WO2021222576A1

WO2021222576A1 - Pag1 fusion proteins and methods of making and using same

Info

Publication number: WO2021222576A1
Application number: PCT/US2021/029907
Authority: WO
Inventors: Carl Alexander Kamb; Agnes HAMBURGER; Breanna DIANDRETH; Mark DARIS; Kiran Deshmukh; Dora Toledo WARSHAVIAK
Original assignee: A2 Biotherapeutics, Inc.
Priority date: 2020-05-01
Filing date: 2021-04-29
Publication date: 2021-11-04

Abstract

Inhibitory receptors comprising a PAG1 intracellular domain, immune cells comprising same, and methods of making and using same are provided. Some inhibitory receptors of the disclosure comprise hinge and/or transmembrane domains of PAG1 or LILRB1. The inhibitory receptor may be a chimeric antigen receptor or an engineered TCR. The inhibitory receptors are useful in treating diseases and disorders that include hematological malignancies, solid tumors, and others, where they can be used to increase the specificity and/or control the activity of an activator receptor, e.g. any conventional chimeric antigen receptor or engineered TCR.

Description

PAG1 FUSION PROTEINS AND METHODS OF MAKING AND USING SAME

CROSS REFERENCE TO REUATED APPUICATIONS

[0001] This application claims the benefit to U.S. Provisional Patent Application Serial No. 63/018,881 filed on May 1, 2020, the contents of which are hereby incorporated by reference in their entirety.

REFERENCE TO SEQUENCE FISTING

[0002] This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “A2BI_016_01WO_SeqList_ST25.txt” created on April 28, 2021 and having a size of ~ 443 kilobytes. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.

BACKGROUND

[0003] Chimeric antigen receptor (CAR) or T cell receptor (TCR) T cell therapy is proving to be an effective therapeutic approach to various diseases, particularly hematological malignancies, but also other cancers. CAR or TCR NK cells may also have clinical applications. Conventional CARs provide a stimulatory signal to the engineered immune cell (e.g. a T cell or an NK cell). In CAR-T cells, this results in killing activity towards the target cell identified by the antigen-binding domain of the CAR. Inhibitory CARs (iCARs) have been developed as a means to control cell activity or restrict the activity of an activator CAR or TCR to specific cell types. Federov et al. Sci. Transl. Med. 5(215):215ral72 (2013). Inhibitory CARs generally have the intracellular domain of an inhibitory signaling molecule (such as PD-1 or CTLA-4) fused to an antigen-binding domain (e.g., a single-chain variable fragment, scFv) through a transmembrane region, and optionally a hinge region. While numerous alternative iCAR architectures have been described in the art, there remains an unmet need for novel alternative inhibitory receptors and identification of particular inhibitory receptor architectures having superior performance, along with associated compositions and methods of use thereof. SUMMARY

[0004] The disclosure relates generally to the inhibitory receptors that employ the intracellular domain of the protein phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1) or a functional variant thereof, and optionally hinge, a transmembrane domain, and/or one or more further intracellular domains. The transmembrane domain may be the transmembrane domain of PAG1. The hinge, transmembrane domain, and/or a further intracellular domain may be from leukocyte immunoglobulin like receptor B 1 (LILRB 1), PAG1 or a combination thereof. Other hinges and transmembrane domains are also contemplated. The inhibitory receptors may have multiple intracellular domains, and may be formed from one or more distinct polypeptide chains.

[0005] In one aspect, the disclosure provides an inhibitory receptor comprising at least a first polypeptide, the first polypeptide comprising one or more of: a) a phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1) intracellular domain or a functional fragment or variant thereof; b) a transmembrane domain; and c) an extracellular ligand binding domain or a portion thereof. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 37-432 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises a sequence identical to amino acid residues 37-432 of SEQ ID NO: 1.

[0006] In some embodiments, the PAG1 intracellular domain comprises a truncated PAG1 intracellular domain. In some embodiments, the truncated PAG1 intracellular domain comprises an N-terminal truncation, a C-terminal truncation, or both. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 41-432 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 93-432 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 112-432 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 41-326 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 41-371 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 41-400 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 112-326 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 217-326 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 intracellular domain comprises amino acid residues 41-432 of SEQ ID NO: 1, residues 93-432 of SEQ ID NO: 1, residues 112-432 of SEQ ID NO: 1, residues 41-326 of SEQ ID NO: 1, residues 41-371 of SEQ ID NO: 1, residues 41-400 of SEQ ID NO: 1, residues 112-326 of SEQ ID NO: 1, or residues 217-326 of SEQ ID NO: 1.

