molecule 5 (CEA), mesothelin (MSLN), and erb-b2 receptor tyrosine kinase 2 (HER2), and a peptide antigen thereof. In some embodiments, the activator antigen is selected from the group consisting of transferrin receptor (TFRC), HLA-E, CD33, PSMA, CD19 (CD19), CLL-1, CD53, SPN, ITGA4, SELPLG, and CLEC12A.Extracellular Ligand Binding Domain

[0112] The disclosure provides a first receptor, comprising a first extracellular ligand binding domain specific to a target antigen. In some embodiments, the target antigen comprises a cancer cell-specific antigen.

[0113] In some embodiments, the cancer cell-specific antigen is a target antigen listed in Table 1 or a target antigen -derived peptide antigen complexed with MHC-I, and the ligand binding domain of the first receptor recognizes and binds to the CEA antigen.

[0114] 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.

[0115] In some embodiments, the cancer cell-specific antigen is MSLN or a MSLN-derived peptide antigen complexed with MHC-I, and the ligand binding domain of the first receptor recognizes and binds to the MSLN antigen.

[0116] In some embodiments, the cancer cell-specific antigen is HER2 or a HER2-derived peptide antigen complexed with MHC-I, and the ligand binding domain of the first receptor recognizes and binds to the HER2 antigen.

[0117] Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure. In some embodiments, the ligand binding domain is an antigen binding domain. Exemplary antigen binding domains include, inter alia, scFv, SdAb, Vβ-only domains, and TCR antigen binding domains derived from the TCR α and β chain variable domains.

[0118] Any type of antigen binding domain is envisaged as within the scope of the instant disclosure.

[0119] The term “antibody,” as used herein, refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.

[0120] The terms “antibody fragment” or “antibody binding domain” refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.

[0121] The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0122] “Heavy chain variable region” or “VH” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0123] Unless specified, as used herein a scFv can have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0124] In some embodiments, the antigen binding domain of the activator and / or inhibitory receptor comprises an scFv. In some embodiments, the scFv comprises a VL and VH region joined by a linker. In some embodiments, the linker comprises a glycine serine linker, for example GGGGSGGGGSGGGGSGG (SEQ ID NO: 1014). In some embodiments, the scFv further comprises a signal sequence at the N terminus of the scFv. Exemplary signal sequences include MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 1015), which is encoded by ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAG GTGCCAGATGT (SEQ ID NO: 1016).

[0125] 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.

[0126] The term “recombinant antibody” refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.CEA

[0127] In some embodiments, the first extracellular ligand binding domain comprises a TCR ligand binding domain that binds to a 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 CEA MHC-I HL A- A* 02 antigens are described in Parkhurst et al., Molecular Therapy 2011 19(3): P620-626, the contents of which are incorporated herein by reference. An illustrative TCR extracellular ligand binding domain that recognizes amino acids 691-699 of SEQ ID NO: 1000 (IMIGVLVGV) complexed with HLA-A*02 MHC-I comprises a TCR alpha domain of TRAV8-l*01 and TRAJ6*01, and a TCR beta domain of TRBV26*01, TRBDl*01, TRBJ2- 7*01 and TRBC2.

[0128] Exemplary CDRs for that recognize a CEA MHC-I HLA-A*02 antigen comprising IMIGVLVGV (SEQ ID NO: 1017) are shown in Table 2 below.Table 2 CDRs for MHC-I HLA-A*02 + CEA (IMIGVLVGV (SEQ ID NO: 1017))

[0129] In some embodiments, the first extracellular ligand binding domain comprises complement determining regions (CDRs) selected from SEQ ID NOs: 3-12 or sequences having at least 85% or at least 95% identity thereto.

[0130] 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 a CDR-1 of TSITA (SEQ ID NO: 3), a CDR-2 of IRSNER (SEQ ID NO: 4) and a 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 0 chain variable domain comprises a CDR-1 of KGHPV (SEQ ID NO: 9), a CDR-2 of FQNQEV (SEQ ID NO: 10), and a CDR-3 of ASSLGLGDYEQ (SEQ ID NO: 11) or ASSLGTGDYEQ (SEQ ID NO: 12), or sequences having at least 85% or at least 95% identity thereto. In some embodiments, the TCR α chain variable domain comprises a CDR-1 of SEQ ID NO: 9, a CDR-2 of SEQ ID NO: 10 and a CDR-3 of SEQ ID NO: 11 or SEQ ID NO: 12; and the TCR α chain variable domain comprises a CDR-1 of SEQ ID NO: 3, a CDR- 2 of SEQ ID NO: 4 and a CDR-3 comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or sequences having at least 85% or at least 95% identity thereto.

[0131] In some embodiments, the TCR alpha and TCR beta chains are separated by a P2A self-cleaving peptide (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 1018)) and a GSG linker.

[0132] In some embodiments, the first receptor comprises 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% identicalto a sequence or subsequence of any one of SEQ ID NOS: 16-31 or 36-51. In some embodiments, the first receptor comprises a sequence or subsequence of any one of SEQ ID NOS: 16-31 or 36-51.

[0133] 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 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 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.

[0134] 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 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 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.

[0135] 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.

[0136] 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 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 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.

[0137] 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 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 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.

[0138] 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.

[0139] In some embodiments, the extracellular ligand binding domain of the first receptor is an scFv. In some embodiments, the scFv domain binds to CEA. In some embodiments, the scFv is the ligand binding domain of a CAR.

[0140] In some embodiments, a CEA scFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 55), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 56), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 57) or WDFAHYFQTMDY (SEQ ID NO: 58), a CDR- L1 of KASQNVGTNVA (SEQ ID NO: 59) or KASAAVGTYVA (SEQ ID NO: 60), a CDR- L2 of SASYRYS (SEQ ID NO: 61) or SASYRKR (SEQ ID NO: 62), and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 63) or sequences having at least 85% or at least 95% identity thereto. In some embodiments, a CEA scFv comprises a CDR-H1 of EFGMN (SEQ ID NO:55), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 56), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 57) or WDFAHYFQTMDY (SEQ ID NO: 58), a CDR- L1 of KASQNVGTNVA (SEQ ID NO: 59) or KASAAVGTYVA (SEQ ID NO: 60), a CDR- L2 of SASYRYS (SEQ ID NO: 61) or SASYRKR (SEQ ID NO: 62) and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 63). In some embodiments, a CEA scFv comprises a CDR- H1 of EFGMN (SEQ ID NO: 55), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO:56), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 57), a CDR-L1 of KASQNVGTNVA (SEQ ID NO: 59), a CDR-L2 of SASYRYS (SEQ ID NO: 61) and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 63). In some embodiments, a CEA scFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 55), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 56), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 57), a CDR-L1 of KASAAVGTYVA (SEQ ID NO: 60), a CDR-L2 of SASYRKR (SEQ ID NO: 62), and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 63). In some embodiments, a CEA scFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 56), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 56), a CDR-H3 of WDFAHYFQTMDY (SEQ ID NO: 58), a CDR-L1 of KASAAVGTYVA (SEQ ID NO: 60), a CDR-L2 of SASYRKR (SEQ ID NO: 62), and a CDR-L3 of HQYYTYPLFT (SEQ ID NO: 63).

[0141] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (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 variable light (VL) portion comprising a set of light chain complementarity determiningregions selected from the group consisting of SEQ ID NOS: 59-63; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative 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 variable heavy (VH) portion comprising a set of heavy chain complementarity determining regions (HC-CDRs) comprising SEQ ID NOS: 55-57 and a variable light (VL) portion comprising a set of light chain complementarity determining regions comprising SEQ ID NOS: 59, 61 and 63; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative 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 variable heavy (VH) portion comprising a set of heavy chain complementarity determining regions (HC-CDRs) comprising SEQ ID NOS: 55-57 and a variable light (VL) portion comprising a set of light chain complementarity determining regions comprising SEQ ID NOS: 59, 61, and 63.

[0142] Exemplary scFv that recognize CEA are shown in Table 5 below. Underlining indicates CDR sequences.

[0143] In some embodiments, a 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, a CEA scFv comprises, or consists essentially of, a sequence selected from the group consisting of SEQ ID NOs: 64-70. Further illustrative anti-CEA antibody sequences are provided in Stewart et al. Cancer Immunol. Immunother. 47:299-306 (1999); WO 1999 / 043817 A1;US 2002 / 0018750 A1; US 2011 / 0104148 A1; US 2016 / 0108131 A1; US20160075795A1; US 2019 / 0185583 A1; US 2020 / 0123270 A1; WO 2020 / 259550 A1; WO 2021 / 053587 A1; WO 2021 / 110647 A1; the contents of which are incorporated by reference herein for the purpose of providing anti-CEA VH, VL, scFv, and / or ligand binding domain sequences.

[0144] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy (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 variable light (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 variable heavy (VH) portion comprising SEQ ID NO: 144, and a variable light (VL) portion comprising SEQ ID NO: 148. In some embodiments, the extracellular ligand binding domain of the first receptor further comprises a linker between VH and VL portions.

[0145] 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 an 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 an scFv sequence of SEQ ID NO: 68.

[0146] In some embodiments, the CEA CAR comprises SEQ ID NO: 68, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 146, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 161, and SEQ ID NO: 79. In some embodiments, the CEA CAR comprises SEQ ID NO: 52.

[0147] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) amino acid residues in a CDR of the antigen binding domains provided herein are substituted with another amino acid. The substitution can be “conservative” in the sense of being a substitution within the same family of amino acids. The naturally occurring amino acids can be divided into the following four families and conservative substitutions will take place within those families: (1) amino acids with basic side chains: lysine, arginine, histidine; (2) amino acids with acidic side chains: aspartic acid, glutamic acid; (3) amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine; and (4) amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine. By varying the amino acid sequence of the CDRs of an antibody by addition, deletion or substitution of amino acids, various effects such as increased binding affinity for the target antigen can be obtained.MSLN

[0148] In some embodiments, the cancer cell-specific antigen is MSLN or a MSLN-derived peptide antigen complexed with MHC-I, and the ligand binding domain of the first receptor recognizes and binds to the MSLN antigen.

[0149] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv antigen binding domain.

[0150] In some embodiments, the extracellular ligand binding domain of the first receptor is an scFv. In some embodiments, the scFv domain binds to MSLN. In some embodiments, the scFv is the ligand binding domain of a CAR. Illustrative scFv domains specific to MSLN are shown in

[0151] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an antigen binding domain having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity or at least 99% identity to a sequence of SEQ ID NOS: 1019-1085. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an antigen binding domain comprising a sequence of SEQ ID NOS: 1019-1085.

[0152] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a binding domain having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity or at least 99% identity to a sequence of SEQ ID NO: 1041. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a binding domain comprising a sequence of SEQ ID NO: 1041.

[0153] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv antigen binding domain having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity or at least 99% identity to any one of SEQ ID NOs: 1019-1022. In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv antigen binding domain comprising a sequence of any one of SEQ ID NOs: 1019-1022 or 1023. In some embodiments, the extracellular ligand binding domain of the first receptor consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1019-1022 or 1023.TABLE 3 Sequences ofMSLN complementary determining regions (CDRs)

[0154] In Table 3, the light chain (LC) CDRs paired with the indicated heavy chain (HC) CDRs are indicated in the left column.

[0155] In some embodiments, the extracellular ligand binding domain of the first receptor comprises the HC CDR1, the HC CDR2, and the HC CDR3 set forth in Table 3 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 3) or sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the LC CDR1, the LC CDR2, and the LC CDR3 set forth in Table 3 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 3) or sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the HC CDR1, the HC CDR2, and the HC CDR3 set forth in Table 3 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 3). In some embodiments, the extracellular ligand binding domain of the first receptor comprises the LC CDR1, the LC CDR2, and the LC CDR3 set forth in Table 3 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 3).

[0156] In some embodiments, the extracellular ligand binding domain of the first receptor comprises the HC CDR1, HC CDR2, HC CDR3, LC CDR1, the LC CDR2, and the LC CDR3 set forth in Table 3 (e.g., the HC CDR1, HC CDR2 and HC CDR3 set forth in line 1, and the LC CDR 1, the LC CDR2, and the LC CDR 3 in line A) In some embodiments, the extracellular ligand binding domain of the first receptor comprises the HC CDR1, the HC CDR2, and the HC CDR3 set forth in Table 3 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 3) or sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor comprises the LC CDR1, the LC CDR2, and the LC CDR3 set forth in Table 3 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 3) or sequences having at most 1, 2, or 3 substitutions, deletions, or insertion relative to the CDRs of Table 3. In some embodiments, an extracellular ligand binding domain of the first receptor comprises one or more HC CDRs set forth in Table 3 and one or more LC CDRs set forth in Table 3. In some embodiments, the extracellular ligand binding domain of the first receptor comprises (i) the HC CDR1, the HC CDR2, and the HC CDR3 set forth in one line of Table 3 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 3) and (ii) the LC CDR1, the LC CDR2, and the LC CDR3 set forth in one line of Table 3 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 3). In each case, the HC CDRs can be paired with any of the LC CDRs, as the heavy chains and light chains share similarity, with routine testing to confirm desired expression and binding activity; however, preferred pairing between heavy and light chains of some embodiments are indicated in the right hand column of Table 3.

[0157] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising a sequence of SGDYYWS, a HC CDR2 comprising a sequence of YIYYSGSTYYNPSLKS, and HC CDR3 comprising a sequence ofCARED VVKGAFDIW, or CDR sequences having at most 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising a sequence of SGDYYWS, a HC CDR2 comprising a sequence of YIYYSGSTYYNPSLKS, and HC CDR3 comprising a sequence of CARED VVKGAFDIW. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a LC CDR1 comprising a sequence of RASQSISSYLN, a LC CDR2 comprising a sequence of AASSLQS, and a LC CDR3 comprising a sequence of QQSYSTPLT, or CDR sequences having at most 1, 2 or 3 aminoacid substitutions, insertions or deletions relative thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a LC CDR1 comprising a sequence of RASQSISSYLN, a LC CDR2 comprising a sequence of AASSLQS, and a LC CDR3 comprising a sequence of QQSYSTPLT. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising a sequence of SGDYYWS, a HC CDR2 comprising a sequence of YIYYSGSTYYNPSLKS, HC CDR3 comprising a sequence of CARED VVKGAFDIW, a LC CDR1 comprising a sequence of RASQSISSYLN, a LC CDR2 comprising a sequence of AASSLQS, and a LC CDR3 comprising a sequence of QQSYSTPLT, or CDR sequences having at most 1, 2 or 3 amino acid substitutions, insertions or deletions relative thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a HC CDR1 comprising a sequence of SGDYYWS, a HC CDR2 comprising a sequence of YIYYSGSTYYNPSLKS, HC CDR3 comprising a sequence of CARED VVKGAFDIW, a LC CDR1 comprising a sequence of RASQSISSYLN, a LC CDR2 comprising a sequence of AASSLQS, and a LC CDR3 comprising a sequence of QQSYSTPLT.

[0158] In some embodiments, the extracellular ligand binding domain of the first receptor comprises an scFv. In some embodiments, the scFv comprises a heavy chain comprising CDRs selected from the sequences of GYTMN, LITPYNGASSYNQKFRG and GGYDGRGFDY. In some embodiments, the heavy chain comprises sequences of GYTMN, LITPYNGASSYNQKFRG and GGYDGRGFDY. In some embodiments, the scFv comprising a light chain comprising CDRs selected from the sequences of SASSSVSYMH, DTSKLAS and QQWSGYPLT. In some embodiments, the light chain comprises sequences of SASSSVSYMH, DTSKLAS and QQWSGYPLT.

[0159] Sequences of illustrative heavy and light chains of antigen binding domains that are specific to MSLN are set forth in Table 4 and Table 5 below. Light chains paired with heavy chains in preferred embodiments are indicated at right in Table 4.TABLE 4 Sequences of heavy chain variable fragments (VH)TABLE 5 Sequences of light chain variable fragments (VL)

[0160] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable heavy region (VH) sequence set forth in Table 4. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VH sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VH set forth in Table 4. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a variable light region (VL) sequence set forth in Table 5. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VL sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VL set forth set forth in Table 5.

[0161] In some embodiments, a the extracellular ligand binding domain of the first receptor comprises a VH that (i) comprises the HC CDR1, the HC CDR2, and the HC CDR3 sequences set forth in Table 3 (e.g., the HC CDR 1, the HC CDR2, and the HC CDR 3 of line #1, line #2, line #3, etc. of Table 5) and (ii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VH sequence set forth in Table 4. In some embodiments, extracellular ligand binding domain of the first receptor (i) comprises the LC CDR1, the LC CDR2, and the LC CDR3 sequences set forth in one line Table 3 (e.g., the LC CDR 1, the LC CDR2, and the LC CDR 3 of line A, line B, or line C of Table 5) and a VL sequence set forth in Table 5 and (ii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VL set forth set forth in Table 5.

[0162] In some embodiments, the extracellular ligand binding domain of the first receptor comprises (i) a VH sequence set forth in Table 4 or a VH sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VH set forth in Table 4, and (ii) a VL sequence set forth in Table 5 or a VL that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VL set forth set forth in Table 5. In each case, the VH can be paired with any of the VLs, as the heavy chains and light chains share similarity, with routine testing to confirm desired expression and binding activity; however, the preferred pairing between Table 4 and Table 5 is indicated in the “LC” column of Table 4, corresponding to the # column of Table 5.

[0163] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VH sequence of, or a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VH sequence of

[0164] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VL sequence ofor a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VL sequence of

[0165] In some embodiments, the extracellular ligand binding domain of the first receptor comprises a VH sequence ofand a VL sequence ofor sequences that have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments the VH and VL are separated for a linker, for example a linker comprising a sequence of. The VH and VL can be in any orientation, for example VH, linker, VL; or alternatively, VL, linker VH.