[0007] In some embodiments, the transmembrane domain comprises a PAG1 transmembrane domain, a LILRB1 transmembrane domain, a CD8a transmembrane domain, or a functional fragment or variant of any of these. In some embodiments, the PAG1 transmembrane domain comprises a sequence of SEQ ID NO: 11, or a sequence having at least 95% identical thereto. In some embodiments, the PAG1 transmembrane domain comprises a sequence identical to SEQ ID NO: 11. In some embodiments, the LILRB1 transmembrane domain comprises a sequence of SEQ ID NO: 88, or a sequence having at least 95% identity thereto. In some embodiments, the LILRB1 transmembrane domain comprises a sequence identical to SEQ ID NO: 88. In some embodiments, the CD8a transmembrane domain comprises a sequence of SEQ ID NO: 13, or a sequence having at least 95% identity thereto. In some embodiments, the CD8a transmembrane domain comprises a sequence identical to SEQ ID NO: 13.

[0008] In some embodiments, the inhibitory receptors provided herein comprise a polypeptide comprising a hinge domain. In some embodiments, the hinge domain comprises a CD8 hinge domain, a LILRB1 hinge domain, a PAG1 hinge domain or a combination, functional fragment or variant of any of these. In some embodiments, the LILRB1 hinge domain comprises a sequence of SEQ ID NO: 16, SEQ ID NO: 87, or a sequence having at least 95% identity thereto. In some embodiments, the LILRB 1 hinge domain comprises a sequence of SEQ ID NO: 17, or a sequence having at least 95% identity thereto. In some embodiments, the LILRB 1 hinge domain comprises a sequence of SEQ ID NO: 15 or a sequence having at least 95% identity thereto. In some embodiments, the LILRB 1 hinge domain comprises a sequence identical to SEQ ID NO: 16, SEQ ID NO: 87, SEQ ID NO: 17, SEQ ID NO: 15, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, or SEQ ID NO: 182. In some embodiments, the PAG1 hinge domain comprises a sequence of SEQ ID NO: 14 or a sequence having at least 95% identity thereto. In some embodiments, the PAG1 hinge domain comprises a sequence identical to SEQ ID NO: 14. In some embodiments, the CD8a hinge domain comprises a sequence of SEQ ID NO: 18, or a sequence having at least 95% identity thereto. In some embodiments, the hinge domain comprises both PAG1 and LILRB1 hinge sequences. In some embodiments, the hinge domain comprises a sequence of SEQ ID NO: 86, or a sequence having at least 95% identity thereto.

[0009] In some embodiments, the first polypeptide comprises a LILRB 1 hinge domain, a LILRB 1 transmembrane domain and the PAG1 intracellular domain or a functional fragment or variant of any of these. In some embodiments, the LILRB 1 hinge domain and the LILRB 1 transmembrane domain comprise a sequence of SEQ ID NO: 48, or a sequence having at least 95% identity thereto.

[0010] In some embodiments, the first polypeptide comprises a PAG1 transmembrane domain and a PAG1 intracellular domain, or a functional fragment or variant of either of these. In some embodiments, the PAG1 transmembrane domain and PAG1 intracellular domain comprises a sequence with at least 95% identity to SEQ ID NO: 47.

[0011] In some embodiments, the first polypeptide comprises a CD8a hinge domain, a CD8a transmembrane domain, and a PAG1 intracellular domain, or functional fragment or variant of any of these. In some embodiments, the CD8a hinge domain and the CD8a transmembrane domain comprise a sequence of SEQ ID NO: 49, or a sequence having at least 95% identity thereto.

[0012] In some embodiments, the first polypeptide comprises a CD8a hinge domain, a PAG1 transmembrane domain and the PAG1 intracellular domain, or a functional fragment or variant of any of these. In some embodiments, the first polypeptide comprises a sequence of SEQ ID NO: 86, or a sequence having at least 95% identity thereto.