[0166] In some embodiments, the sequence of the first receptor isHER.2

[0167] In some embodiments, the cancer cell-specific antigen is HER2 or HER2-derived peptide antigen complexed with MHC-I, and the ligand binding domain of the first receptor recognizes and binds to the HER2 antigen.TABLE 6 HER2 antigen binding domain complement determining regions (CDRs)

[0168] In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of GFNIKDTYIH (1174), ARIYPTNGYTRYADSVKG (1177), and SRWGGDGFYAMD[Y / V] (1181) or (1182); and the VL region comprises one or more CDR sequences selected from the group consisting of RASQDVNTAVA (1163), SASFLY (1168), and QQHYTTPP (1171).

[0169] In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of NYGMN (1176), WINTSTGESTFADDFKG (1179) and WEVYHGYVPY (1183); and the VL region comprises one or more CDR sequences selected from the group consisting of KASQDVYNAVA (1165), SASSRYT (1169), and QQHFRTPFT (1173).

[0170] In some embodiments, the VH region comprises CDR sequences of GFNIKDTYIH (1175), ARIYPTNGYTRYADSVKG (1178), and SRWGGDGFYAMDY (1181) or SRWGGDGFYAMDV (1182); and the VL region comprises CDR sequences of RASQDVNTAVA (1163), SASFLY (1168) and QQHYTTPP (1171).

[0171] In some embodiments, the VH region comprises CDR sequences of NYGMN (1176), WINTSTGESTFADDFKG (1179) and WEVYHGYVPY (1183); and the VL region comprises CDR sequences of KASQDVYNAVA (1165), SASSRYT (1169), and QQHFRTPFT (1173)In some embodiments, the full length VH and VL regions comprise the sequences disclosed in Table 6. In some embodiments, the binding domain comprises the full length VH region and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises from N-terminal to C-terminal the full length VH regions and the full length VL region. In some embodiments, the polypeptide comprises from N-terminal to C- terminal the full length VL region and the full length VH region. In some embodiments, the full length VH and VL comprises the sequence selected from Table 7. In some embodiments, the binding domain comprises SEQ ID NO: 1143 and SEQ ID NO: 1144. In some embodiments, the binding domain comprises SEQ ID NO: 1145 and SEQ ID NO: 1146. In some embodiments, the binding domain comprises SEQ ID NO: 1147 and SEQ ID NO: 1148. In some embodiments, the binding domain comprises SEQ ID NO: 1145 and SEQ ID NO: 1149. In some embodiments, the binding domain comprises SEQ ID NO: 1150 and SEQ ID NO: 1149.TABLE 7 VH and VL Domains for HER2 CAR Constructsembodiments, the HER2 scFv domain is selected from a sequence listed in Table 7. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 1151. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 1152. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 1153. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 1154. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 1155. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 1156. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 1157. In some embodiments, the HER2scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 1158. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 1159. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 1160. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 1161. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 1162.TABLE 8 HER2 ScFv Domains for HER2 CAR Constructs

[0173] In some embodiments, the HER2 scFv domain comprises a sequence of any one of SEQ ID NOS: 1151, 1153, 1155, 1157, 1159 or 1161, or a sequence having at least 90%, at least 95%, at least 97%, at least 99% or is identical thereto.Chimeric Antisen Receptors (CARs)

[0174] The disclosure provides a first, activator receptor and immune cells comprising same. In some embodiments, the first receptor is a chimeric antigen receptor.

[0175] The term “chimeric antigen receptors (CARs)” as used herein, can refer to artificial receptors derived from T-cell receptors and encompasses engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs can be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specificembodiments, CARs direct specificity of the cell to a tumor associated antigen, for example. Illustrative CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region. In some embodiments, CARs further comprise a hinge domain. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to a CD3 transmembrane domain and endodomain. The specificity of other CAR designs can be derived from ligands of receptors (e.g., peptides). In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3, 4-1BB, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, cytokines, and cytokine receptors.

[0176] In some embodiments, the extracellular ligand binding domain of the first receptor is fused to the extracellular domain of a CAR.

[0177] In some embodiments, the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo. Illustrative hinges can be isolated or derived from IgD and CD8 domains, for example IgGl. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0178] 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 is identical to a sequence of(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 CD8α hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

[0179] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(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 is identical to a sequence of(SEQ ID NO: 74). In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 74.

[0180] The CARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. For example, a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0181] The transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions can be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0182] In some embodiments of the CARs of the disclosure, the CARs comprise a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 75). In some embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 75. In someembodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 76). In some embodiments, theCD28 transmembrane domain is encoded by SEQ ID NO: 76. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 157. In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 157.

[0183] In some embodiments of the CARs of the disclosure, the CARs comprise an IL- 2Rbeta transmembrane domain. In some embodiments, the IL-2Rbeta transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 77). In some embodiments, the IL- 2Rbeta transmembrane domain comprises or consists essentially of SEQ ID NO: 77. In some embodiments, the IL-2Rbeta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 78). In some embodiments, the IL-2Rbetatransmembrane domain is encoded by SEQ ID NO: 78.

[0184] The cytoplasmic domain or otherwise the intracellular signaling domain of the CARs of the instant disclosure is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term “effector function” refers to a specialized function of a cell. Thus, the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired functions of the CAR-T cells of the instant disclosure. The term intracellular signaling domain is thusmeant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.

[0185] Examples of intracellular signaling domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0186] Accordingly, the intracellular domain of CARs of the instant disclosure comprises at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures that there is T-cell receptor (TCR) signaling necessary to activate the effector functions of the CAR T-cell. In some embodiments, the at least one cytoplasmic activation is a CD247 molecule (CD3ζ) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX-activating protein of 12 kDa (DAP 12) activation domain.

[0187] In some embodiments, the CD3ζ activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 79).

[0188] In some embodiments, the CD3ζ activation domain comprises or consists essentially of SEQ ID NO: 79. In some embodiments, the CD3ζ activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 80). In some embodiments, theCD3ζ activation domain is encoded by SEQ ID NO: 80. In some embodiments, the CD3ζ activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 163. In some embodiments, the CD3ζ activation domain is encoded by SEQ ID NO: 163.

[0189] It is known that signals generated through the TCR alone are often insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0190] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. In some embodiments, the IT AM contains a tyrosine separated from a leucine or an 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 illustrative IT AM containing cytoplasmic domain is the CD3ζ activation domain. Further examples of IT AM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from

[0191] In some embodiments, the CD3ζ activation domain comprising a single IT AM comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR ( SEQ ID NO : 81 ) . In SOme embodiments, the CD3ζ activation domain comprises SEQ ID NO: 81. In some embodiments, the CD3ζ activation domain comprising a single IT AM consists essentially of an amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR ( SEQ ID NO : 81 ) . In some embodiments, the CD3ζ activation domain comprising a single ITAM is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 82). In some embodiments, the CD3ζ activation domain is encoded by SEQ ID NO: 82.

[0192] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise the CD3ζ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the instant disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3ζ chain portion and a co-stimulatory domain. The co-stimulatory domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include the co- stimulatory domain is selected from the group consisting of IL-2RP, Fc Receptor gamma (FcRγ), Fc Receptor beta (FcRP), 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 superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional variants thereof. In some embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is isolated or derived from CD28.

[0193] In some embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 83). In some embodiments, the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 83). In some embodiments, the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 84). In some embodiments, the CD28 co-stimulatorydomain is encoded by SEQ ID NO: 84. In some embodiments, the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ IDNO: 160. In some embodiments, the CD28 co-stimulatory domain is encoded by SEQ ID NO: 160.

[0194] In some embodiments, the co-stimulatory domain is isolated or derived from 4-1BB. In some embodiments, the 4- IBB co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of (SEQ ID NO: 161). Insome embodiments, the 4- IBB co-stimulatory domain comprises or consists essentially of(SEQ ID NO: 161). In some embodiments, the 4- IBB co-stimulatory domain s encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of(SEQ ID NO: 162).

[0195] In some embodiments, the intracellular domain of the CAR comprises a CD28 co- stimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain. In some embodiments, the intracellular domain of the CAR comprises a sequence of(SEQ ID NO: 158), or a sequence having atleast 80% identity, at least 90% identity, at least 95% identity, 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, at least 99% identity thereto. In some embodiments, the intracellular domain of the CAR is encoded by SEQ ID NO: 159.

[0196] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure can be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length can form the linkage. A glycine-serine doublet provides an example of a suitable linker. An illustrative linker comprises a sequence of

[0197] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure can be linked to each other in a random or specified order. Optionally, ashort oligo- or polypeptide linker, for example between 2 and 10 amino acids in length can form the linkage. A glycine-serine doublet provides an example of a suitable linker.Inhibitory Receptors

[0198] The disclosure provides a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen that has been lost in a cancer cell, such as an allelic variant of a gene. The non-target allelic variant can be lost in the cancer cell through any mechanism, such as, without limitation, epigenetic changes that effect non-target allelic variant expression, mutations to the gene encoding the non-target allelic variant, disruption of cellular signaling that regulates expression of the non-target allelic variant, chromosome loss, partial or complete deletion of the genomic locus, gene silencing through modification of nucleic acids or heterochromatin, or loss of expression through other mechanisms. In variations of the compositions and methods disclosed herein, the cells or subject treated can exhibit a loss of expression of the non-target allelic variant because of non-genetic changes. Accordingly the disclosure provides compositions and methods for killing cells and / or treating subject lacking expression of the non-target antigen from any cause, including but not limited to, loss of heterozygosity.

[0199] The major histocompatibility complex (MHC) is a central molecular genetic control element of the immune system. In humans, the MHC is called the human leukocyte antigen (HLA) complex and the alpha subunit is located on the short arm of human chromosome 6. Two distinct structural and functional classes of HLA molecules exist, class I and class II antigens. HLA-I antigens are expressed on all nucleated cells and platelets, and HLA-II antigens are expressed on antigen-presenting cells such as macrophages and B cells. HLA-I genes consist of HLA- A, -B, and -C loci that are closely related. The HLA-I molecules bind to specific peptides derived from intracellular proteins and display these peptides on the cell surface, guiding immune cells to sites of disease or infection. Because of its key role as an initiator and controller of the immune response, HLA is a highly polymorphic gene complex. HLA- A, -B, and -C loci all possess thousands of alleles, and homozygosity is, consequently, rare. Like many loci across the cancer genome, HLA is commonly involved in LOH.

[0200] LOH occurs when a heterozygous genetic locus loses 1 of its 2 parental alleles. LOH is commonly observed in solid tumor malignancies; about 20% of genomic loci have undergone LOH in an average tumor (Beroukhim 2010), with enrichment in high-risk patients with TP53 mutations, high mutation burden, and metastatic disease across multiple tumor types (Montesion 2021). Importantly, loss of genetic material through LOH is irreversible. Thus, LOH is a frequent,predictable, and irreversible genetic change that unequivocally distinguishes tumor cells from normal cells.

[0201] The non-target antigen can be a protein, or an antigen peptide thereof in a complex with a major histocompatibility complex class I (MHC-I), where the non-target antigen comprises a polymorphism. Because the non-target antigen is polymorphic, loss of a single copy of the gene encoding the non-target antigen, which can occur through loss of heterozygosity in a cancer cell, yields a cancer cell that retains the other polymorphic variant of gene, but has lost the non-target antigen. For example, a subject having HLA-A*02 and HLA-A*01 alleles at the HLA locus can have a cancer in which only the HLA-A*02 allele is lost. In such a subject, the HLA-A*01 protein remains present, but is not recognized by the inhibitory receptor of immune cells encountering the cancer cell, because the inhibitor receptor is designed to be specific to the HLA-A*02 (or other non-target antigen). In normal non-malignant cells, the HL A- A* 02 (or other non-target antigen) is present and inhibits activation of the engineered immune cell. In cancer cells having loss of heterozygosity, the HLA-A*02 allelic variant (or other non-target antigen) is lost. Immune cells engineered to express the inhibitory receptor do not receive an inhibitory signal from the inhibitory receptor, as the inhibitory receptor only responds to the HL A- A* 02 (or other non-target antigen), which is absent on cancer cells. By this mechanism, the immune cell is selectively activated, and selectively kills, cancer cells expressing target antigens (e.g., CEA, MSLN, or HER2) but having lost HLA-A*02 (or another non-target antigen) due to loss-of- heterozygosity. HLA-A is used here as an example. Similar polymorphic variation occurs in the population at other MHC genes and in other non-MHC genes as well.

[0202] The disclosure provides a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen selected from TNFRSF11 A, ACHRB, ITGAE, TRPV1, and SREC, or an antigen peptide thereof in a complex with a major histocompatibility complex class I (MHC-I), wherein the non-target antigen comprises a polymorphism, and immune cells comprising same.

[0203] The disclosure provides a second receptor, comprising an extracellular ligand binding domain specific to a non-target antigen selected from intercellular adhesion molecule 1 (ICAM1), catechol-O-methyltransf erase (COMT), C-X-C motif chemokine ligand 16 (CXCL16), leucine rich repeat neuronal 4 (LRRN4) and uroplakin 3B (UPK3B), or an antigen peptide thereof in a complex with a major histocompatibility complex class I (MHC- I), wherein the non-target antigen may comprise a nonsynonymous, extracellular-domainpolymorphism (e.g., in an extracellular domain of ICAM1, COMT, CXCL16), and immune cells comprising same.

[0204] Non-target major histocompatibility complex class I MHC-I (or pMHC-I) antigens comprising any of HLA-A, HLA-B, HLA-C or HLA-E are envisaged as within the scope of the disclosure. In some embodiments, the non-target antigen comprises a Major Histocompatibility Complex (MHC) protein. In some embodiments, the MHC is MHC class I. In some embodiments, the MHC class I protein comprises a human leukocyte antigen (HLA) protein. In some embodiments, the non-target antigen comprises an allele of an HLA Class I protein selected from the group consisting of HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the HLA-A allele comprises HLA-A*01, HLA-A*02, HLA-A*03 or HLA-A* 11. In some embodiments, the HLA-B allele comprises HLA-B*07. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0205] In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the non-target antigen comprises an allele of HLA-A. In some embodiments, the allele of HLA-A comprises HLA-A*01, HLA-A*02, HLA-A*03, or HLA-A*11. In some embodiments, the non-target antigen comprises HLA-A*69. In some embodiments, the non- target antigen comprises a human leukocyte antigen A*02 allele (HLA-A*02).

[0206] In some embodiments, the non-target antigen comprises an allele of HLA-B. In some embodiments, the allele of HLA-B comprises HLA-B*07.

[0207] In some embodiments, the non-target antigen comprises HLA-C. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0208] Illustrative inhibitory receptors are described in PCT / US2020 / 045228 filed on September 6, 2020, PCT / US2020 / 064607, filed on December 11, 2020, PCT / US2021 / 029907, filed on April 29, 2021 and PCT / US2020 / 059856 filed on November 10, 2020, the contents of each of which are incorporated herein by reference.

[0209] In some embodiments, the second receptor is an inhibitory chimeric antigen receptor (inhibitory receptor).

[0210] In some embodiments, the second receptor is an inhibitory receptor. In some embodiments, the second receptor is humanized.

[0211] 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 thereto. In some embodiments, the second receptor comprises SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 90% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 91%identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 92% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 93% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 94% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 95% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 95% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 96% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 97% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 98% identical to SEQ ID NO: 164. In some embodiments, the second receptor comprises a sequence at least 99% identical to SEQ ID NO: 164.

[0212] 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0213] The disclosure provides a second receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can discriminate between single amino-acid variant alleles of a non-target antigen. This ability to discriminate between allelic variants of a non- target antigen allows the second receptor to inhibit activation of immune cells comprising the second receptor in the presence of non-target cells that express that the allele recognized by the ligand binding domain. However, activation of immune cells is not inhibited in the presence of target cells that have lost the allele, for example cancer cells that have lost one allele of a gene through loss of heterozygosity.

[0214] The disclosure provides a second receptor, which is an inhibitory receptor, comprising an extracellular ligand binding that can discriminate between different levels of expression of a non-target antigen. This allows the second receptor to inhibit activation of immune cells comprising the second receptor in the presence of non-target cells that express the ligand for the second receptor, but to allow activation of immune cells in the presence of cancer cells that express low levels, or have no expression, of the ligand for the second receptor.Inhibitor Ligands

[0215] In some embodiments, the non-target antigen is not expressed by the target cells, and 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 aplurality 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, normal, or healthy cells), that express both the target and the non-target antigen.

[0216] Any cell surface molecule expressed by the non-target cells that is not expressed by target cells can be a suitable non-target antigen for the second receptor extracellular ligand binding domain. For example, a cell adhesion molecule, a cell-cell signaling molecule, an extracellular domain, a molecule involved in chemotaxis, a glycoprotein, a G protein-coupled receptor, a transmembrane, a receptor for a neurotransmitter or a voltage gated ion channel can be used as a non-target antigen.

[0217] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in a complex with a major histocompatibility complex class I (MHC-I).

[0218] Non-target MHC-1 (pMHC) antigens comprising any ofHLA-A, HLA-B, HLA-C, or HLA-E are envisaged as within the scope of the disclosure.

[0219] 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 ofHLA-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.

[0220] In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the non-target antigen comprises an allele ofHLA-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*02. In some embodiments, the non- target antigen comprises HLA-A* 69.

[0221] In some embodiments, the non-target antigen comprises an allele HLA-B. In some embodiments, the allele of HLA-B comprises HLA-B*11. In some embodiments, the HLA-B allele comprises HLA-B*07. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0222] In some embodiments, the non-target antigen comprises an allele of HLA-C. In some embodiments, the HLA-C allele comprises HLA-C*07.

[0223] In some embodiments, the non-target antigen is selected from the group consisting of a polymorphic variant of TNFRSF11A, ACHRB, ITGAE, TRPV1, and SREC. In some embodiments, the non-target antigen is an antigen peptide comprising a polymorphic residueof TNFRSF11A, ACHRB, ITGAE, TRPV1, or SREC, in a complex with a major histocompatibility complex class I (MHC-I). In some embodiments, the non-target antigens lost in cancer cells due to loss of heterozygosity include ICAM1, COMT and CXCL16. In some embodiments, the non-target antigen is selected from the group consisting of a polymorphic variant of ICAM1, COMT and CXCL16. In some embodiments, the non-target antigen is an antigen peptide comprising a polymorphic residue of ICAM1, COMT or CXCL16 in a complex with a major histocompatibility complex class I (MHC-I).

[0224] 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 the specified HLA alleles, for use in embodiments described herein. Such scFvs include, for example and without limitation, the following mouse and humanized scFv antibodies that bind HLA alleles in a peptide-independent way in SEQ ID NOs: 91-102, 250-276, or 278-345.

[0225] 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 SEQ ID NOS: 91-102, 250-260, 262, 264, 266, 268, 270, 272, 274, 276, and 278-345, or the group of sequences set forth in SEQ ID NOs: 91-102, 250-276, or 278-345, 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 SEQ ID NOs: 91-102, 250-276, or 278-345. 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 as set forth in SEQ ID NOs: 91-102, 250-276, or 278-345, 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 as set forth in SEQ ID NOs: 91-102, 250-276, or 278-345, 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 as set forth in SEQ ID NOs: 91-102, 250-276, or 278-345, 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 NOS: 260, 262, 264, 266, 268, 270, 272, 274 or 276, 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 NOS: 250-259, 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, 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.

[0226] Illustrative heavy chain and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR-L1, CDR-L2 and CDR-L3, respectively) for HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07 and HLA-C*07 ligand binding domains are shown in Table 9 below.TABLE 9 CDRs corresponding to HLA antigen binding domainsHLA-A*03 CDRs

[0227] 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 CDR sequences as set forth in Table 9.

[0228] In some embodiments, the non-target antigen comprises HLA-B. In some embodiments, the ligand binding domain of the second, inhibitory receptors comprise an HLA-B*07 ligand binding domain comprising CDR sequences as set forth in Table 9.

[0229] In some embodiments, the non-target antigen comprises HLA-C. In some embodiments, the ligand binding domain of the second, inhibitory receptors comprise an HLA-C*07 ligand binding domain comprising CDR sequences as set forth in Table 9.

[0230] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds 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.

[0231] 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 Table 9; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the HLA-A*01 CDRs of Table 9.

[0232] 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 Table 9; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the HLA-A*02 CDRs of Table 9.

[0233] 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, of SEQ ID NOS: 109-114 or SEQ ID NOs: 103, 2074, 105, 2075, 107, 108; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of SEQ ID NOS: 103-108 or SEQ ID NOS: 109-114.

[0234] In some embodiments, the extracellular ligand binding domain of the second receptor specifically binds to HLA-A*03. In some embodiments, the extracellular ligand binding domain of the second receptor comprises HLA-A*03 complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 as disclosed Table 9; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the HLA-A*03 CDRs of Table 9.

[0235] 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 Table 9; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the HLA-A* 11 CDRs of Table 9.

[0236] 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 Table 9; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the HLA-B*07 CDRs of Table 9.

[0237] 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 Table 9; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the HLA-C*07 CDRs of Table 9.

[0238] In further embodiments of any of the ligand binding domains, each CDR sequence can have 1, 2, 3 or more substitutions, insertions, or deletions. CDR sequences can tolerate substitutions, deletions, or insertions. Using sequence alignment tools, routine experimentation, and known assays, those of skill in the art can generate and test variant sequences having 1, 2, 3, or more substitutions, insertions, or deletions in CDR sequences without undue experimentation.

[0239] In some embodiments, the non-target antigen comprises HLA-A*02, and the ligand binding domain of the second receptor comprises an HLA-A*02 ligand binding domain. In some embodiments, the ligand binding domain binds HLA-A*02 independent 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 a 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 a sequence of any one of SEQ ID NOs: 91-102.

[0240] In some embodiments, the HLA-A*02 scFv comprises the complementarity determined regions (CDRs) of any one of SEQ ID NOS: 103-114. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOS: 103-114. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOS: 103-114. In some embodiments, the heavy chain of the antigen binding domain comprises theheavy chain CDRs of any one of SEQ ID NOS: 103-114, and wherein the light chain of the antigen binding domain comprises the light chain CDRs of any one of SEQ ID NOS: 103- 114. In some embodiments, the HLA-A*02 antigen binding domain comprises a heavy chain and a light chain, and the heavy chain comprises CDRs selected from SEQ ID NOs: 106-108 and 112-14 and the light chain comprises CDRs selected from SEQ ID NOs: 103-15 and 109- 111.

[0241] In some embodiments, the HLA-A*02 antigen binding domain comprises a heavy chain and a light chain, and the heavy chain comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOS: 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.

[0242] 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 wherein the light chain of comprises a sequence identical to the light chain portion of any one of SEQ ID NOS: 91-102.

[0243] In some embodiments, the HLA-A*02 scFv comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to any one of SEQ ID NOs: 91-102. In some embodiments, the HLA- A*02 scFv comprises a sequence identical to any one of SEQ ID NOs: 91-102.

[0244] 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 SEQ ID NOs: 91-102, 250-276, or 278-345,, or a sequence at least 90%, at least 95% or at least 99% identical to thereto. In some embodiments, the HLA-A*01 scFv comprises HLA-A*1 CDR sequences as set forth in Table 9.

[0245] 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 SEQ ID NOs: 91-102, 250-276, or 278-345, or a sequence at least 90%, at least 95% or at least 99% identical to thereto. In some embodiments, the HLA-A*03 scFv comprises HLA-A*03 CDR sequences as set forth in Table 9.

[0246] 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 anscFv domain comprising a sequence selected from the group of sequences set forth in SEQ ID NOs: 91-102, 250-276, or 278-345,, or a sequence at least 90%, at least 95% or at least 99% identical to thereto. In some embodiments, the HLA-A*11 scFv comprises HLA-A*11 CDR sequences as set forth in Table 9.

[0247] 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 SEQ ID NOs: 91-102, 250-276, or 278-345,, or a sequence at least 90%, at least 95% or at least 99% identical to thereto. In some embodiments, the HLA-B*07 scFv comprises HLA-B*07 CDR sequences as set forth in Table 9.

[0248] 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 SEQ ID NOs: 91-102, 250-276, or 278-345,, or a sequence at least 90%, at least 95% or at least 99% identical to thereto. In some embodiments, the HLA-C*07 scFv comprises HLA-C*07 CDR sequences as set forth in Table 9.Inhibitory Chimeric Antigen Receptors

[0249] The disclosure provides a second receptor that is an inhibitory chimeric antigen receptor. The inhibitory receptor can comprise an extracellular ligand binding domain that binds to and recognizes the non-target antigen or a peptide derivative thereof in a MHC-I complex.

[0250] Illustrative inhibitory receptors are described in PCT / US2020 / 045228 filed on September 6, 2020, PCT / US2020 / 064607, filed on December 11, 2020,PCT / US2021 / 029907, filed on April 29, 2021 and PCT / US2020 / 059856 filed on November 10, 2020, the contents of each of which are incorporated herein by reference.

[0251] The term “inhibitory receptor,” as used herein refers to a ligand binding domain that is fused to an intracellular signaling domain capable of transducing an inhibitory signal that inhibits or suppresses the immune activity of an immune cell. Inhibitory receptors have immune cell inhibitory potential, and are distinct and distinguishable from CARs, which are receptors with immune cell activating potential. For example, CARs are activating receptors as they include intracellular stimulatory and / or co-stimulatory domains. Inhibitory receptors are inhibiting receptors that contain intracellular inhibitory domains.

[0252] As used herein “inhibitory signal” refers to signal transduction or changes in protein expression in an immune cell resulting in suppression of an immune response (e.g., decrease in cytokine production or reduction of immune cell activation). Inhibition or suppression of an immune cell can selective and / or reversible, or not selective and / or reversible. Inhibitory receptors are responsive to non-target antigens (e.g. HLA-A*02). For example, when a non- target antigen (e.g. HLA-A*02) binds to or contacts the inhibitory receptor, the inhibitory receptor is responsive and activates an inhibitory signal in the immune cell expressing the inhibitory receptor upon binding of the non-target antigen by the extracellular ligand binding domain of the inhibitory receptor.

[0253] Inhibitory receptors of the disclosure can comprise an extracellular ligand binding domain. Any type of ligand binding domain that can regulate the activity of a receptor in a ligand dependent manner is envisaged as within the scope of the instant disclosure.

[0254] In some embodiments, the ligand binding domain is an antigen binding domain. Illustrative antigen binding domains include, inter alia, scFv, SdAb, VP-only domains, and TCR antigen binding domains derived from the TCR α and β chain variable domains.

[0255] Any type of antigen binding domain is envisaged as within the scope of the instant disclosure.

[0256] In some embodiments, the extracellular ligand binding domain of the second receptor is an scFv.

[0257] In some embodiments, the extracellular ligand binding domain of the second receptor is fused to the extracellular domain of an inhibitory CAR.

[0258] In some embodiments, the inhibitory receptors of the present disclosure comprise an extracellular hinge region. Illustrative hinges can be isolated or derived from IgD and CD8 domains, for example IgGl. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0259] The inhibitory receptors of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the inhibitory receptor. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0260] The transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions can be isolated or derived from (i.e.comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and the intracellular domain of the inhibitory receptor. A glycine-serine doublet provides a particularly suitable linker.

[0261] The disclosure provides an inhibitory receptor comprising an intracellular domain. The intracellular domain of the inhibitory receptors of the instant disclosure is responsible for inhibiting activation of the immune cells comprising the inhibitory receptor, which would otherwise be activated in response to activation signals by the first receptor. In some embodiments, the inhibitory intracellular domain comprises an immunoreceptor tyrosine- based inhibitory motif (ITIM). In some embodiments, the inhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immune inhibitory receptors expressed on the surface of T cells, and play a pivotal role in attenuating or terminating T cell responses.

[0262] In some embodiments, an inhibitory intracellular domain is isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1. In some embodiments, the TRAIL receptor comprises TR10A, TR10B or TR10D.

[0263] In some embodiments, an inhibitory intracellular domain is isolated from phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1). In some embodiments, an inhibitory intracellular domain is isolated from leukocyte immunoglobulin like receptor B 1 (LILRB1).

[0264] In some embodiments, the inhibitory domain is isolated or derived from a human protein, for example a human TRAIL receptor, CTLA-4, PD-1, PAG1 or LILRB1 protein.

[0265] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region or a combination thereof.

[0266] In some embodiments, the inhibitory domain is isolated or derived from killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2),killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin like receptor Bl (LIR1, also called LIR-1 and LILRB1), programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin like receptor KI (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N and C terminal SH2 domains), or ZAP70 KI K369A (kinase inactive ZAP70).

[0267] In some embodiments, the second, inhibitory receptor comprises an inhibitory domain. In some embodiments, the second, inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to an intracellular domain of an inhibitory receptor. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of an inhibitory receptor.

[0268] In some embodiments, the second, inhibitory receptor comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain or a portion thereof isolated or derived isolated or derived from the same protein, for example an ITIM containing protein. In some embodiments, the second, inhibitory receptor comprises a hinge region isolated or derived from isolated or derived from the same protein as the intracellular domain and / or transmembrane domain, for example an ITIM containing protein.LILRB 1 Inhibitory receptors

[0269] The disclosure provides a second, inhibitory receptor comprising a LILRB 1 inhibitory domain, and optionally, a LILRB 1 transmembrane and / or hinge domain, or functional variants thereof. The inclusion of the LILRB 1 transmembrane domain and / or the LILRB 1 hinge domain in the inhibitory receptor can increase the inhibitory signal generated by the inhibitory receptor compared to a reference inhibitory receptor having another transmembrane domain or another hinge domains. The second, inhibitory receptor comprising the LILRB 1 inhibitory domain can be a CAR or TCR, as described herein. Any suitable ligand binding domain, as described herein, can be fused to the LILRB 1 -based second, inhibitory receptors.

[0270] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB 1), also known as Leukocyte immunoglobulin-like receptor Bl, as well as ILT2, LIR1, MIR7, PIRB, CD85J, ILT-2 LIR-1, MIR-7 and PIR-B, is a member of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB 1 protein belongs to the subfamily B class of LIR receptors. These receptors contain two to four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-basedinhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. LILRB1 is thought to regulate inflammatory responses, as well as cytotoxicity, and to play a role in limiting auto-reactivity. Multiple transcript variants encoding different isoforms of LILRB1 exist, all of which are contemplated as within the scope of the instant disclosure.

[0271] In some embodiments of the inhibitory receptors described herein, the inhibitory receptor comprises one or more domains isolated or derived from LILRB1. In some embodiments of the receptors having one or more domains isolated or derived from LILRB1, the one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 115. In some embodiments, the one or more domains of LILRB1 comprise an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 115. In some embodiments, the one or more domains of LILRB1 consist of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 115. In some embodiments, the one or more domains of LILRB1 consist of an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 115.

[0272] In some embodiments of the receptors having one or more domains isolated or derived from LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 116.

[0273] In some embodiments of the receptors having one or more domains of LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is identical to a sequence or subsequence of SEQ ID NO: 116.

[0274] In various embodiments, an inhibitory receptor is provided, comprising a polypeptide, wherein the polypeptide comprises one or more of: an LILRB1 hinge domain or functional variant thereof; an LILRB1 transmembrane domain or a functional variant thereof; and an LILRB1 intracellular domain or an intracellular domain comprising at least one, or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0275] As used herein an “immunoreceptor tyrosine-based inhibitory motif” or “ITIM” refers to a conserved sequence of amino acids with a consensus sequence of S / I / V / LxYxxI / V / L (SEQ ID NO: 984), or the like, that is found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-possessing inhibitory receptors interact with their ligand, the ITIM motif is phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or the inositol- phosphatase called SHIP.

[0276] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), at least two ITIMs, at least 3 ITIMs, at least 4 ITIMs, at least 5 ITIMs or at least 6 ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0277] 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).

[0278] In further particular embodiments, the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0279] In some embodiments, the intracellular domain comprises both 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.

[0280] In some embodiments, the intracellular domain comprises both 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.

[0281] In some embodiments, the intracellular domain comprises both 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.

[0282] 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.

[0283] In some embodiments, the intracellular domain comprises the ITIMs 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.

[0284] 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.

[0285] 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).

[0286] LILRB1 intracellular domains or functional variants thereof of the disclosure can have at least 1, at least 2, at least 4, at least 4, at least 5, at least 6, at least 7, or at least 8 ITIMs. In some embodiments, the LILRB 1 intracellular domain or functional variant thereof has 2, 3, 4, 5, or 6 ITIMs.

[0287] In particular embodiments, the intracellular domain comprises two, three, four, five, or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0288] In particular embodiments, the intracellular domain comprises at least three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0289] In particular embodiments, the intracellular domain comprises three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected fromNLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0290] In particular embodiments, the intracellular domain comprises four immunoreceptor tyrosine-based inhibitory 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).

[0291] In particular embodiments, the intracellular domain comprises five immunoreceptor tyrosine-based inhibitory 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).

[0292] In particular embodiments, the intracellular domain comprises six immunoreceptor tyrosine-based inhibitory 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] In particular embodiments, the intracellular domain comprises at least seven immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 117), VTYAEV (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 120).

[0294] The LILRB1 protein has four immunoglobulin (Ig) like domains termed DI, D2, D3 and D4. In some embodiments, the LILRB1 hinge domain comprises an LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB 1 D3D4 domain comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to SEQ ID NO: 127. In some embodiments, the LILRB 1 D3D4 domain comprises or consists essentially of SEQ ID NO: 127.

[0295] In some embodiments, the polypeptide comprises the LILRB 1 hinge domain or functional variant thereof. In embodiments, the LILRB 1 hinge domain or functional variant thereof comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or identical to SEQ ID NO: 134, SEQ ID NO: 127, or SEQ ID NO: 128. In embodiments, the LILRB 1 hinge domain or functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 134, SEQ ID NO: 127, or SEQ ID NO: 128.

[0296] In some embodiments, the LILRB 1 hinge domain comprises a sequence identical to SEQ ID NO: 134, SEQ ID NO: 127, or SEQ ID NO: 128.

[0297] In some embodiments, the LILRB 1 hinge domain consists essentially of a sequence identical to SEQ ID NO: 134, SEQ ID NO: 127, or SEQ ID NO: 128.

[0298] In some embodiments, the transmembrane domain is a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB 1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% to SEQ ID NO: 135. In some embodiments, the LILRB 1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 135. In some embodiments, the LILRB 1 transmembrane domain comprises a sequence identical to SEQ ID NO: 135. In embodiments, the LILRB 1 transmembrane domain consists essentially of a sequence identical to SEQ ID NO: 135.

[0299] In some embodiments, the transmembrane domain can be attached to the extracellular region of the second, inhibitory receptor, e.g., the antigen binding domain or ligand binding domain, via a hinge, e.g., a hinge from a human protein. For example, in some embodiments, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, a CD8α hinge or an LILRB1 hinge.

[0300] In some embodiments, the second, inhibitory receptor comprises an inhibitory domain. In some embodiments, the second, inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory domain is isolated or derived from LILR1B.Inhibitory Receptors Comprising Combinations of LILRB1 Domains

[0301] In some embodiments, the LILRB1-based inhibitory receptors of the disclosure comprise more than one LILRB1 domain or functional equivalent thereof. For example, in some embodiments, the inhibitory receptor comprises an LILRB1 transmembrane domain and intracellular domain, or an LILRB1 hinge domain, transmembrane domain and intracellular domain.

[0302] In particular embodiments, the inhibitory receptor comprises an LILRB1 hinge domain or functional fragment thereof, and the LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to SEQ ID NO: 129. In some embodiments, the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 129. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 129.

[0303] In further embodiments, the inhibitory receptor comprises: the LILRB1 transmembrane domain or a functional variant thereof, and an LILRB1 intracellular domainand / or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory 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 the LILRB1 transmembrane domain or a functional variant thereof, and an LILRB1 intracellular domain and / or an intracellular domain comprising at least two ITIM, 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).

[0304] In some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and intracellular domain. In some embodiments, the polypeptide comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to SEQ ID NO: 130. In some embodiments, the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 130. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 130.

[0305] In preferred embodiments, the inhibitory receptor comprises: an LILRB1 hinge domain or functional variant thereof; an LILRB1 transmembrane domain or a functional variant thereof; and an LILRB1 intracellular domain and / or an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from LYAAV (SEQ ID NO: 117), VTYAE (SEQ ID NO: 118), VTYAQL (SEQ ID NO: 119), and SIYATL (SEQ ID NO: 11).