[0013] In some embodiments, the first polypeptide comprises a PAG1 hinge domain, a PAG1 transmembrane domain and a PAG1 intracellular domain or a functional fragment or variant of any of these. In some embodiments, the first polypeptide comprises a sequence of SEQ ID NO: 1, or a sequence having at least 95% identity thereto. In some embodiments, the first polypeptide comprises a sequence of SEQ ID NO: 1.

[0014] In some embodiments, the extracellular ligand binding domain comprises an antigen binding domain. In some embodiments, the antigen binding domain is specific to an antigen that is lost in a cancer cell through loss of heterozygosity. In some embodiments, the antigen binding domain is specific to a minor histocompatibility antigen (MiHA). In some embodiments, the antigen binding domain is specific to an antigen that is lost in a cancer cell through loss of Y chromosome. In some embodiments, the antigen binding domain is specific to an antigen that is lost in tumors via epigenetic or similar mechanisms of transcriptional silencing. In some embodiments, the antigen binding domain is specific to a major histocompatibility class I allele. In some embodiments, the major histocompatibility class I allele comprises an HLA-A, an HLA-B, an HLA-C allele, an HLA-E allele, and HLA-F allele or an HLA- G allele. In some embodiments, the HLA-A allele comprises an HLA-A*02 allele. [0015] In some embodiments, the antigen binding domain comprises an antibody fragment, a nb only domain, a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb). In some embodiments, the antigen binding domain comprises a heavy chain and a light chain of an antibody, wherein the heavy chain comprises heavy chain complementarity determining regions (CDRs) of any one of SEQ ID NOs: 25-27 or 31-33, and wherein the light chain comprises light chain CDRs of any one of SEQ ID NOs: 22-24 or 28-30. In some embodiments, the antigen binding domain comprises a heavy chain and a light chain of an antibody, wherein the heavy chain comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 35-46, and wherein the light chain comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOs: 35-46 or 125.

[0016] In some embodiments, the scFv comprises CDRs of any one of SEQ ID NOs: 22-33. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOs: 35-46 or 125. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOs: 35-46 or 125.

[0017] In some embodiments, the receptor comprises a second polypeptide. In some embodiments, the second polypeptide comprises an extracellular ligand binding domain or a portion thereof. In some embodiments, the extracellular ligand binding domain of the first polypeptide comprises a first chain of an antibody and the extracellular ligand binding domain of the second polypeptide comprise a second chain of said antibody. [0018] In some embodiments, the antibody comprises a Fab fragment of an antibody. In some embodiments, the first polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody and the second polypeptide comprises an antigen binding fragment of the light chain of the antibody. In some embodiments, the first polypeptide comprises an antigen-binding fragment of the light chain of the antibody and the second polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody. In some embodiments, the heavy chain of the antibody comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 35-46 or 125, and wherein the light chain of the antibody comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 35-46 or 125.

[0019] In some embodiments, the receptor comprises an extracellular fragment of a T cell receptor (TCR). In some embodiments, the first polypeptide comprises an antigen binding fragment of the alpha chain of the TCR and the second polypeptide comprises an antigen-binding fragment of the beta chain of the TCR. In some embodiments, the first polypeptide comprises an antigen-binding fragment of the beta chain on the TCR and the second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR.

[0020] In some embodiments, the extracellular ligand binding domain binds to an antigen that is present on a normal cell but is not present on a cancer cell.

[0021] In one aspect, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding the inhibitory receptor described herein. In another aspect, the disclosure provides a vector comprising the polynucleotide described herein.

[0022] In one aspect, the disclosure provides an immune cell comprising the inhibitory receptor described herein, the polynucleotide described herein, or the vector of described herein.

[0023] In some embodiments, activation of the immune cell is reduced when the cell is contacted with the extracellular ligand or a cell expressing the extracellular ligand on its surface. In some embodiments, the extracellular ligand is an antigen.