[0306] 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.

[0307] 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.

[0308] 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 10 Polypeptide sequences for illustrative LILRB 1-based inhibitory receptorsPolynucleotides and Vectors

[0309] The disclosure provides polynucleotides encoding the sequence(s) of the first and second receptors of the disclosure. The disclosure further provides T cells comprising the polynucleotides and vectors described herein.

[0310] In some embodiments, the sequence of the first and / or second receptor is operably linked to a promoter. In some embodiments, the sequence encoding the first receptor is operably linked to a first promoter, and the sequence encoding the second receptor is operably linked to a second promoter.

[0311] The disclosure provides vectors comprising the polynucleotides described herein.

[0312] 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 encoded shRNA, for example a B2M shRNA.

[0313] In some embodiments, both receptors are encoded by a single vector. In some embodiments the vector further comprises an encoded shRNA, for example a B2M shRNA.

[0314] In some embodiments, the first and second receptors are encoded by a single vector. Methods of encoding multiple polypeptides using a single vector will be known to persons of ordinary skill in the art, and include, inter alia, encoding multiple polypeptides under control of different promoters, or, if a single promoter is used to control transcription of multiple polypeptides, use of sequences encoding internal ribosome entry sites (IRES) and / or self- cleaving peptides. Illustrative self-cleaving peptides include T2A, P2A, E2A and F2A self- cleaving peptides. In some embodiments, the T2A self-cleaving peptide comprises a sequence of (SEQ ID NO: 1239). In some embodiments, theP2A self-cleaving peptide comprises a sequence of(SEQ ID NO: 1240). In some embodiments, the E2A self-cleaving peptide comprises a sequence of(SEQ ID NO: 1241). In some embodiments, the F2A selfcleaving peptide comprises a sequence of(SEQ ID NO: 1242). In some embodiments, the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 1243). Any of the foregoing can also include an N terminal GSG linker. For example, a T2A self-cleaving peptide can also comprise a sequence of(SEQ ID NO: 1244), which can be encoded by a sequence of(SEQ ID NO: 1245).

[0315] In some embodiments, the vector is an expression vector, i.e. for the expression of the first and / or second receptor in a suitable cell.

[0316] Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0317] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector encodes for a first receptor specific for a target antigen, a second receptor specific for a non-target antigen, and a B2M shRNA.

[0318] In some embodiments, the lentiviral vector encodes for a first receptor specific for CEA, a second receptor specific for HLA-A*02, and a B2M shRNA.

[0319] In some embodiments, the lentiviral vector encodes for a first receptor specific for MSLN, a second receptor specific for HLA-A*02, and a B2M shRNA.

[0320] In some embodiments, the lentiviral vector encodes for a first receptor specific for HER2, a second receptor specific for HLA-A*02, and a B2M shRNA.

[0321] The expression of natural or synthetic nucleic acids encoding receptors is typically achieved by operably linking a nucleic acid encoding the receptor or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0322] The polynucleotides encoding the receptors can be cloned into a number of types of vectors. For example, the polynucleotides can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0323] Further, the expression vector can be provided to cells, such as immune cells, in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0324] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0325] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 base pairs (bp) upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0326] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-lα). 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, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, a U6 promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0327] In some embodiments, the promoter is a U6 promoter.

[0328] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0329] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0330] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0331] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. An illustrative colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0332] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays can be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELIS As and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.Engineered T Cells

[0333] The disclosure provides engineered T cells comprising the receptors, shRNAs, vectors, and polynucleotides described herein.

[0334] The starting cells for the engineered T cells can be obtained from the subject themselves (autologous cells). In some embodiments, the cells are obtained before the subject starts any treatment.

[0335] Methods for adoptive cell therapy using autologous cells includes isolating immune cells from patient blood, performing a series of modifications on the isolated cells including transducing the cells with one or more vectors encoding the dual receptor system described herein, and administering the cells to a patient. Providing immune cells from a subject suffering from or at risk for cancer or a hematological malignancy requires isolation of immune cell from the patient’s blood, and can be accomplished through methods known in the art, for example, by leukapheresis. During leukapheresis, blood from a subject is extracted and the peripheral blood mononuclear cells (PBMCs) are separated, and the remainder of the blood is returned to the subject’s circulation. The PBMCs are stored either frozen or cryopreserved as a sample of immune cells and provided for further processing steps, such as, e.g. the modifications described herein.

[0336] The disclosure provides enrichment and / or depletion steps that can be, for example, washing and fractionating methods known in the art for preparation of subject PBMCs for downstream procedures, e.g. the modifications described herein. For example, without limitation, methods can include devices to remove gross red blood cells and platelet contaminants, systems for size-based cell fractionation for the depletion of monocytes and the isolation of lymphocytes, and / or systems that allow the enrichment of specific subsets of Tcells, such as, e.g. CD4+, CD8+, CD25+, or CD62L+ T cells. Following the enrichment steps, a target sub-population of immune cells will be isolated from the subject PMBCs for further processing. Those skilled in the art will appreciate that enrichment steps, as provided herein, can also encompass any newly discovered method, device, reagent or combination thereof.

[0337] The disclosure provides activation steps that can be any method known in the art to induce activation of immune cells, e.g. T cells, required for their ex vivo expansion. Immune cell activation can be achieved, for example, by culturing the subject immune cells in the presence of dendritic cells, culturing the subject immune cells in the presence of artificial antigen-presenting cells (AAPCs), or culturing the immune cells in the presence of irradiated K562-derived AAPCs. Other methods for activating subject immune cells can be, for example, culturing the immune cells in the presence of isolated activating factors and compositions, e.g. beads, surfaces, or particles functionalized with activating factors.Activating factors can include, for example, antibodies, e.g. anti-CD3 and / or anti-CD28 antibodies. Activating factors can also be, for example, cytokines, e.g. interleukin (IL)-2 or IL-21. Activating factors can also be costimulatory molecules, such as, for example, CD40, CD40L, CD70, CD80, CD83, CD86, CD137L, ICOSL, GITRL, and CD134L. Those skilled in the art will appreciate that activating factors, as provided herein, can also encompass any newly discovered activating factor, reagent, composition, or combination thereof that can activate immune cells.

[0338] The disclosure provides genetic modification steps for modifying the subject immune cells. In some embodiments, the genetic modification comprises transducing the immune cell with a vector comprising a shRNA described herein complementary to B2M or HLA-A. In some embodiments, the genetic modification comprises modifying the genome of the immune cells to induce mutations in B2M or HLA-A using CRISPR / Cas mediated genome engineering. In some embodiments, the method comprises transducing the immune cell with one or more vectors encoding the activator and inhibitory receptors, thereby producing immune cells expressing the activator and inhibitory receptors.

[0339] The disclosure provides expansion steps for the genetically modified subject immune cells. Genetically modified subject immune cells can be expanded in any immune cell expansion system known in the art to generate therapeutic doses of immune cells for administration. For example, bioreactor bags for use in a system comprising controller pumps, and probes that allow for automatic feeding and waste removal can be used for immune cell expansion. Cell culture flasks with gas-permeable membranes at the base can beused for immune cell expansion. Any such system known in the art that enables expansion of immune cells for clinical use is encompassed by the expansion step provided herein. Immune cells are expanded in culture systems in media formulated specifically for expansion. Expansion can also be facilitated by culturing the immune cell of the disclosure in the presence of activation factors as described herein. Those skilled in the art will appreciate that expansion steps, as provided herein, can also encompass any newly discovered culture systems, media, or activating factors that can be used to expand immune cells.

[0340] The disclosure provides formulation and cry opreservation steps for the expanded genetically modified subject immune cells. Formulation steps provided include, for example, washing away excess components used in the preparation and expansion of immune cells of the methods of treatment described herein. Any pharmaceutically acceptable formulation medium or wash buffer compatible with immune cells known in the art can be used to wash, dilute / concentration immune cells, and prepare doses for administration. Formulation medium can be acceptable for administration of the engineered cells, such as, for example crystalloid solutions for intravenous infusion.

[0341] Cryopreservation can optionally be used to store immune cells long-term.Cry opreservation can be achieved using known methods in the art, including for example, storing cells in a cryopreservation medium containing cryopreservation components.Cry opreservation components can include, for example, dimethyl sulfoxide or glycerol. Immune cells stored in cry opreservation medium can be cryopreserved by reducing the storage temperature to -80°C to -196 °C.

[0342] In some embodiments, the engineered T cells comprise: (a) a first receptor comprising an extracellular ligand binding domain specific to the target antigen and an intracellular domain comprising at least one signal transduction element that activates and / or costimulates the engineered T cell; and (b) a second receptor comprising an extracellular ligand binding domain specific to the non-target antigen and an intracellular domain comprising at least one signal transduction element that inhibits the engineered T cell.

[0343] In some embodiments, the engineered T cells comprise: (a) a first receptor comprising an extracellular ligand binding domain specific to CEA and an intracellular domain comprising at least one signal transduction element that activates and / or costimulates the engineered T cell; and (b) a second receptor comprising an extracellular ligand binding domain specific to the non-target antigen and an intracellular domain comprising at least one signal transduction element that inhibits the engineered T cell.

[0344] In some embodiments, the engineered T cells comprise: (a) a first receptor comprising an extracellular ligand binding domain specific to MSLN and an intracellular domain comprising at least one signal transduction element that activates and / or co-stimulates the engineered T cell; and (b) a second receptor comprising an extracellular ligand binding domain specific to the non-target antigen and an intracellular domain comprising at least one signal transduction element that inhibits the engineered T cell.

[0345] In some embodiments, the engineered T cells comprise: (a) a first receptor comprising an extracellular ligand binding domain specific to HER2 and an intracellular domain comprising at least one signal transduction element that activates and / or co-stimulates the engineered T cell; and (b) a second receptor comprising an extracellular ligand binding domain specific to the non-target antigen and an intracellular domain comprising at least one signal transduction element that inhibits the engineered T cell.

[0346] In some embodiments, the disclosure provides for a population of cells comprising engineered T cells. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that express an activator receptor. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that express an inhibitory receptor. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that express both an activator receptor and inhibitory receptor. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that express an B2M shRNA. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that have reduced B2M expression compared to non-engineered T cells. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that present a reduced amount of MHC-I at the cell surface compared to non-engineered T cells. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that express an activator receptor, an inhibitory receptor, and a B2M shRNA.

[0347] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered Tcells that express a CEA activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA.

[0348] In some embodiments, at least 10% of the population of cells are engineered T cells that express a CEA activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 20% population of cells are engineered T cells that express a CEA activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 30% population of cells are engineered T cells that express a CEA activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 40% of the population of cells are engineered T cells that express a CEA activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 50% of the population of cells are engineered T cells that express a CEA activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 60% of the population of cells are engineered T cells that express a CEA activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 70% of the population of cells are engineered T cells that express a CEA activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 80% of the population of cells are engineered T cells that express a CEA activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 90% of the population of cells are engineered T cells that express a CEA activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA.

[0349] In some embodiments, the about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that express a MSLN activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA.

[0350] In some embodiments, at least 10% of the population of cells are engineered T cells that express a MSLN activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 20% of the population of cells are engineered T cells that express MSLN activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 30% of the population of cells are engineered T cells that express a MSLN activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 40% of the population of cells are engineered T cells that express s a MSLN activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 50% of the population of cells are engineered T cells that express a MSLN activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In someembodiments, at least 60% of the population of cells are engineered T cells that express a MSLN activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 70% of the population of cells are engineered T cells that express a MSLN activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 80% of the population of cells are engineered T cells that express a MSLN activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 90% of the population of cells are engineered T cells that express a MSLN activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA.

[0351] In some embodiments, the about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the population of cells are engineered T cells that express a HER2 activator receptor, an HL A- A* 02 inhibitory receptor, and a B2M shRNA.

[0352] In some embodiments, at least 10% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 20% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 30% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 40% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 50% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 60% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 70% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 80% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA. In some embodiments, at least 90% of the population of cells are engineered T cells that express a HER2 activator receptor, an HLA-A*02 inhibitory receptor, and a B2M shRNA.

[0353] Methods of measuring expression of proteins in a cell are known in the art. For example, expression of cell surface receptors can be measured by using cell surface receptor specific antibodies and flow cytometry methods. Expression of cell surface proteins such asM2M can be measured using B2M specific antibodies and flow cytometry. Since B2M form and obligate complex with MHC-I proteins and MHC-I protein trafficking to the cell surface is dependent on B2M proteins, measuring MHC-I expression at the cell surface can be used as a surrogate for B2M expression.

[0354] 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.

[0355] The disclosure provides engineered T cells comprising a first receptor comprising a sequence of SEQ ID NO: 52, and second receptor comprising a sequence of SEQ ID NO: 164, or sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0356] The disclosure provides engineered T cells comprising a first receptor comprising a sequence of, and second receptor comprising a sequence of SEQ ID NO: 164, or sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0357] The disclosure provides engineered T cells comprising a first receptor comprising a sequence of, and second receptor comprising a sequence of SEQ ID NO: 164, or sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0358] In some embodiments, the engineered T cells comprise an shRNA encoded by a sequence comprising (SEQ ID NO:179) or a sequence having at least 80%, at least 90%, or at least 95% identity thereto. In some embodiments, the engineered T cells comprise first receptor comprising a sequence of SEQ ID NO: 52, a second receptor comprising a sequence of SEQ ID NO: 164, and a sequence encoding an shRNA comprising a sequence of SEQ ID NO: 179. In some embodiments, the first receptor and second receptor are encoded by a single polynucleotide, and wherein the sequences encoding the first and second receptors are separated by a sequence encoding a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises a T2A self-cleaving polypeptide comprising a sequence of(SEQ ID NO: 181).

[0359] The disclosure provides engineered T cells comprising a polypeptide comprising a 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.

[0360] The disclosure provides engineered T cells comprising a polynucleotide comprising a 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.

[0361] As used herein, a “T-cell” refers to a type of lymphocyte that originates from a bone marrow precursor that develops in the thymus gland. There are several distinct types of T- cells which develop upon migration to the thymus, which include, helper CD4+ T-cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T-cells and stem memory T-cells. Different types of T-cells can be distinguished by the ordinarily skilled artisan based on their expression of markers. Methods of distinguishing between T-cell types will be readily apparent to the ordinarily skilled artisan.

[0362] In some embodiments, the first receptor and the second receptor together specifically activate the engineered T cells in the presence of the target cell.

[0363] In some embodiments, the engineered T cell is an alpha beta (αβ) T cell. In some embodiments, the engineered T cell is a gamma delta (γδ) T cell. In some embodiments, the engineered T cell is an invariant T 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.

[0364] In some embodiments, the engineered T cell is non-natural. In some embodiments, the engineered T cell is isolated.

[0365] Methods transforming populations of engineered T cell, such as T cells, with the vectors of the instant disclosure will be readily apparent to the person of ordinary skill in the art. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi), according to manufacturer’s instructions. T cells can be cultured at a density ofcells / mL in X-Vivo 15 media supplemented with 5% human A / B serum and 1% Pen / strep in the presence of CD3 / 28 Dynabeads (1 : 1 cell to bead ratio) and 300 Units / mL of IL-2 (Miltenyi). After 2 days, T cells can be transduced with viral vectors, such as lentiviral vectors using methods known in the art. In 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 combinations of IL-7 / 15 / 21 for an additional 5 days prior to enrichment. Methods of isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells, will be readily apparent to the person of ordinary skill in the art. Although this method outlines a potential approach it should be noted that these methodologies are rapidly evolving. For example, excellent viral transduction of peripheralblood mononuclear cells can be achieved after 5 days of growth to generate a >99% CD3+ highly transduced cell population.

[0366] Methods of activating and culturing populations of T cells comprising the TCRs, CARs, inhibitory receptors or vectors encoding same, will be readily apparent to the person of ordinary skill in the art.

[0367] Whether prior to or after genetic modification of T cells to express a CAR, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10040846; and U.S. Pat. Appl. Pub. No. 2006 / 0121005.

[0368] In some embodiments, T cells of the instant disclosure are expanded and activated in vitro. Generally, the T cells of the instant disclosure are expanded in vitro by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, such as by contact with an anti-CD3 antibody. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(l-2):53-63, 1999).Immune Cells with Reduced MHC Class I Polypeptide Expression

[0369] In some embodiments, the engineered T cell described herein are modified to inactivate, or reduce or eliminate expression or function of an endogenous gene encoding an allele of an endogenous MHC class I polypeptide. In some embodiments, the gene encoding the MHC class I polypeptide is 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 envisaged as within the scope of the instant 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. Insome 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.

[0370] In some embodiments, the genetically engineered T cells described herein are modified to reduce or eliminate expression of the B2M gene product. The beta-2 microglobulin (B2M) gene encodes a protein that associates with the major histocompatibility complex (MHC) class I, i.e. MHC-I complex. The MHC-I complex is required for presentation of antigens on the cell surface. The MHC -I complex is disrupted and non-functional when the B2M is deleted (Wang D et al. Stem Cells Transl Med. 4: 1234- 1245 (2015)). Furthermore, the B2M gene can be disrupted with high efficiency using gene editing techniques known in the art (Ren et al. Clin. Cancer Res. 23:2255-2266 (2017)). Reducing or eliminating B2M can reduce, or eliminate functional MHC I on the surface of the engineered T cell.

[0371] The disclosure provides gene editing systems for editing an endogenous target gene in an immune cell. The disclosure provides interfering RNAs specific to sequences of target genes. Gene editing systems such as CRISPR / Cas systems, TALENs and zinc fingers can be used to generate double strand breaks, which, through gene repair mechanisms such as homology directed repair or non-homologous end joining (NHEJ), can be used to introduce mutations. NHEJ after resection of the ends of the break, or improper end joining, can be used to introduce deletions. In some embodiments, the target gene comprises a gene encoding a subunit of the MHC-I complex.

[0372] Target gene sequences include, but are not limited to, promoters, enhancers, introns, exons, intron / exon junctions, transcription products (pre-mRNA, mRNA, and splice variants), and / or 3’ and 5’ untranslated regions (UTRs). Any gene element or combination of gene elements can be targeted for the purpose of genetic editing in the immune cells described herein. Modifications to the target genes can be accomplished using any method known in the art to edit the target gene that results in altered or disrupted expression or function the target gene or gene product.

[0373] In some embodiments, modifying the gene encoding the MHC class I polypeptide comprises deleting all or a portion of the gene. In some embodiments, modifying the gene encoding the MHC class I polypeptide comprises introducing a mutation in 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.

[0374] Gene sequences for the target genes described herein are known in the art. The sequences can be found at public databases, such as NCBI GenBank or the NCBI nucleotide database. 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 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 11 provides non-limiting illustrative sequences of HLA-A allele and B2M gene sequences targeted for modification as described herein.Table 11 Illustrative Target Gene Sequences

[0375] The person of ordinary skill in the art will appreciate that T can be substituted for U to convert an RNA sequence to a DNA sequence and vice versa, and both are envisaged as target gene sequences of the disclosure.

[0376] In some embodiments, the gNAs specifically target a coding DNA sequence that is shared by more than 1000 HLA-A*02 alleles. In some embodiments, the gNAs that specifically target a coding DNA sequence in greater than 1000 HLA-A*02 alleles comprise a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity or is identical to a sequence selected from SEQ ID NOs: 400-465.

[0377] The sequences in SEQ ID NOs: 618-869 or 1248-1367 are presented as DNA sequences. The skilled artisan will understand that thymine (T) can be replaced with uracil (U) in any DNA sequence including those set forth in SEQ ID NOs: 618-869 or 1248-1367 , to arrive at the corresponding RNA sequence.

[0378] The sequences disclosed in SEQ ID NOs: 1248-1367 include the corresponding genomic sequences, inclusive of the PAM sequence. The skilled artisan will understand that the targeting sequence of the gRNA does not include three 3 ’ terminal nucleotides of the sequences in SEQ ID NOs: 1248-1367 , which represent the corresponding PAM site for the gRNA.

[0379] The disclosure provides gNAs comprising a targeting sequence specific to the B2M gene. In some embodiments, the gNAs specifically target the coding sequence (CDS) sequence of the B2M gene. In some embodiments, the gNA comprises a sequence that targets the B2M gene promoter sequence.

[0380] In some embodiments the gNA comprise a targeting sequence and a gNA scaffold sequence. In some embodiments, the targeting sequence comprises a sequence set forth in SEQ ID NOs: 618-731, or a sequence shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity thereto.

[0381] In some embodiments, the targeting sequence is complementary to a sequence of the B2M gene. In some embodiments, the B2M gene comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity to the B2M sequence set forth in Table 11.

[0382] The sequences set forth in Table 22 are presented as DNA sequences. In all sequences set forth in SEQ ID NOs: 732-869, thymine (T) can be replaced by uracil (U) to arrive at the sequence of the target mRNA sequence.

[0383] An illustrative sequence encoding a B2M shRNA comprises a sequence of(SEQ ID NO:179), or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto. A further illustrative sequence encoding a B2M shRNA comprises a sequence of(SEQ ID NO:180), or a sequence having at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

[0384] In some embodiments, the interfering RNAs comprise a sequence complementary to a sequence of an HLA-A*02 mRNA. In some embodiments, the interfering RNA is capable of inducing RNAi-mediated degradation of the 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.

[0385] In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises a first sequence, having from 5’ to 3’ end a sequence complementary to the HLA-A*02 mRNA; and a second sequence, having from 5’ to 3’ end a sequence complementary to the first sequence, wherein the first sequence and second sequence form the shRNA

[0386] Illustrative target HLA sequences complementary to the first sequence are shown in SEQ ID NOs: 870-955.

[0387] In some embodiments, the 5’ flank sequence is selected from the sequences set forth in SEQ ID NOs: 956-959. Illustrative flank sequences are shown in SEQ ID NOs: 960-961.

[0388] In some embodiments, the first and second sequence are present on a single stranded polynucleotide, wherein the first sequence and second sequence are separated by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, wherein the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides form a loop region in the shRNA. In some embodiments, the loop region comprises a sequence selected from the sequences set forth in SEQ ID NOs: 962-975.

[0389] shRNAs of the disclosure can be generated exogenously by chemical synthesis, by in vitro transcription, or by cleavage of longer double-stranded RNA with Dicer or another appropriate nuclease with similar activity. Chemically synthesized siRNAs, produced from protected ribonucleoside phosphoramidites using a conventional DNA / RNA synthesizer, can be obtained from commercial suppliers such as Millipore Sigma (Houston, Tex.), Ambion Inc. (Austin, Tex.). Invitrogen (Carlsbad, Calif.), or Dharmacon (Lafayette, Colo.). siRNAs can be purified by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof, for example. Alternatively, siRNAs can be used with little if any purification to avoid losses due to sample processing.

[0390] In some embodiments, shRNAs of the disclosure can be produced using an expression vector into which a nucleic acid encoding the double stranded RNA has been cloned, for example under control of a suitable promoter.Pharmaceutical Compositions

[0391] The disclosure provides pharmaceutical compositions comprising engineered T cells comprising the first and second receptors of the disclosure and a pharmaceutically acceptable diluent, carrier or excipient.

[0392] Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; and preservatives.

[0393] Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise of immune cells combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteraladministration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations can further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. Parenteral formulations also include aqueous solutions which can contain excipients such as salts, carbohydrates and buffering agents. Illustrative parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Formulations for parenteral administration can be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0394] In some embodiments, the engineered T cell is formulated with a commercially available animal protein-free, serum-free cry opreservation medium. In some embodiments, the engineered T cell is in 20% HSA (human serum albumin). In some embodiments, the engineered T cell is in 25% HSA (human serum albumin). In some embodiments, the engineered T cell is in 30% HSA (human serum albumin). In some embodiments, the engineered T cell is cryopreserved.

[0395] In some embodiments, the cells in the pharmaceutical composition comprise a population of engineered T cells and a population of non-engineered cells. The combination of engineered T cells and non-engineered cells is the amount of total cells in the pharmaceutical composition.

[0396] In some embodiments, the pharmaceutical composition comprises a plurality of engineered T cells. In some embodiments, the plurality of engineered T cells comprises an activator receptor and an inhibitory receptor. In some embodiments, the plurality of engineered T cells comprises an activator receptor, an inhibitory receptor, and a B2M shRNA. The percentage of transduced cells that express the activator receptor, the inhibitory receptor, and / or a shRNA in a population of cells can be measured by methods known in the art, for example by flow cytometry. Total cells in a sample and engineered T cells positive for the activator receptor, the inhibitory receptor, and / or a shRNA can be tallied and the percentage of transduced cells can be calculated. Insertion of a transgene into the genome can be measured by methods known in the art, for example by ddPCR.

[0397] In some embodiments, the formulated composition comprising the immune cells is suitable for administration via injection. In some embodiments, the formulated composition comprising the immune cells is suitable for administration via infusion.

[0398] The pharmaceutical compositions of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the immune cells with the pharmaceutical carrier(s) or excipient(s), such as liquid carriers.

[0399] Aqueous suspensions can further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also contain stabilizers.

[0400] The compositions of the present disclosure can additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the immune cells of the compositions of the present disclosure.

[0401] The formulation or composition can also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the immune cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents.

[0402] The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.Dosage and Administration

[0403] The engineered T cells or the pharmaceutical compositions of the present disclosure can be formulated in a therapeutically effective dose and administered in a number of ways depending upon whether local or systemic treatment is desired.

[0404] An effective amount or therapeutically effective dose means an amount which provides a therapeutic or prophylactic benefit. The dosage administered will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In some embodiments, an effective amount of cells or composition comprising those cells are administrated parenterally. In some embodiments, administration can be an intravenous administration. In some embodiments, administration can be directly done by injection within a tumor.

[0405] When calculating the therapeutically effective dose of cells in a pharmaceutical composition, only the amount of engineered T cells and not the total cell amount in a dose are taken into account. For example, a therapeutically effective dose can be 0.5 × 106engineered T cells but the total amount of cells that is administered is 1 × 106cells, meaning 0.5× 106cells are not engineered T cells. The therapeutically effective dose is defined as the number of viable and double receptor-positive engineered T cells in a dose. For example, the double receptor-positive engineered T cells express both the activator and the inhibitor receptor.

[0406] In some embodiments, a therapeutically effective dose comprises about 0.5× 106engineered T cells, about 1 × 106engineered T cells, about 2× 106engineered T cells, about 3× 106engineered T cells, 4× 106engineered T cells, about 5× 106engineered T cells, about 6× 106engineered T cells, about 7× 106engineered T cells, about 8× 106engineered T cells, about 9× 106engineered T cells, about 1 × 107, about 2×107, about 3 × 107, about 4×107, about 5× 107, about 6× 107, about 7× 107, about 8× 107, about 9× 107, about 1 × 108engineered T cells, 1.2× 108engineered T cells, about 1.4× 108engineered T cells, about 1.6× 108engineered T cells, about 1.8×108engineered T cells, about 2×108engineered T cells, about 2.2× 108engineered T cells, about 2.4×108engineered T cells, about 2.6×108engineered T cells, about 2.8× 108engineered T cells, about 3× 108engineered T cells, about 3.2× 108engineered T cells, about 3.4× 108engineered T cells, about 3.6×108engineered T cells, about 3.8×108engineered T cells, about 4× 108engineered T cells, about 4.2× 108engineered T cells, about 4.4× 108engineered T cells, about 4.6× 108engineered T cells, about 4.8×108engineered T cells, about 5× 108engineered T cells, about 5.2× 108engineered T cells, about 5.4× 108engineered T cells, about 5.6×108engineered T cells, about 5.8×108engineered T cells, about 6×108engineered T cells, about 6.2× 108engineered T cells, about 6.4× 108engineered T cells, about 6.6× 108engineered T cells, about 6.8× 108engineered T cells, about 7× 108engineered T cells, about 7.2× 108engineered T cells, about 7.4× 108engineered T cells, about 7.6× 108engineered T cells, about 7.8× 108engineered T cells, about 8× 108engineered T cells, about 8.2× 108engineered T cells, about 8.4× 108engineered T cells, about 8.6× 108engineered T cells, about8.8×108engineered T cells, about 9×108engineered T cells, about 9.2×108engineered T cells, about 9.4×108engineered T cells, about 9.6×108engineered T cells, about 9.8×108engineered T cells, , about 1×109engineered T cells, about 1.2×109engineered T cells, about 1.4×109engineered T cells, about 1.6×109engineered T cells, about 1.8×109engineered T cells, about 2×109engineered T cells, about 2.2×109engineered T cells, about 2.4×109engineered T cells, about 2.6×109engineered T cells, about 2.8 ×109engineered T cells, about 3×109engineered T cells, about 3.2×109engineered T cells, about 3.4×109engineered T cells, about 3.6×109engineered T cells, about 3.8×109engineered T cells, about 4×109engineered T cells, about 4.2×109engineered T cells, about 4.4×109engineered T cells, about 4.6×109engineered T cells, about 4.8×109engineered T cells, about 5×109engineered T cells, about 5.2×109engineered T cells, about 5.4×109engineered T cells, about 5.6×109engineered T cells, about 5.8×109engineered T cells, about 6×109engineered T cells, about 6.2×109engineered T cells, about 6.4×109engineered T cells, about 6.6×109engineered T cells, about 6.8×109engineered T cells, about 7×109engineered T cells, about 7.2×109engineered T cells, about 7.4×109engineered T cells, about 7.6×109engineered T cells, about 7.8×109engineered T cells, about 8×109engineered T cells, about 8.2×109engineered T cells, about 8.4×109engineered T cells, about 8.6×109engineered T cells, about 8.8×109engineered T cells, about 9×109engineered T cells, about 9.2×109engineered T cells, about 9.4×109engineered T cells, about 9.6×109engineered T cells, about 9.8×109engineered T cells, about 1×1010engineered T cells, 1.2×1010engineeredT cells, 1.4×1010engineeredT cells, 1.6×1010engineered T cells, 1.8×1010engineered T cells, about 2×1010engineered T cells, about 2.2×1010engineered T cells, about 2.4×1010engineered T cells, about 2.6×1010engineered T cells, about 2.8×1010engineered T cells, about 3×1010engineered T cells, about 3.2×1010engineered T cells, about 3.4×1010engineered T cells, about 3.6xlO10engineered T cells, about 3.8xlO10engineered T cells, about 4×1010engineered T cells, about 4.2×1010engineered T cells, about 4.4×1010engineered T cells, about 4.6×1010engineered T cells, about 4.8×1010engineered T cells, about 5×1010engineered T cells, 5.3 ×1010engineered T cells, 5.4×1010engineered T cells, 5.6×1010engineered T cells, 5.8×1010engineered T cells, about 6×1010engineered T cells, about 6.2×1010engineered T cells, about 6.4×1010engineered T cells, about 6.6×1010engineered T cells, about 6.8×1010engineered T cells, about 7×1010engineered T cells, about 7.2×1010engineered T cells, about 7.4×1010engineered T cells, about 7.6×1010engineered T cells, about 7.8×1010engineered T cells, about 8×1010engineered T cells, about 8.2×1010engineered T cells, about 8.4×1010engineered T cells, about 8.6×1010engineered T cells, about 8.8×1010engineered T cells,about 9×1010engineered T cells, about 9.2×1010engineered T cells, about 9.4×1010engineered T cells, about 9.6×1010engineered T cells or about 9.8×1010engineered T cells.

[0407] In some embodiments, a therapeutically effective dose comprises about 0.5×106engineered T cells to about 9.8×1010engineered T cells, about l×106engineered T cells to about 9.6×1010engineered T cells, about 1.2×106engineered T cells to about 9.4×1010engineered T cells, about 1.4×106engineered T cells to about 9.2×1010engineered T cells, about 1.6×106engineered T cells to about 9×1010engineered T cells, about 1.8×106engineered T cells to about 8.8×1010engineered T cells, about2×106engineered T cells to about 8.6×1010engineered T cells, 2.4×106engineered T cells to about 8.4×1010engineered T cells, about 2.6×106engineered T cells to about 8.2×1010engineered T cells, about 2.8×106engineered T cells to about 8×1010engineered T cells, about 3×106engineered T cells to about 7.8×1010engineered T cells, about 3.2×106engineered T cells to about 7.6×1010engineered T cells, about 3.4×106engineered T cells to about 7.4×1010engineered T cells, about 3.6×106engineered T cells to about 7.2×1010engineered T cells, about 3.8×106engineered T cells to about 7×1010engineered T cells, about 4×106engineered T cells to about 6.8×1010engineered T cells, about 4.2×106engineered T cells to about 6.6×1010engineered T cells, about 4.4×106engineered T cells to about 6.4×1010engineered T cells, about 4.6×106engineered T cells to about 6.2×1010engineered T cells, about 4.8×106engineered T cells to about 6×1010engineered T cells, about 5×106engineered T cells to about 5.8×1010engineered T cells, about 5.2×106engineered T cells to about 5.6×1010engineered T cells, about 5.4×106engineered T cells to about 5.4×1010engineered T cells, about 5.6×106engineered T cells to about 5.2×1010engineered T cells, about 5.8×106engineered T cells to about 5 ×1010engineered T cells, about 6×106engineered T cells to about 4.8×1010engineered T cells, about 6.2×106engineered T cells to about 4.6×1010engineered T cells, about 6.4×106engineered T cells to about 4.4×1010engineered T cells, about 6.6×106engineered T cells to about 4.2×1010engineered T cells, about 6.8×106engineered T cells to about 4×1010engineered T cells, about 7×106engineered T cells to about 3.8×1010engineered T cells, about 7.2×106engineered T cells to about 3.6×1010engineered T cells, about 7.4×106engineered T cells to about 3.4×1010engineered T cells, about 7.6×106engineered T cells to about 3.2×1010engineered T cells, about 7.8×106engineered T cells to about 3 ×1010engineered T cells, about 8×106engineered T cells to about 2.8×1010engineered T cells, about 8.2×106engineered T cells to about 2.6×1010engineered T cells, about 8.4×106engineered T cells to about 2.4×1010engineered T cells, about 8.6×106engineered T cells to about 2.2×1010engineered T cells, about 8.8×106engineered T cells to about 2×1010engineered T cells, about 9×106engineered T cells to about 1.8×1010engineered T cells, about 9.2×106engineered T cells to about 1.6×1010engineered T cells, about 9.4×106engineered T cells to about 1.4×1010engineered T cells, about 9.6×106engineered T cells to about 1.2×1010engineered T cells, about 9.8×106engineered T cells to about 1×1010engineered T cells, about 1×107engineered T cells to about 9.8×109engineered T cells, about 1.2×107engineered T cells to about 9.6×109engineered T cells, about 1.4×107engineered T cells to about 9.4×109engineered T cells, about 1.6×107engineered T cells to about 9.2×109engineered T cells, about 1.8×107engineered T cells to about 9×109engineered T cells, about 2×107engineered T cells to about 8.8×109engineered T cells, about 2.2×107engineered T cells to about 8.6×109engineered T cells, about 2.4×107engineered T cells to about 8.4×109engineered T cells, about 2.6×107engineered T cells to about 8.2×109engineered T cells, about 2.8×107engineered T cells to about 8×109engineered T cells, about 3×107engineered T cells to about 7.8×109engineered T cells, about 3.2×107engineered T cells to about 7.6×109engineered T cells, about 3.4×107engineered T cells to about 7.4×109engineered T cells, about 3.6×107engineered T cells to about 7.2×109engineered T cells, about 3.8×107engineered T cells to about 7×109engineered T cells, about 4×107engineered T cells to about 6.8×109engineered T cells, about 4.2×107engineered T cells to about 6.6×109engineered T cells, about 4.4×107engineered T cells to about 6.4×109engineered T cells, about 4.6×107engineered T cells to about 6.2×109engineered T cells, about 4.8× 107engineered T cells to about 6×109engineered T cells, about 5×107engineered T cells to about 5.8×109engineered T cells, about 5.2×107engineered T cells to about 5.6×109engineered T cells, about 5.4×107engineered T cells to about 5.4×109engineered T cells, about 5.6×107engineered T cells to about 5.2×109engineered T cells, about 5.8×107engineered T cells to about 5×109engineered T cells, about 6×107engineered T cells to about 4.8×109engineered T cells, about 6.2×107engineered T cells to about 4.6×109engineered T cells, about 6.4×107engineered T cells to about 4.4×109engineered T cells, about 6.6×107engineered T cells to about 4.2×109engineered T cells, about 6.8×107engineered T cells to about 4×109engineered T cells, about 7×107engineered T cells to about 3.8×109engineered T cells, about 7.2×107engineered T cells to about 3.6×109engineered T cells, about 7.4×107engineered T cells to about 3.4×109engineered T cells, about 7.6×107engineered T cells to about 3.2×109engineered T cells, about 7.8×107engineered T cells to about 3×109engineered T cells, about 8×107engineered T cells to about 2.8×109engineered T cells, about 8.2×107engineered T cells to about 2.6×109engineered T cells, about 8.4×107engineered T cells to about 2.4×109engineered T cells, about 8.6×107engineered T cells to about 2.2×109engineered T cells, about 8.8×107engineered T cells to about 2×109engineered T cells, about 9×107engineered T cells to about 1.8×109engineered T cells, about 9.2×107engineered T cells to about 1.6×109engineered T cells, about 9.4×107engineered T cells to about 1.4×109engineered T cells, about 9.6×107engineered T cells to about 1.2×109engineered T cells, about 9.8×107engineered T cells to about 1×109engineered T cells, about l×108engineered T cells to about 9.8×108engineered T cells, about 1.2×108engineered T cells to about 9.6×108engineered T cells, about 1.4×108engineered T cells to about 9.4×108engineered T cells, about 1.6×108engineered T cells to about 9.2×108engineered T cells, about 1.8×108engineered T cells to about 9×108engineered T cells, about 2×108engineered T cells to about 8.8×108engineered T cells, about 2.2×108engineered T cells to about 8.6×108engineered T cells, about 2.4×108engineered T cells to about 8.4×108engineered T cells, about 2.6×108engineered T cells to about 8.2×108engineered T cells, about 2.8×108engineered T cells to about 8×108engineered T cells, about 3×108engineered T cells to about 7.8×108engineered T cells, about 3.2×108engineered T cells to about 7.6×108engineered T cells, about 3.4×108engineered T cells to about 7.4×108engineered T cells, about 3.6×108engineered T cells to about 7.2×108engineered T cells, about 3.8×108engineered T cells to about 7×108engineered T cells, about 4×108engineered T cells to about 6.8×108engineered T cells, about 4.2×108engineered T cells to about 6.6×108engineered T cells, about 4.4×108engineered T cells to about 6.4×108engineered T cells, about 4.6×108engineered T cells to about 6.2×108engineered T cells, about 4.8×108engineered T cells to about 6×108engineered T cells, about 5×108engineered T cells to about 5.8×108engineered T cells, about 5.2×108engineered T cells to about 5.6×108engineered T cells, or about 5.4×108engineered T cells to about 5.4×108engineered T cells.