[0024] In some embodiments, the immune cell comprises a second receptor, wherein the second receptor is an activatory receptor. In some embodiments, the activatory receptor activates the immune cell or promotes activation of the immune cell. In some embodiments, the activatory receptor is a chimeric antigen receptor (CAR) or an engineered T Cell Receptor (TCR). In some embodiments, the activatory receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to a tumor specific antigen (TSA). In some embodiments, the TSA is selected from the group consisting of EGFR, mesothelin (MSLN) and cancer embryonic antigen (CEA or CEACAM5). In some embodiments, the antigen binding domain binds to a transferrin receptor (TFRC) antigen. In some embodiments, the activatory receptor comprises an antigen binding domain that binds to a pan-HLA antigen.

[0025] In some embodiments, the immune cell is a T cell, a B cell, an NK cell or a macrophage. In some embodiments, the immune cell is isolated. In some embodiments, the immune cell is non-natural.

[0026] In one aspect, the disclosure provides a method, comprising introducing the polynucleotide described herein, or the vector described herein into a cell. In some embodiments, the cell expresses the polynucleotide. In some embodiments, the immune cell is a T cell, a B cell, an NK cell or a macrophage. In some embodiments, immune cell activation is reduced when the cell is contacted with the extracellular ligand or a cell expressing the extracellular ligand on its surface. In some embodiments, the extracellular ligand is an antigen. In some embodiments, the cell expressing the extracellular ligand on its surface is not a cancer cell.

[0027] In one aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a plurality of immune cells comprising a chimeric antigen receptor (CAR) or engineered T-cell receptor (TCR), and comprising the inhibitory receptor described herein. In some embodiments, the disease is cancer.

[0028] In some embodiments, the inhibitory receptor increases the specificity of the immune cells for a target cell or tissue compared to immune cells that express the CAR or TCR but do not express the inhibitory receptor.

[0029] In some embodiments, the immune cells comprise a second activatory receptor. In some embodiments, the activatory receptor activates the immune cell or promotes activation of the immune cell. In some embodiments, the activatory receptor is a chimeric antigen receptor (CAR) or an engineered T Cell Receptor (TCR). In some embodiments, the activatory receptor comprises an antigen binding domain.

[0030] In some embodiments, the antigen binding domain binds to a tumor specific antigen (TSA). In some embodiments, the TSA is selected from the group consisting of EGFR, mesothelin (MSLN) and cancer embryonic antigen (CEA or CEACAM5). In some embodiments, the antigen binding domain binds to a transferrin receptor (TFRC) antigen. In some embodiments, the antigen binding domain binds to a pan-HLA antigen.

[0031] In some embodiments, the immune cells are T cells. In some embodiments, the plurality of immune cells are isolated. In some embodiments, the plurality of immune cells are non-natural.

[0032] In some embodiments, the immune cells comprising the inhibitory receptor have reduced side effects when compared to immune cells that express the CAR or TCR but do not express the inhibitory receptor.

[0033] In one aspect, the disclosure provides a pharmaceutical composition, comprising a therapeutically effective amount of the immune cell described herein and a pharmaceutically acceptable carrier or diluent. In another aspect, the disclosure provides the immune cell described herein for use as a medicament. In a further aspect, the disclosure provides the immune cell described herein for use in a method of treatment of a subject in need thereof.

[0034] In one aspect, the disclosure provides a kit, comprising the polynucleotide described herein or the vector described herein, or the immune cell described herein. In some embodiments, the kit further comprises instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0036] FIG. 1 is a plot showing Jurkat cell assays of scFv-PAGl constructs. The ability of scFv-PAGl constructs comprising an NY-ESO-1 pMHC scFv extracellular domain to inhibit MAGE-A3 pMHC chimeric antigen receptor (CAR) activation of an NFAT Luciferase reporter was assayed.

[0037] FIG.2 is a plot showing Jurkat cell assays of scFv-PAGl constructs. The ability of scFv-PAGl constructs comprising an NY-ESO-1 pMHC scFv extracellular domain to inhibit MAGE-A3 pMHC T cell receptor (TCR) activation of an NFAT Luciferase reporter was assayed.

[0038] FIG.3 is a plot showing Jurkat cell assays of scFv-PAGl constructs. The ability of scFv-PAGl constructs comprising an NY-ESO-1 pMHC scFv extracellular domain and a CD8 hinge, a CD8 hinge and transmembrane domain, or a LILRB1 hinge and transmembrane domain to inhibit MAGE-A3 TCR activation was assayed.

[0039] FIG. 4 is a plot showing Jurkat cell assays of scFv-PAGl constructs. ScFv- PAG1 constructs comprising an NY-ESO-1 pMHC scFv extracellular domain and N- or C-terminal truncations of the PAG1 intracellular domain were assayed for their ability to inhibit MAGE-A3 pMHC CAR.

[0040] FIG. 5 is a plot showing Jurkat cell assays of scFv-PAGl constructs. ScFv- PAG1 constructs comprising an NY-ESO-1 pMHC scFv extracellular domain, a LILRB 1 hinge and transmembrane domain, and N- or N- and C-terminal truncations of the PAG1 intracellular domain were assayed for their ability to inhibit MAGE-A3 pMHC CAR.

[0041] FIG. 6 is a plot showing Jurkat cell assays of scFv-PAGl constructs. ScFv- PAGl constructs comprising an NY-ESO-1 pMHC scFv extracellular domain, a LILRB 1 hinge and transmembrane domain, and truncations or modifications of the PAG1 intracellular domain at amino acid residues 217-326 were assayed for their ability to inhibit MAGE-A3 pMHC CAR.

DETAILED DESCRIPTION

[0042] The immune system works by balancing activation and inhibition. A classic example is the natural killer (NK) cell whose functional state, either quiescence or cytototoxic attack on other cells, depends on integration of activating stimuli using a slew of receptors (e.g., the killer activating receptor (KAR) family) and inhibitory signals derived from a variety of other receptors, many of whom recognize self antigens, including MHC class I proteins. These two opposing functions act as accelerators and brakes on immune cell activation, and the set point of this system determines that threshold for activation. Together, activatory and inhibitory signals comprise an “AND NOT” logic gate, a complex molecular machine that receives various inputs whereby the immune cell is in the “OFF” state when the inhibitory (blocker) receptor inputs dominate, or when activator inputs or both inputs are absent, and in the “ON” state if the activator inputs prevail. Design of a simple but effective AND NOT gate enables immune cells, including T cells, to be engineered such that these cells respond decisively to a chosen pair of activation and inhibition inputs. This integration of activation and inhibition inputs can increase the specificity of immune cell therapies for particular tissues or cell types, and reduce side effects of immune cell- based therapies.

[0043] Integration of activation and inhibition inputs can occur through co-expression of activatory and inhibitory receptors in immune cells, such as immune cells used for immunotherapies. Exemplary applications of the inhibitory receptors described herein include cancer therapy. Cancer therapies, including immunotherapies, work best when focused on molecular targets that differ in expression or function between normal cells and tumor cells. Unfortunately, there are few absolute discriminators between cancer cells and non-cancer cells. Perhaps the best known and most black-and-white targets are the class of somatic mutant proteins known as neo-antigens. These proteins are variants of normal polypeptides that have undergone mutation during somatic growth of the tumors and contains sequence variants that are totally absent in normal tissues. However, one of the main limitations of this class of cancer targets is its frequency among cancers. The most frequent neo-antigens occur at a rate of approximately 5% (e.g. , G12D KRAS). This feature, combined with the fact that the vast majority of neo antigens are cytoplasmic, and therefore inaccessible to conventional larger-molecule therapeutics, including antibodies, means that targeting neo-antigens is an ineffective strategy for many cancers.

[0044] There thus exists a need for additional classes of therapeutic targets. One such class are the tumor-selective antigens (TSAs). TSAs include proteins that have wild- type sequence and limited expression in normal tissues, but are frequently expressed by various tumor types. Examples of TSAs in solid tumors include EGFR, mesothelin (MSLN) and cancer embryonic antigen (CEA or CEACAM5). These proteins are expressed on the surface of many cancer types, and have restricted normal tissue expression. For example, CEA is expressed almost exclusively in the gut epithelium. However, safety and tolerability remain serious challenges for therapeutic agents that target TSAs, because even the limited expression on normal cells can trigger severe toxicity that caps the dose of the therapeutic agent.