[0408] In some embodiments, the therapeutically effective dose can be split in two, three, four, or more sub doses. In some embodiments, the therapeutically effective dose can be split in two sub doses. In some embodiments the sub doses can be delivered at the same time, for example on the same day. In some embodiments the sub doses can be administered spaced apart from each other, for example on consecutive days. In some embodiments, the sub doses can be administered on consecutive days. In some embodiments, the sub doses can be administered 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days apart.

[0409] In some embodiments, a therapeutically effective dose comprises about 0.5× 108engineered T cells to about 9× 1010engineered T cells. In some embodiments, a therapeutically effective dose comprises about 0.5 × 108engineered T cells to about 1 × 1010engineered T cells. In some embodiments, a therapeutically effective dose comprises about 0.5 × 108engineered T cells to about 5× 109engineered T cells. In some embodiments, a therapeutically effective dose comprises about 0.5 × 108engineered T cells to about 1 × 109engineered T cells. The term “about” as referred to in a therapeutically dose, can be, for example, ± 0.5× 106engineered T cells, ± 0.5× 107engineered T cells, or ± 0.5× 108engineered T cells.

[0410] In some embodiments, a therapeutically effective dose of the engineered T cells described herein are administered to the subject in need thereof. In general, administration can be parenteral. In some embodiments, the engineered T cells of the disclosure are administered by intravenous injection. In some embodiments, the engineered T cells of the disclosure are administered by intraperitoneal injection.

[0411] Methods for administration of cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al and U.S. Pat. No. 4,690,915 to Rosenberg.

[0412] Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[0413] The pharmaceutical composition in some embodiments contains the engineered T cells in amounts effective to treat or prevent a cancer, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over days, weeks or months, depending on the condition, the treatment can be repeated until a desired suppression of cancer signs or symptoms occurs. However, other dosage regimens can be useful and can be determined. The desired dosage can be delivered by a single bolus administration or infusion of the composition or by multiple bolus administrations or infusions of the composition.

[0414] For purposes of the disclosure, an assay, which comprises, for example, comparing the extent to which target cells are lysed or one or more cytokines are secreted by immune cells expressing the receptors, upon administration of a given dose of such immune cells to amammal, among a set of mammals of which is each given a different dose of the immune cells, can be used to determine a starting dose to be administered to a mammal.Methods of Conditioning

[0415] Administration of a lymphodepleting agent prior to administration of an adoptive immune cell therapy reduces the number of endogenous lymphocytes. The endogenous lymphocytes that are reduced can include, but are not limited to, endogenous regulatory T cells, B cells, natural killer cells, CD4+ T cells, CD8+ T cells, or any combination thereof, which can inhibit the anti-tumor effect of adoptively transferred T cells. Endogenous lymphocytes can compete with adoptively transferred T cells for access to antigens and supportive cytokines.

[0416] In embodiments, a lymphodepleting chemotherapy is administered to the subject prior to (i.e., a preconditioning lymphodepletion (PCLD)), or concurrently with the engineered T cells. In an example, the lymphodepleting chemotherapy is administered to the subject prior to administration of the engineered T cells (i.e., a PCLD regimen). For example, the lymphodepleting chemotherapy ends 1-4 days (e.g., 1, 2, 3, or 4 days) prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 1 day prior to adoptive cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 2 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 3 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 4 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 5 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 6 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 7 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 8 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 9 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 10 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 11 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 12 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 13 days prior to engineered T cell infusion. In some embodiments, the lymphodepleting chemotherapy ends 14 days prior to engineered T cell infusion.

[0417] In embodiments, multiple doses of engineered T cells are administered, e.g., as described herein. In embodiments, a lymphodepleting chemotherapy is administered to thesubject prior to or concurrently with administration (e.g., infusion) of the engineered T cells described herein. Examples of lymphodepletion include, but can not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc. Examples of lymphodepleting agents include, but are not limited to, antithymocyte globulin, anti-CD3 antibodies, anti-CD4 antibodies, anti- CD8 antibodies, anti-CD52 antibodies, anti-CD2 antibodies, TCRαβ blockers, anti-CD20 antibodies, anti-CD19 antibodies, Bortezomib, rituximab, anti-CD 154 antibodies, rapamycin, CD3 immunotoxin, fludarabine, cyclophosphamide, busulfan, melphalan, Mabthera, Tacrolimus, alefacept, alemtuzumab, 0KT3, 0KT4, 0KT8, 0KT11, fmgolimod, anti-CD40 antibodies, anti-BR3 antibodies, Campath-IH, anti-CD25 antibodies, calcineurin inhibitors, mycophenolate, and steroids, which can be used alone or in combination. As a further example, a lymphodepletion regimen can include, administration of alemtuzumab, cyclophosphamide, benduamustin, rituximab, pentostatin, and / or fludarabine.

[0418] In some embodiments, the lymphodepleting agent is cyclophosphamide and fludarabine. In some embodiments, administration of cyclophosphamide and fludarabine reduces endogenous lymphocytes. In some embodiments, administration of cyclophosphamide and fludarabine increases the availability of a homeostatic cytokine. In some embodiments, administration of cyclophosphamide and fludarabine enhances an effector function of T cells administered after the conditioning. In some embodiments, administration of cyclophosphamide and fludarabine enhances antigen presenting cell activation and / or availability.

[0419] In one embodiment, the disclosure provides a method of conditioning a patient in need of a T cell therapy comprising administering to the patient a dose of cyclophosphamide and of fludarabine. Illustrative Dosing Schedules for a Conditioning regimen in combination with engineered T cells are provided in Table 12.Table 12 Illustrative Dosing Schedules for engineered T cells with Conditioning

[0420] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 1 day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 2 days. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 3 days. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 4 days. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 5 days.

[0421] In some embodiments, the method comprises administering to the patient a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day for 1 day. In some embodiments, the method comprises administering to the patient a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day for 2 days. In some embodiments, the method comprises administering to the patient a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day for 3 days. In some embodiments, the method comprises administering to the patient a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day for 4 days. In some embodiments, the method comprises administering to the patient a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day for 5 days.

[0422] In some embodiments, the method comprises administering the dose of cyclophosphamide and the dose of fludarabine on the same day. In some embodiments, the method comprises administering the dose of cyclophosphamide and the dose of fludarabine on different days. In some embodiments, the method comprises administering the dose of cyclophosphamide and the dose of fludarabine on consecutive days.

[0423] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 3 days and a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 2 days and a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day.

[0424] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 4 days and a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 3 days and a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 2 days and a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide between about 250 mg / m2 / day and about 750 mg / m2 / day for 3 days and a dose of fludarabine between about 20 mg / m2 / day and about 50 mg / m2 / day for 4 days. In some embodiments, the cyclophosphamide and the fludarabine are started on the same day. In some embodiments, the cyclophosphamide and the fludarabine are started on different days, with the cyclophosphamide preceding the fludarabine or the cyclophosphamide following the fludarabine.

[0425] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 250 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 300 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 350 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about400 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 450 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 500 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, themethod comprises administering to the patient a dose of cyclophosphamide of about 550 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 600 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 650 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 700 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 750 mg / m2 / day for 3 days and a dose of fludarabine of about 25 mg / m2 / day.

[0426] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 250 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 300 mg / m2 / day for 3 days and a dose of fludarabine of about 30mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 350 mg / m2 / day for 3 days and a dose of fludarabine of about 30mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about400 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 450 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 500 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 550 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 600 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 650 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 700 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 750 mg / m2 / day for 3 days and a dose of fludarabine of about 30 mg / m2 / day.

[0427] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 250 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 300 mg / m2 / day for 3 days and a dose of fludarabine of about 30mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 350 mg / m2 / day for 3 days and a dose of fludarabine of about 30mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about400 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 450 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 500 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 550 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 600 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 650 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 700 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 750 mg / m2 / day for 3 days and a dose of fludarabine of about 35 mg / m2 / day.

[0428] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 250 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 300 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 350 mg / m2 / day for 3 days and a dose of fludarabine of about 30mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 400 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 450 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the methodcomprises administering to the patient a dose of cyclophosphamide of about 500 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 550 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 600 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 650 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 700 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 750 mg / m2 / day for 3 days and a dose of fludarabine of about 40 mg / m2 / day.

[0429] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 250 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 300 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 350 mg / m2 / day for 3 days and a dose of fludarabine of about 30mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 400 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 450 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 500 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 550 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 600 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 650 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 700 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day. In someembodiments, the method comprises administering to the patient a dose of cyclophosphamide of about 750 mg / m2 / day for 3 days and a dose of fludarabine of about 50 mg / m2 / day.

[0430] In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide and a dose of fludarabine daily for two days. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide and a dose of fludarabine daily for three days. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide and a dose of fludarabine daily for four days. In some embodiments, the method comprises administering to the patient a dose of cyclophosphamide and a dose of fludarabine daily for more than two days.

[0431] Lymphodepletion regimen can be administered in one or more cycles until the desired outcome of reduced circulating immune cells. In some embodiments, the lymphodepletion comprises administering an agent that specifically targets, and reduces or eliminates CD52+ cells in the subject, and the immune cells are modified to reduce or eliminate CD52 expression.

[0432] In some embodiments, an immune stimulating therapy is administered to the subject prior to, concurrently with, or after administration (e.g. infusion) of adoptive immune cells. In some embodiments, the immune stimulating therapy comprises homeostatic cytokines. In some embodiments, the immune stimulating therapy comprises immune-stimulatory molecules. In some embodiments, the immune stimulating therapy comprises IL-2, IL-7, IL- 12, IL-15, IL-21, IL-9, or a functional fragment thereof. In some embodiments, the immune stimulating therapy comprises IL-2, IL-7, IL-12, IL-15, IL-21, IL-9, or combinations thereof. In some embodiments, the immune stimulating therapy comprises IL-2, or a functional fragment thereof.

[0433] In some embodiments, recombinant IL-2, or a functional fragment thereof, is administered to the subject. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,000,000 IUS per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 100,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 200,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 300,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 400,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 500,000 IUs per dose. In some embodiments a therapeutically effective dose of IL-2 comprises about 600,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 700,000 IUs per dose. In some embodiments, a therapeutically effectivedose of IL-2 comprises about 800,000 IUS per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 900,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,000,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,100,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,200,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,300,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,400,000 IUs per dose. In some embodiments a therapeutically effective dose of IL-2 comprises about 1,500,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,600,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,700,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,800,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 1,900,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,000,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,100,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,200,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,300,000 IUs per dose. In some embodiments a therapeutically effective dose of IL-2 comprises about 2,400,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,500,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,600,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,700,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,800,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 2,900,000 IUs per dose. In some embodiments, a therapeutically effective dose of IL-2 comprises about 3,000,000 IUs per dose.

[0434] In some embodiments, the IL-2 is administered to the subject via subcutaneous injections. In some embodiments, the IL-2 is administered to the subject via intravenous injections. In some embodiments, the IL-2 is administered to the subject via intradermal injections. In some embodiments, the IL-2 is administered to the subject via intramuscular injections. In some embodiments, the IL-2 is administered to the subject via intrathecal injections.

[0435] In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 250 / 25 PCLD regimen comprising cyclophosphamide at a dose of 250 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.25 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising to SEQ ID NO: 2070, and a B2M shRNA comprising to SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.25 ×108engineered T cells comprising an MLSN activator receptor comprising to SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.5 * 108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 1 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 2×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 4 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprisingSEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 8 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 750 / 30 PCLD regimen comprising fludarabine at a dose of 30 mg / m2 / day for 4 days and cyclophosphamide at a dose of 750 mg / m2 / day for 3 days, followed by 2 days of rest, then infusion with 8 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 60 / 25 PCLD regimen comprising cyclophosphamide at a dose of 60 mg / m2 / day for 2 days (Days -7 and -6) followed by fludarabine at a dose of 25 mg / m2 / day for 5 days (Days -5 to -1) followed by at least 24 hours of rest, then infusion with 8 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days.

[0436] In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 250 / 25 PCLD regimen comprising cyclophosphamide at a dose of 250 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.5 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.5 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 1 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 2 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 4×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 6 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 14 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 750 / 30 PCLD regimen comprising fludarabine at a dose of 30 mg / m2 / day for 4 days and cyclophosphamide at a dose of 750 mg / m2 / day for 3 days, followed by 2 days of rest, then infusion with 14 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 60 / 25 PCLD regimen comprising cyclophosphamide at a dose of 60 mg / m2 / day for 2 days (Days -7 and -6) followed by fludarabine at a dose of 25 mg / m2 / day for 5 days (Days -5 to -1) followed by at least 24 hours of rest, then infusion with 14 ×108engineered T cells comprising an MLSN activator receptor comprising SEQ ID NO: 2066, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days.

[0437] In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 250 / 25 PCLD regimen comprising cyclophosphamide at a dose of 250 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.25 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.25 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days,followed by 2 days of rest, and then infusion with 0.5 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 1 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 2×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 4 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 8 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 750 / 30 PCLD regimen comprising fludarabine at a dose of 30 mg / m2 / day for 4 days and cyclophosphamide at a dose of 750 mg / m2 / day for 3 days, followed by 2 days of rest, then infusion with 8 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, anHLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 60 / 25 PCLD regimen comprising cyclophosphamide at a dose of 60 mg / m2 / day for 2 days (Days -7 and -6) followed by fludarabine at a dose of 25 mg / m2 / day for 5 days (Days -5 to -1) followed by at least 24 hours of rest, then infusion with 8 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days.In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 250 / 25 PCLD regimen comprising cyclophosphamide at a dose of 250 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.5 x 108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.5 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 1 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 2 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO:179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 4* 108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 6 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 14 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 750 / 30 PCLD regimen comprising fludarabine at a dose of 30 mg / m2 / day for 4 days and cyclophosphamide at a dose of 750 mg / m2 / day for 3 days, followed by 2 days of rest, then infusion with 14 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 164, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 60 / 25 PCLD regimen comprising cyclophosphamide at a dose of 60 mg / m2 / day for 2 days (Days -7 and -6) followed by fludarabine at a dose of 25 mg / m2 / day for 5 days (Days -5 to -1) followed by at least 24 hours of rest, then infusion with 14 ×108engineered T cells comprising an CEA activator receptor comprising SEQ ID NO: 52, an HLA-A*02 inhibitor receptor comprising SEQ IDNO: 164, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days.