[0045] Another class of underappreciated targets for cancer that are, like neo-antigens, absolute discriminators of tumor versus normal cells are protein isoforms lost through genetic deletion. The most common form of deletion in cancer cells involves heterozygous loss of genetic material, called loss of heterozygosity (LOH). LOH can encompass large swaths of a cancer cell genome, averaging about 20% across all cancers. Thus, loci that encode polymorphic proteins that can be targeted by therapeutics are candidates, in principle, for patient-specific targeted cancer therapies. Such a therapeutic strategy depends on devising a system that can recognize and respond to the loss of proteins from tumor cells.

[0046] The inhibitory receptors described herein can be used to address the safety and tolerability issues of immunotherapies that target tumor selective antigens. In some embodiments, the system employs an inhibitory receptor that responds to an inhibitory (blocking) signal emanating from normal cells, and an activating signal that must be present on tumor cells. In principle, the activating signal can be present on all normal cells, so long as the inhibitory signal is also coincidentally expressed on normal cells. This design ensures that cytotoxicity is delivered only to the tumors cells and not normal cells.

[0047] A large family of ligand-gated inhibitory receptors are known to function within immune cells (e.g., inhibitory killer-cell immunoglobulin-like receptors, KIRs). While several proteins derived from KIRs and other negative regulators of the immune response, notably checkpoint inhibitors such as CTLA-4 and PD-1, have been proposed as inhibitory CARs (iCARs), none of these constructs has proven so far to have robust behavior when combined with activatory signals such as those provided by CARs or engineered TCRs. It is thus unclear which native inhibitory receptors can be co-opted as part of a therapeutically useful activatory and inhibitory receptor pair, or whether non-receptor molecules, when fused to, for instance, an scFv ligand-binding domain (LBD), can be engineered to function as the inhibitory part of the activatory and inhibitory receptor pair. Such inhibitory receptors must be ligand-gated, to ensure proper regulation within the body and, as a result, selectively target immune cell activity to the appropriate cells or tissues within the body. In addition, for safety reasons, inhibitory receptors should be potent enough to mediate a wide therapeutic window. Finally, inhibitory receptors should be modular, and capable of acting in concert with a variety of activators, such as CARs and TCRs.

[0048] The inventors have found that fusions of the phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1) protein that include the PAG1 intracellular (ICD) and an extracellular ligand binding domain, such as a single-chain variable fragment (scFv), can inhibit CAR or TCR signaling in a ligand dependent manner. These fusion proteins function as a potent ligand-regulated inhibitory receptors that are capable of inhibiting activation signals emanating from either TCRs or CARs when tested in T cells. [0049] Wild type PAG1 is known to associate with immune function, but does not contain a ligand binding domain (LBD). Indeed, the PAG1 extracellular domain is only 10-15 amino acids, depending on the species. Therefore it is not obvious that PAG1, despite its known involvement in TCR signaling, could be rendered ligand-dependent, and therefore serve as an inhibitory receptor capable of blocking CAR or TCR activation signals.

[0050] The invention describes polypeptides and receptors that comprise a PAG1 intracellular domain, and optionally a PAG1 transmembrane domain and/or extracellular domain or functional fragments or derivatives thereof fused to an extracellular ligand binding domain at the N-terminus. These receptors function as potent ligand-dependent inhibitory receptors, and can function to inhibit both CAR and TCR activators. These PAG 1 -based inhibitory receptors can counteract activation signals that emanate from either TCRs or CARs in order to provide further specificity over T cell activation, which allows for safer therapies as well as new antigen recognition strategies (e.g., detecting the absence of antigen).

Definitions

[0051] The term “stimulation” refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.

[0052] The term “stimulatory molecule” or “stimulatory domain” refers to a molecule or portion thereof that, when natively expressed by a T-cell, provides the primary cytoplasmic signaling sequence(s) that regulate activation of the TCR complex in a stimulatory way for at least some aspect of the T-cell signaling pathway. TCR alpha and/or TCR beta chains of wild type TCR complexes do not contain stimulatory domains and require association with CD3 subunits such as CD3 zeta to initiate signaling. In one aspect, the primary stimulatory signal is initiated by, for instance, binding of a TCR/CD3 complex with an a major histocompatibility complex (MHC) bound to peptide, and which leads to mediation of a T-cell response, including, but not limited to, proliferation, activation, differentiation, and the like. One or more stimulatory domains, as described herein, can be fused to the intracellular portion of any one or more subunits of the TCR complex, including TCR alpha, TCR beta, CD3 delta, CD3 gamma and CD3 epsilon.

[0053] As used herein, a “domain capable of providing a stimulatory signal” refers to any domain that, either directly or indirectly, can provide a stimulatory signal that enhances or increases the effectiveness of signaling mediated by the TCR complex to enhance at least some aspect of T-cell signaling. The domain capable of providing a stimulatory signal can provide this signal directly, for example with the domain capable of providing the stimulatory signal is a primary stimulatory domain or co-stimulatory domain. Alternatively, or in addition, the domain capable of providing the stimulatory signal can act indirectly. For example, the domain can be a scaffold that recruits stimulatory proteins to the TCR, or provide an enzymatic activity, such as kinase activity, that acts through downstream targets to provide a stimulatory signal.

[0054] As used herein, a “domain capable of providing an inhibitory signal” refers to any domain that, either directly or indirectly, can provide an inhibitory signal that inhibits or decreases the effectiveness signaling mediated by the TCR complex or by a receptor such as a chimeric antigen receptor (CAR). The domain capable of providing an inhibitory signal can reduce, or block, totally or partially, at least some aspect of T- cell signaling or function. The domain capable of providing an inhibitory signal can provide this signal directly, for example with the domain capable of providing the inhibitory signal provides a primary inhibitory signal. Alternatively, or in addition, the domain capable of providing the inhibitory signal can act indirectly. For example, the domain can recruit additional inhibitory proteins to the TCR, or can provide an enzymatic activity that acts through downstream targets to provide an inhibitory signal. [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 to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

[0056] In general, “sequence identity” or “sequence homology” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0057] As used herein, a “subsequence” refers to a length of contiguous amino acids or nucleotides that form a part of a sequence described herein. A subsequence may be identical to a part of a full length sequence when aligned to the full length sequence, or less than 100% identical to the part of the full length sequence to which it aligns (e.g., 90% identical to 50% of the full sequence, or the like).

[0058] 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).

[0059] A polynucleotide is “operably linked” to another polynucleotide when it is placed into a functional relationship with the other polynucleotide. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. A peptide is “operably linked” to another peptide when the polynucleotides encoding them are operably linked, preferably they are in the same open reading frame.

[0060] A “promoter” is a sequence of DNA needed to turn a gene on or off. Promoters are located immediately upstream and/or overlapping the transcription start site, and are usually between about one hundred to several hundred base pairs in length.

[0061] 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.

[0062] 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%.

Phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1)

[0063] The disclosure provides inhibitory receptors comprising an intracellular domain isolated or derived from PAG1. In some embodiments, the inhibitory receptor comprises an intracellular domain, a transmembrane domain, and optionally, a portion of an extracellular domain (e.g., a hinge), that are isolated or derived from PAG1. Numerous inhibitory receptors comprising PAG1 intracellular domains, engineered cells, and uses thereof are contemplated herein. [0064] Phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1), also known as CBP and PAG, is a type III transmembrane adaptor protein that binds to the non-receptor tyrosine kinase Csk. Without wishing to be bound by theory, a key event in T cell activation by antigen binding to the T cell receptor (TCR) complex is phosphorylation of ITAM tyrosines in the TCR subunits by the Src family kinases Lck and FynT. Both Lck and FynT are negatively regulated through phosphorylation of conserved C-terminal regulatory tyrosine residues by tyrosine kinases, including Csk. Csk, like other Src family tyrosine kinases, has SH3, SH2 and kinase domains, but lacks C-terminal regulatory tyrosine and autophosphorylation sites, as well as the N-terminal lipid modification sequences that targets other Src family members to membranes. PAG1 is a ubiquitously expressed transmembrane protein, and likely interacts with actin filaments through an Ezrin/radixin/moesin (ERM) binding protein of 50 kDa (EBP50)-ERM protein complex. Without wishing to be bound by theory, it is thought that PAG1 regulates Csk through the Csk SH2 domain, which is required for membrane translocation of Csk to lipid rafts. PAG1 binds not only to the SH2 domain of Csk, but is also thought to interact with FynT and EBP50. Stimulation of T cells has been found to result in dephosphorylation of PAG1, dissociation of PAG 1 -Csk and PAGl-FynT complexes, and decreased Csk content in lipid rafts.