[0438] In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 250 / 25 PCLD regimen comprising cyclophosphamide at a dose of 250 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.25 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.25 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.5 * 108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 1 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 2×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 4 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 8 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 750 / 30 PCLD regimen comprising fludarabine at a dose of 30 mg / m2 / day for 4 days and cyclophosphamide at a dose of 750 mg / m2 / day for 3 days, followed by 2 days of rest, then infusion with 8 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing less than 50 kg a 60 / 25 PCLD regimen comprising cyclophosphamide at a dose of 60 mg / m2 / day for 2 days (Days -7 and -6) followed by fludarabine at a dose of 25 mg / m2 / day for 5 days (Days -5 to -1) followed by at least 24 hours of rest, then infusion with 8 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days.In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 250 / 25 PCLD regimen comprising cyclophosphamide at a dose of 250 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.5 ×108engineered T cells comprising an HER2 activator receptorcomprising SEQ IDNO: 2071, anHLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 0.5 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 1 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ IDNO: 2071, anHLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 2 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, and then infusion with 4×108engineered T cells comprising an HER2 activator receptor comprising SEQ IDNO: 2071, anHLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2 days of rest, then infusion with 6 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 300 / 30 PCLD regimen comprising cyclophosphamide at a dose of 300 mg / m2 / day and fludarabine at a dose of 30 mg / m2 / day for 3 consecutive days, followed by 2days of rest, then infusion with 14 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 750 / 30 PCLD regimen comprising fludarabine at a dose of 30 mg / m2 / day for 4 days and cyclophosphamide at a dose of 750 mg / m2 / day for 3 days, followed by 2 days of rest, then infusion with 14 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and aB2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days. In some embodiments, the method comprises administering to a subject weighing more than 50 kg a 60 / 25 PCLD regimen comprising cyclophosphamide at a dose of 60 mg / m2 / day for 2 days (Days -7 and -6) followed by fludarabine at a dose of 25 mg / m2 / day for 5 days (Days -5 to -1) followed by at least 24 hours of rest, then infusion with 14 ×108engineered T cells comprising an HER2 activator receptor comprising SEQ ID NO: 2071, an HLA-A*02 inhibitor receptor comprising SEQ ID NO: 2070, and a B2M shRNA comprising SEQ ID NO: 179 and treatment with low-dose IL-2 (1,000,000 IU) administered subcutaneously twice daily, beginning 3 to 24 hours after completion of the Day 0 infusion, for a maximum of 14 doses over 7 days.Methods of Treatment

[0439] Provided herein are methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising engineered T cells comprising the first and second receptor or a pharmaceutical composition of the disclosure. In some embodiments, the engineered T cells express both receptors in the same cell.

[0440] In some embodiments, the methods of the disclosure can be used to treat a cancer in a subject, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent growth of a tumor, eliminate a tumor from a subject, prevent relapse of a tumor, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In certain embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

[0441] Cancer is a disease in which abnormal cells divide without control and spread to nearby tissue. In some embodiments, the cancer comprises a liquid tumor or a solid tumor. Illustrative liquid tumors include leukemias and lymphomas. Cancers can arise in virtually any organ in the body, including epithelial tissues. Any cancer wherein a plurality of the cancer cells express the first, activator, ligand and do not express the second, inhibitor ligand is envisaged as within the scope of the instant disclosure.

[0442] Target antigen 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, biliary cancer, breast cancer, cervical cancer, lung adeno cancer, lung squamous cancer, uterine cancer. Additional cancers and target antigens are listed in Table 1.

[0443] Colorectal cancer (CRC) is the second leading cause of cancer death in the US and the third most common cancer diagnosed among men and women. Approximately 50% to 60% of patients diagnosed with CRC develop distant metastatic disease. Patients with metastatic CRC (mCRC) have a 5-year relative survival of approximately 15% in the US.

[0444] Pancreatic cancer (PANC) is the third leading cause of cancer death in the US and the eleventh most common cancer diagnosed among men and women. Fewer than 20% of patients with PANC are diagnosed at a localized stage and are candidates for surgery; for patients with metastatic disease, the 5-year relative survival is approximately 3% in the US.

[0445] Ovarian cancer (OVCA) is the fifth leading cause of cancer death among women in the US. Over 80% of patients are diagnosed with advanced disease, which is associated with a 5-year relative survival of approximately 30%.

[0446] Mesothelioma (MESO) is a rare malignancy, with approximately 3000 new diagnoses per year in the US. Patients diagnosed with metastatic disease have a 5-year relative survival of approximately 7%. CRC, NSCLC, PANC, OVCA, MESO, colorectal cancer (CRC), nonsmall cell lung cancer (NSCLC), pancreatic cancer (PANC), ovarian cancer (OVCA), mesothelioma (MESO), and other solid tumors.

[0447] In some embodiments, the plurality of cancer cells express the target antigen. In some embodiments, the plurality cancer cells of the subject express CEA. Any cancer whose cells express CEA, i.e. are CEA-positive, is envisaged as within the scope of the instant disclosure. In some embodiments, the CEA positive cancer is CRC, NSCLC, or PANC. Illustrative CEA-positive cancers include, but are not limited to, prostate, ovary, lung, thyroid, gastrointestinal, breast and liver cancers. Further CEA-positive cancers include colorectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung cancer, head and neckcancer, gallbladder cancer, diffuse large B cell cancer 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. In some embodiments, the cancer comprises NSLC adenocarcinoma. The compositions and methods disclosure herein can be used to treat CEA- positive cancers that are relapsed, refractory and / or metastatic. In some embodiments, CEA positive cancers can be treated using the methods described herein.

[0448] In some embodiments, the plurality cancer cells of the subject express MLSN. Any cancer whose cells express MSLN, i.e. are MSLN-positive, is envisaged as within the scope of the instant disclosure. In some embodiments, the MSLN positive cancer is CRC, NSCLC, PANC, OVCA, or MESO. In some embodiments, the MSLN positive cancer is pilomatrixoma, basal cell carcinoma, benign nevus, squamous cell carcinoma of the skin, malignant melanoma, merkel cell carcinoma, squamous cell carcinoma of the larynx, squamous cell carcinoma of the pharynx, oral squamous cell carcinoma (floor of the mouth), pleomorphic adenoma of the parotid gland, warthin tumor of the parotid gland, basal cell adenoma of the salivary gland, squamous cell carcinoma of the lung, adenocarcinoma of the lung, small cell carcinoma of the lung, mesothelioma epitheloid, mesothelioma other types, thymoma, squamous cell carcinoma of the vagina, squamous cell carcinoma of the vulva, squamous cell carcinoma of the cervix, endometrioid endometrial carcinoma, endometrial serous carcinoma, carcinosarcoma of the uterus, endometrial carcinoma, high grade G3, endometrial clear cell carcinoma, endometrioid carcinoma of the ovary, serous carcinoma of the ovary, mucinous carcinoma of the ovary, clear cell carcinoma of the ovary, carcinosarcoma of the ovary, brenner tumor, Invasive breast carcinoma of no special type, lobular carcinoma of the breast, medullary carcinoma of the breast, tubular carcinoma of the breast, mucinous carcinoma of the breast, phyllodes tumor of the breast, adenomatous polyp low-grade dysplasia, adenomatous polyp high-grade dysplasia, adenocarcinoma of the colon, adenocarcinoma of the small intestine, gastric adenocarcinoma diffuse type, gastric adenocarcinoma intestinal type, gastric adenocarcinoma mixed type, adenocarcinoma of the esophagus, squamous cell carcinoma of the esophagus, squamous cell carcinoma of the anal canal, cholangiocarcinoma, hepatocellular carcinoma, ductal adenocarcinoma of the pancreas, pancreatic / Ampullary adenocarcinoma, acinar cell carcinoma of the pancreas, gastrointestinal stromal tumor (GIST), non-invasive papillary urothelial carcinoma pTa G2 low grade, non-invasive papillary urothelial carcinoma pTa G2 high grade, non-invasive papillary urothelial carcinoma pTa G3, urothelial carcinoma pT2-4 G3, small cell neuroendocrine carcinoma of the bladder, sarcomatoid urothelial carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, clear cell (tubulo) papillary renal cell carcinoma, chromophobe renal cell carcinoma, Oncocytoma, adenocarcinoma of the prostate Gleason 3 + 3, adenocarcinoma of the prostate, Gleason 4 + 4, adenocarcinoma of the prostate Gleason 5 + 5, adenocarcinoma of the prostate (recurrence), small cell neuroendocrine carcinoma of the prostate, seminoma embryonal carcinoma of the testis, yolk sack tumor, teratoma, squamous cell carcinoma of the penis, adenoma of the thyroid gland, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, anaplastic thyroid carcinoma, adrenal cortical adenoma, adrenal cortical carcinoma, phaeochromocytoma, appendix neuroendocrine tumor (NET), colorectal neuroendocrine tumor (NET), ileum neuroendocrine tumor (NET), lung neuroendocrine tumor (NET), pancreas neuroendocrine tumor (NET), colorectal neuroendocrine carcinoma (NEC), gallbladder neuroendocrine carcinoma (NEC), pancreas neuroendocrine carcinoma (NEC), Hodgkin Lymphoma, non-Hodgkin Lymphoma, small lymphocytic lymphoma B-cell type (B-SLL / B-CLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma, T-cell Non Hodgkin lymphoma, mantle cell lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma (DLBCL) in the testis, Burkitt lymphoma, tenosynovial giant cell tumor, granular cell tumor, leiomyoma, angiomyolipoma, angiosarcoma, dermatofibrosarcoma protuberans, ganglioneuroma, kaposi sarcoma, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor (MPNST), myofibrosarcoma, neurofibroma, sarcoma not otherwise specified (NOS), paraganglioma, primitive neuroectodermal tumor (PNET), rhabdomyosarcoma schwannoma, synovial sarcoma, osteosarcoma, or chondrosarcoma. Additional MSLN positive cancers are listed in Weidmann, S. et al. Mesothelin Expression in Human Tumors: A Tissue Microarray Study on 12,679 Tumors. Biomedicines. 2021 Apr 7;9(4):397, the contents of which are incorporated by reference herein. In some embodiments, MSLN positive cancers can be treated using the methods described herein. In some embodiments, subjects treated with the methods described herein are germline heterozygous HLA-A*02 adults with recurrent unresectable, locally advanced, or metastatic CRC, NSCLC, PANC, OVCA, MESO, or other solid tumors that express MSLN and have lost HLA-A*02 expression.

[0449] In some embodiments, the plurality cancer cells of the subject express HER2. Any cancer whose cells express HER2, i.e. are HER2-positive, is envisaged as within the scope of the instant disclosure. In some embodiments, the HER2 positive cancer is CRC, NSCLC,PANC, OVCA, or MESO. In some embodiments, the HER2 positive cancer is bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma (extrahepatic), cholangiocarcinoma (intrahepatic), colorectal cancer, esophageal cancer, esophagogastric junction cancer, gallbladder cancer, gastric adenocarcinoma, a gastrointestinal stromal tumor, glioblastoma multiforme high grade glioma, glioma (low grade), head and neck carcinoma, hepatocellular carcinoma, intestinal (small) malignancy, kidney cancer, melanoma, melanoma (uveal), neuroendocrine tumor, oligodendroglioma, ovarian (epithelial) cancer, ovarian (non-epithelial) cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcomas (peritoneal, retroperitoneal), sarcomas (soft tissues), solitary fibrous tumor, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer.

[0450] In some embodiments, the HER2 positive cancer Additional HER2 positive cancers are listed in Yan M. et al. HER2 expression status in diverse cancers: review of results from 37,992 patients. Cancer Metastasis Rev. 2015 Mar;34(l): 157-64, the contents of which are incorporated by reference herein. In some embodiments, HER2 positive cancers can be treated using the methods described herein.

[0451] Provided herein are methods of treating target antigen positive cancer in a subject having a target antigen positive + tumor, the tumor having loss of heterozygosity at an MHC class I locus. In some embodiments, the methods comprise administering to the subject an effective amount of the immune cells or pharmaceutical compositions described herein. In some embodiments, the methods comprise (a) determining 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 target antigen in a plurality of cancer cells of the subject; and (c) administering to the subject an effective amount of the engineered T cells or pharmaceutical compositions of the 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 HLA-A, HLA-B, or HLA-C non-target antigen, and the plurality of cancer cells are also target antigen positive.

[0452] In some embodiments, where the cancer is known to be CEA+, the methods comprise (a) determining 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 the engineered T cells or pharmaceutical compositions of the 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.

[0453] In some embodiments, where the cancer is known to be MSLN+, the methods comprise (a) determining 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 the engineered T cells or pharmaceutical compositions of the 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.

[0454] In some embodiments, where the cancer is known to be HER2+, the methods comprise (a) determining 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 the engineered T cells or pharmaceutical compositions of the 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.

[0455] 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.

[0456] The disclosure provides methods of treating a cancer in a subject comprising measuring the expression level of the non-target antigen in a plurality of cancer cells, and treating the subject when the expression level of the non-target antigen in the plurality of cancer cells is less than the expression level of the non-target antigen in the plurality of cancer cells is less than the expression level of the non-target antigen a plurality of healthy cells.

[0457] Administration of the engineered T cells or pharmaceutical compositions described herein can reduce the size of a tumor in the 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%, relative to the size of the tumor before administration of the engineered T cells or pharmaceutical compositions. In some embodiments, the tumor is eliminated.

[0458] Administration of the engineered T cells or pharmaceutical compositions described herein can arrest the growth of a tumor in the subject. For example, the engineered T cells or pharmaceutical compositions can kill tumor cells, so that the tumor stops growing, or is reduced in size. In some cases, the engineered T cells or pharmaceutical compositions can prevent formation of additional tumors, or reduce the total number of tumors in the subject.

[0459] Administration of the engineered T cells or pharmaceutical compositions described herein can result in selective killing of a cancer cell but not a wild-type cell in the subject. Insome embodiments, about 60% of the cells killed are cancer cells, about 65% of the cells killed are cancer cells, about 70% of the cells killed are cancer cells, about 75% of the cells killed are cancer cells, about 80% of the cells killed are cancer cells, about 85% of the cells killed are cancer cells, about 90% of the cells killed are cancer cells, about 95% of the cells killed are cancer cells, or about 100% of the cells killed are cancer cells.

[0460] Administration of the engineered T cells or pharmaceutical compositions described herein can result in the killing of about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or all of the cancer cells of the subject.

[0461] In some embodiments, the method of treatment comprises determining the HLA germline type of the subject. In some embodiments, the HLA germline type is determined in bone marrow.

[0462] In some embodiments, the method of treatment comprises determining the level of expression of target antigen. In some embodiments, the level of expression of target antigen is determined in tumor tissue samples from the subject. In some embodiments, the expression level of target antigen is determined using next generation sequencing. In some embodiments, the expression level of target antigen is determined using RNA sequencing. In some embodiments, the level of target antigen is determined using immunohistochemistry.

[0463] In some embodiments, the method of treatment comprises determining the level of expression of CEA. In some embodiments, the level of expression of CEA is determined in tumor tissue samples from the subject. In some embodiments, the expression level of CEA is determined using next generation sequencing. In some embodiments, the expression level of CEA is determined using RNA sequencing. In some embodiments, the level of CEA is determined using immunohistochemistry.

[0464] In some embodiments, the method of treatment comprises determining the level of expression of MSLN. In some embodiments, the level of expression of MSLN is determined in tumor tissue samples from the subject. In some embodiments, the expression level of MSLN is determined using next generation sequencing. In some embodiments, the expression level of MSLN is determined using RNA sequencing. In some embodiments, the level of MSLN is determined using immunohistochemistry.

[0465] In some embodiments, the method of treatment comprises determining the level of expression of HER2. In some embodiments, the level of expression of HER2 is determined in tumor tissue samples from the subject. In some embodiments, the expression level of HER2 is determined using next generation sequencing. In some embodiments, the expression levelof HER2 is determined using RNA sequencing. In some embodiments, the level of HER2 is determined using immunohistochemistry.

[0466] In some embodiments, the method of treatment comprises administering a therapeutically effective dose of engineered T cells comprising an HLA-A*02 inhibitory receptor to a subject in need thereof, wherein the subject is determined to be HLA germline HLA-A*02 heterozygous and have cancer cells with loss of HLA-A*02. In some embodiments, the method of treatment comprises administering a therapeutically effective dose of engineered T cells comprising an HLA-A*01 inhibitory receptor to a subject in need thereof, wherein the subject is determined to be HLA germline HLA-A*01 heterozygous and have cancer cells with loss of HLA-A*01. In some embodiments, the method of treatment comprises administering a therapeutically effective dose of engineered T cells comprising an HLA-A*03 to a subject in need thereof, wherein the subject is determined to be HLA germline HLA-A*03 heterozygous and have cancer cells with loss of HLA-A*03. In some embodiments, the method of treatment comprises administering a therapeutically effective dose of engineered T cells comprising an HLA-A*07 inhibitory receptor to a subject in need thereof, wherein the subject is determined to be HLA germline HLA-A*07 heterozygous and have cancer cells with loss of HLA-A*07. In some embodiments, the method of treatment comprises administering a therapeutically effective dose of engineered T cells comprising an HLA-C*07 inhibitory receptor to a subject in need thereof, wherein the subject is determined to be HLA germline HLA-C*07 heterozygous and have cancer cells with and loss of HLA- C*07. In some embodiments, the method of treatment comprises administering a therapeutically effective dose of engineered T cells comprising an HLA-B*07 inhibitory receptor in a subject in need thereof, wherein the subject is determined to be HLA germline HLA-B*07 heterozygous and have cancer cells with loss of HLA-B*07.

[0467] In various embodiments, the disclosure provides methods of treatment of heterozygous HLA-A*02 patients with malignancies that express target antigen and have lost HLA-A*02 expression; and / or of treatment of heterozygous HLA-A*02 adult patients with recurrent unresectable or metastatic solid tumors that express target antigen and have lost HLA-A*02 expression.

[0468] In various embodiments, the disclosure provides methods of treatment of heterozygous HLA-A*02 patients with malignancies that express CEA and have lost HLA- A*02 expression; and / or of treatment of heterozygous HLA-A*02 adult patients with recurrent unresectable or metastatic solid tumors that express CEA and have lost HLA-A*02 expression.

[0469] In various embodiments, the disclosure provides methods of treatment of heterozygous HLA-A*02 patients with malignancies that express HER2 and have lost HLA- A*02 expression; and / or of treatment of heterozygous HLA-A*02 adult patients with recurrent unresectable or metastatic solid tumors that express HER2 and have lost HLA-A*02 expression.

[0470] Administration of the engineered T cells or pharmaceutical compositions described herein can result in fewer side effects for the subject than administration of an otherwise equivalent immune cell comprising the first activator receptor but no second inhibitory receptor. For example, administering the engineered T cells or pharmaceutical compositions described herein can reduce dose limited toxicity relative to the CEA CAR, or CEA TCR administered without the second inhibitory receptor.

[0471] Methods of genotyping cancer cells and normal cells from a subject for the presence or absence of SNPs will be rea...

Claims

CLAIMSWhat is claimed is:

1. A method of treating a target antigen positive solid tumor in a subject comprising administering to the subject a therapeutically effective amount of engineered T cells, wherein the engineered T cells comprise: a. a first receptor comprising an extracellular ligand binding domain specific to the target antigen and an intracellular domain comprising at least one signal transduction element that activates and / or co-stimulates the engineered T cell; and b. a second receptor comprising an extracellular ligand binding domain specific to a non-target antigen and an intracellular domain comprising at least one signal transduction element that inhibits the engineered T cell.

2. The method of claim 1, wherein the solid tumor exhibits loss of expression of the non-target antigen.

3. The method of claim 1, wherein the non-target antigen is an allelic variant of a polymorphic cell surface protein absent from at least some cells of the solid tumor but present in at least some cells of normal tissue of the subject.

4. The method of claim 1, wherein the second receptor comprises a LILRB1 intracellular domain or a functional variant thereof.

5. The method of any one of claims 1-4, wherein the therapeutically effective amount is between about 0.5× 108engineered T cells and about 36× 108engineered T cells.

6. The method of claim 5, wherein the therapeutically effective amount is about 0.25 × 108engineered T cells.

7. The method of claim 5, wherein the therapeutically effective amount is about 0.5 × 108engineered T cells.

8. The method of claim 5, wherein the therapeutically effective amount is about I × 108engineered T cells.

9. The method of claim 5, wherein the therapeutically effective amount is about 2× 108engineered T cells.

10. The method of claim 5, wherein the therapeutically effective amount is about 4× 108engineered T cells.

11. The method of claim 5, wherein the therapeutically effective amount is about 6× 108engineered T cells.

12. The method of claim 5, wherein the therapeutically effective amount is about 8× 108engineered T cells.

13. The method of claim 5, wherein the therapeutically effective amount is about 14×108engineered T cells.

14. The method of any one of claims 1-13, wherein the engineered T cells are administered after preconditioning lymphodepletion (PCLD).

15. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide and / or fludarabine.

16. The method of claim 15, wherein the lymphodepletion comprises administering cyclophosphamide and fludarabine.

17. The method of claim 14, wherein the lymphodepletion comprises administering: a. fludarabine at 25 mg / m2 / Day and cyclophosphamide at 250 mg / m2 / Day; b. fludarabine at 30 mg / m2 / Day and cyclophosphamide at 500 mg / m2 / Day; c. cyclophosphamide at 60 mg / kg for 2 Days followed by fludarabine at 25 mg / m2; or d. fludarabine at 30 mg / m2 / Day and cyclophosphamide at 750 mg / m2 / Day.

18. The method of claim 14, wherein the lymphodepletion comprises administering: a. fludarabine at 25 mg / m2 / Day and cyclophosphamide at 250 mg / m2 / Day for 3 Days; b. fludarabine at 30 mg / m2 / Day and cyclophosphamide at 500 mg / m2 / Day for 3 Days; or c. cyclophosphamide at 60 mg / kgx2 Days followed by fludarabine at 25 mg / m2for 5 Days.

19. The method of claim 14, wherein the lymphodepletion comprises administering fludarabine at 30 mg / m2 / Day for 4 Days and administering cyclophosphamide at 500 mg / m2 / Day for three Days, wherein the cyclophosphamide treatment is started on Day 2 of the fludarabine.

20. The method of any one of claims 1-19, wherein the engineered T cells are administered after the lymphodepletion.

21. The method of claim 20, wherein the engineered T cells are administered 2 Days after the lymphodepletion.

22. The method of any one of claims 1-21, wherein the method further comprises administration of recombinant IL-2 or a fragment thereof.

23. The method of claim 22, where the recombinant IL-2 is administered at a dose ranging from about 500,000 to about 2,000,000 IU.

24. The method of claim 23, wherein the dose of recombinant IL-2 is about 1,000,000 IU.

25. The method of claims 22-24, wherein the IL-2 is administered starting 3-24 hours after engineered T cells are administered.

26. The method of any one of claims 22-25 wherein the IL-2 administered every twelve hours over 7 Days.

27. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 250 mg / m2 / day and fludarabine 25 mg / m2 / day for 3 days; and wherein the therapeutically effective amount is 0.25 × 108engineered T cells administered 2 days after the preconditioning lymphodepletion.

28. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 250 mg / m2 / day and fludarabine 25 mg / m2 / day for 3 days; and wherein the therapeutically effective amount is 0.5× 108engineered T cells administered 2 days after the preconditioning lymphodepletion.

29. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 days; and wherein the therapeutically effective amount is 0.25 × 108engineered T cells administered 2 days after the preconditioning lymphodepletion.

30. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 days; and wherein the therapeutically effective amount is 0.5× 108engineered T cells administered 2 days after the preconditioning lymphodepletion.

31. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 day; and wherein the therapeutically effective amount is l × 108engineered T cells administered 2 days after the preconditioning lymphodepletion.

32. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 day; and wherein the therapeutically effective amount is 2× 108engineered T cells administered 2 days after the preconditioning lymphodepletion.

33. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 day; and wherein the therapeutically effective amount is 4× 108engineered T cells administered 2 days after the preconditioning lymphodepletion.

34. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 day; and wherein the therapeutically effective amount is 4× 108engineered T cells administered 2 days after the preconditioning lymphodepletion; wherein the method further comprises administration of a recombinant IL-2 or a fragment thereof at a dose of 1,000,000 IU starting 3-24 hours after engineered T cells are administered; and the IL-2 is administered every 12 hours over 7 days.

35. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 day; and wherein the therapeutically effective amount is 6× 108engineered T cells administered 2 days after the preconditioning lymphodepletion; wherein the method further comprises administration of a recombinant IL-2 or a fragment thereof at a dose of 1,000,000 IU starting 3-24 hours after engineered T cells are administered; and the IL-2 is administered every 12 hours over 7 days.

36. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 day; and wherein the therapeutically effective amount is 8× 108engineered T cells administered 2 days after the preconditioning lymphodepletion; wherein the method further comprises administration of a recombinant IL-2 or a fragment thereof at a dose of 1,000,000 IU starting 3-24 hours after engineered T cells are administered; and the IL-2 is administered every 12 hours over 7 days.

37. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 300 mg / m2 / day and fludarabine 30 mg / m2 / day for 3 day; and wherein the therapeutically effective amount is 14× 108engineered T cells administered 2 days after the preconditioning lymphodepletion; wherein the method further comprises administration of a recombinant IL-2 or a fragment thereof at a dose of 1,000,000 IU starting 3-24 hours after engineered T cells are administered; and the IL-2 is administered every 12 hours over 7 days.

38. The method of claim 14, wherein the lymphodepletion comprises administering fludarabine 30 mg / m2 / day for 4 days and cyclophosphamide 750 mg / m2 / day for 3 days; and wherein the therapeutically effective amount is 8× 108engineered T cells administered 2 days after the preconditioning lymphodepletion; wherein the method further comprises administration of a recombinant IL-2 or a fragment thereof at a dose of 1,000,000 IU starting 3-24 hours after engineered T cells are administered; and the IL-2 is administered every 12 hours over 7 days.

39. The method of claim 14, wherein the lymphodepletion comprises administering fludarabine 30 mg / m2 / day for 4 days and cyclophosphamide 750 mg / m2 / day for 3 days; and wherein the therapeutically effective amount is 14× 108engineered T cells administered 2days after the preconditioning lymphodepletion; wherein the method further comprises administration of a recombinant IL-2 or a fragment thereof at a dose of 1,000,000 IU starting 3-24 hours after engineered T cells are administered; and the IL-2 is administered every 12 hours over 7 days.

40. The method of claim 14, wherein the lymphodepletion comprises administering cyclophosphamide 60 mg / kg / day for 2 days followed by fludarabine 25 mg / m2 / day for 5 days; and wherein the therapeutically effective amount is 14× 108engineered T cells administered at least 24 hours after the preconditioning lymphodepletion; wherein the method further comprises administration of a recombinant IL-2 or a fragment thereof at a dose of 1,000,000 IU starting 3-24 hours after engineered T cells are administered; and the IL-2 is administered every 12 hours over 7 days.

41. The method of any one of claims 1-40, wherein the non-target antigen is an allelic variant of a major histocompatibility complex (MHC) protein.

42. The method of any one of claims 1-41, wherein the non-target antigen is an allelic variant of an HLA-A, HLA-B, or HLA-C protein.

43. The method of claim 42, wherein the non-target antigen is HLA-A*02.

44. The method of claim 43, wherein the solid tumor has somatic loss of HLA-A*02.

45. The method of any one of claims 1-44, wherein the solid tumor is associated with colorectal cancer (CRC), non-small cell lung cancer (NSCLC), pancreatic cancer (PANC), ovarian cancer (OVCA), or mesothelioma (MESO).

46. The method of any one of claims 1-45, wherein the solid tumor is associated with disease or disorder selected from Table 1.

47. The method of any one of claims 1-46, wherein the solid tumor expresses the target antigen.

48. The method of any one of claims 1-47, wherein the target antigen is as listed in Table 149. The method of any one of claims 1-48, wherein the solid tumor expresses CEA, MSLN, and / or HER2.

50. The method of any one of claims 1-48, wherein the target antigen is CEA, MSLN, or HER2.

51. The method of any one of claims 1-50, wherein the target antigen is CEA.

52. The method of claim 51, wherein the subject is a heterozygous HLA-A*02 adult; the non-target antigen is HLA-A*02; the engineered T cell is a CAR T cell; and the solid tumor isa recurrent unresectable, locally advanced or metastatic, solid tumor that expresses CEA and has lost HLA-A*02 expression.

53. The method of any one of claims 1-52, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (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 variable light (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 at most 1, 2, or 3 substitutions, deletions, or insertions relative to SEQ ID NOS: 55-58 or SEQ ID NOS: 59-63.

54. The method of any one of claims 1-52, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a set of heavy chain complementarity determining regions (HC-CDRs) comprising SEQ ID NOS: 55-57 and a variable light (VL) portion comprising a set of light chain complementarity determining regions comprising SEQ ID NOS: 59, 61 and 63; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to SEQ ID NOS: 55-57 or SEQ ID NOS: 59, 61 and 63.

55. The method of any one of claims 1-52, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (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 variable light (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.

56. The method of any one of claims 1-55, wherein 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.

57. The method of any one of claims 1-56, 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%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

58. The method of any one of claims 1-50, wherein the target antigen is MSLN.

59. The method of claim 58, wherein the subject is a heterozygous HLA-A*02 adult; the non-target antigen is HLA-A*02; the engineered T cell is a CAR T cell; and the solid tumor is a recurrent unresectable, locally advanced or metastatic, solid tumor that expresses MSLN and has lost HLA-A*02 expression.

60. The method of any one of claims 1-59, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a set of heavy chain complementarity determining regions (HC-CDRs) selected from the group consisting of SEQ ID NOS: 429-534 and a variable light (VL) portion comprising a set of light chain complementarity determining regions selected from the group consisting of SEQ ID NOS: 535-545; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to SEQ ID NOS: 429-534 or SEQ ID NOS: 535-545.

61. The method of any one of claims 1-59, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a set of heavy chain complementarity determining regions (HC-CDRs) comprising SEQ ID NOS: 438, 454, 488 and a variable light (VL) portion comprising a set of light chain complementarity determining regions comprising SEQ ID NOS: 535, 539 and 542; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to SEQ ID NOS: 438, 454, 488 or SEQ ID NOS: 535, 539 and 542.

62. The method of any one of claims 1-59, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NOs: 2000-2061 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NOs: 2062-2065 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

63. The method of any one of claims 1-62, wherein the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 1019-1085, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

64. The method of any one of claims 1-63, wherein the extracellular ligand binding domain of the first receptor comprises an scFv sequence of SEQ ID NO: 2066; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

65. The method of any one of claims 1-50, wherein the target antigen is HER2.

66. The method of claim 65, wherein the subject is a heterozygous HLA-A*02 adult; the non-target antigen is HLA-A*02; the engineered T cell is a CAR T cell; and the solid tumor is a recurrent unresectable, locally advanced or metastatic, solid tumor that expresses HER2 and has lost HLA-A*02 expression.

67. The method of any one of claims 1-66, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a set of heavy chaincomplementarity determining regions (HC-CDRs) selected from the group consisting of SEQ ID NOS: 36-42 and a variable light (VL) portion comprising a set of light chain complementarity determining regions selected from the group consisting of SEQ ID NOS: 30- 35; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to SEQ ID NOS: 30-35 or SEQ ID NOS: 36-42.

68. The method of any one of claims 1-67, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising a set of heavy chain complementarity determining regions (HC-CDRs) comprising SEQ ID NOS: 38, 2068, and 2069 and a variable light (VL) portion comprising a set of light chain complementarity determining regions comprising SEQ ID NOS: 30, 32, and 2067; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to SEQ ID NOS: 38, 2068, and 2069 or SEQ ID NOS: 30, 32, and 2067.

69. The method of any one of claims 1-68, wherein the extracellular ligand binding domain of the first receptor comprises a variable heavy (VH) portion comprising SEQ ID NO: 1143, 1145, 1147, and 1150 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, and a variable light (VL) portion comprising SEQ ID NO: 114, 1148 and 1149 or a sequence having 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

70. The method of any one of claims 1-69, wherein the extracellular ligand binding domain of the first receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 1151, 1153, 1155, 1157, and 1161, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

71. The method of any one of claims 1-70, wherein the extracellular ligand binding domain of the first receptor comprises an scFv sequence of SEQ ID NO: 2071; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

72. The method of any one of claims 1-71, wherein the first receptor is a chimeric antigen receptor (CAR).

73. The method of any one of claims 1-72, wherein the first receptor comprises a hinge domain, a transmembrane domain, and an intracellular domain.

74. The method of claim 73, wherein the hinge domain comprises a CD8α hinge domain.

75. The method of claim 74, wherein the CD8α hinge domain comprises a 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.

76. The method of any one of claims 73-75, wherein the transmembrane domain of the first receptor comprises a CD28 transmembrane domain.

77. The method of claim 76, wherein the CD28 transmembrane domain of the first receptor comprises a 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.

78. The method of any one of claims 72-77, wherein the intracellular domain of the first receptor comprises a CD28 co-stimulatory domain, a 4- IBB co-stimulatory domain, and a CD3ζ activation domain.

79. The method of claim 78, wherein the intracellular domain of the first receptor comprises a 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.

80. The method of any one of claims 1-79, wherein the second receptor comprises a LILRB 1 intracellular domain or a functional variant thereof.

81. The method of claim 80, wherein the LILRB 1 intracellular domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99%, or is identical to SEQ ID NO: 131.

82. The method of any one of claims 1-81, wherein the second receptor comprises a LILRB 1 transmembrane domain or a functional variant thereof.

83. The method of claim 82, wherein the LILRB 1 transmembrane domain or a functional variant thereof comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 135.

84. The method of any one of claims 1-83, wherein the second receptor comprises a LILRB 1 hinge domain or functional variant thereof.

85. The method of claim 84, wherein the LILRB 1 hinge domain comprises a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 134.

86. The method of any one of claims 1-85, wherein the second receptor comprises a LILRB 1 intracellular domain, a LILRB 1 transmembrane domain, a LILRB 1 hinge domain, a functional variant of any of these, or combinations thereof.

87. The method of claim 86, wherein the LILRB 1 hinge domain, LILRB 1 intracellular domain and LILRB 1 transmembrane domain comprises SEQ ID NO: 132 or a sequence at least 90%, at least 95%, at least 97%, at least 99% or is identical to SEQ ID NO: 132.

88. The method of any one of claims 1-87, wherein the extracellular ligand binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 for HLA-A as disclosed in Table 9; or CDRsequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of Table 9.

89. The method of any one of claims 1-87, wherein the extracellular ligand binding domain of the second receptor comprises complementarity determining regions (CDRs) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOS: 103-108 or of SEQ ID NOS: 109114; or CDR sequences having at most 1, 2, or 3 substitutions, deletions, or insertions relative to the CDRs of SEQ ID NOS: 103-108 or SEQ ID NOS: 109-114.

90. The method of any one of claims 1-87, wherein the extracellular ligand binding domain of the second receptor comprises a polypeptide sequence selected from the polypeptide sequence disclosed in SEQ ID NOs: 91-102, 250-276, or 278-345; or a sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.

91. The method of any one of claims 1-87, wherein 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.

92. The method of any one of claims 1-87, wherein the second receptor comprises a sequence of SEQ ID NO: 164, or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

93. The method of any one of claims 1-92, wherein the solid tumor is a target antigen positive / HLA-A*02 negative solid tumor that does not express HLA-A*02.

94. The method of any one of claims 1-93, wherein the first receptor and the second receptor together specifically activate the engineered T cell in the presence of the target antigen positive / HLA-A*02 negative solid tumor cells having loss of heterozygosity.

95. The method of any one of claims 1-94, wherein the engineered T cell comprises a polynucleotide comprising an interfering RNA, the interfering RNA comprising a sequence complementary to a sequence of a B2M mRNA.

96. The method of claim 95, wherein the interfering RNA is capable of inducing RNAi-mediated degradation of the B2M mRNA.

97. The method of claims 96, wherein the interfering RNA is a short hairpin RNA (shRNA).

98. The method of claim 97, wherein the shRNA comprises: a. a first sequence, having from 5’ end to 3’ end a sequence complementary to a sequence of the B2M mRNA; and b. a second sequence, having from 5’ end to 3’ end a sequence complementary to the first sequence,wherein the first sequence and the second sequence form the shRNA.

99. The method of claim 97 or 98, wherein the shRNA is encoded by a sequence comprising a sequence ofor(SEQ ID NO: 180), or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

100. The method of claim 99, wherein the engineered T cell comprises an shRNA encoded by a sequence comprising(SEQ ID NO: 179) or a sequence having at least 80%, at least 90%, or at least 95% identity thereto.

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