[0065] The mechanism of action of PAG1 is distinct from that used by other inhibitor CARs (PD-1, CTLA-1, and the like).

[0066] In some embodiments, the inhibitory receptor comprises at least a PAG1 intracellular domain or a functional fragment or derivative thereof. In some embodiments, the inhibitory receptor comprises a PAG1 intracellular domain and a PAG1 transmembrane domain, or functional fragments or derivatives of either of these domains. In some embodiments, the inhibitory receptor comprises a PAG1 intracellular domain, a PAG1 transmembrane domain, and a PAG1 extracellular domain (or hinge) or functional fragments or derivatives of any of these. In some embodiments, a single polypeptide comprises the PAG1 intracellular domain, the PAG1 transmembrane domain, and optionally, the PAG1 extracellular domain.

[0067] In some embodiments, the inhibitory receptor comprises one or more domains of PAG1 comprising 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: 1. In some embodiments, the one or more domains of PAG1 comprise an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 1. In some embodiments, the inhibitory receptor comprises one or more domains of PAG1 that consist essentially 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: 1. In some embodiments, the one or more domains of PAG1 consist essentially of an amino acid sequence that is identical to a sequence or subsequence of SEQ ID NO: 1.

[0068] An exemplary human PAG1 protein is described NCBI Reference NP_060910.3, the contents of which are incorporated by reference in their entirety herein. An exemplary human PAG1 protein comprises a sequence of:

1 MGPAGSLLGS GQMQITLWGS LAAVAIFFVI TFLIFLCSSC DREKKPRQHS GDHENLMNVP 61 SDKEMFSRSV TSLATDAPAS SEQNGALTNG DILSEDSTLT CMQHYEEVQT SASDLLDSQD 121 STGKPKCHQS RELPRIPPES AVDTMLTARS VDGDQGLGME GPYEVLKDSS SQENMVEDCL 181 YETVKEIKEV AAAAHLEKGH SGKAKSTSAS KELPGPQTEG KAEFAEYASV DRNKKCRQSV 241 NVESILGNSC DPEEEAPPPV PVKLLDENEN LQEKEGGEAE ESATDTTSET NKRFSSLSYK 301 SREEDPTLTE EEISAMYSSV NKPGQLVNKS GQSLTVPEST YTSIQGDPQR SPSSCNDLYA 361 TVKDFEKTPN STLPPAGRPS EEPEPDYEAI QTLNREEEKA TLGTNGHHGL VPKENDYESI 421 SDLQQGRDIT RL (SEQ ID NO: 1).

[0069] In some embodiments of the inhibitory receptors described herein, the receptors comprise at least one PAG1 domain, and the at least one PAG1 domain is 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:

1 atggggcccg cggggagcct gctgggcagc ggacagatgc agatcaccct gtggggaagt

61 ctggctgctg tcgccatttt cttcgtcatc accttcctca tcttcctgtg ctctagttgt

121 gacagggaaa agaagccgcg acagcatagt ggggaccatg agaacctgat gaacgtgcct

181 tcagacaagg agatgttcag ccgttcagtt actagcctgg caacagatgc tcctgccagc

241 agtgagcaga atggggcact caccaatggg gacattcttt cagaggacag tactctgacc

301 tgcatgcagc attacgagga agtccagaca tcggcctcgg atctgctgga ttcccaggac

361 agcacaggga aaccaaaatg tcatcagagt cgggagctgc ccagaatccc tcccgagagc

421 gcagtggata ccatgctcac ggcgagaagt gtggacgggg accaggggct ggggatggaa

481 gggccctatg aagtgctcaa ggacagctcc tcccaagaaa acatggtgga ggactgcttg

541 tatgaaactg tgaaagagat caaggaggtg gctgcagctg cacacctgga gaaaggccac

601 agtggcaagg caaaatctac ttctgcctcg aaagagctcc cagggcccca gactgaaggc

661 aaagctgagt ttgctgaata tgcctcggtg gacagaaaca aaaaatgtcg tcaaagtgtt

721 aatgtagaga gtatccttgg aaattcatgt gatccagaag aggaggcccc accacctgtc

781 cctgttaagc ttctggacga gaatgaaaac cttcaggaga aggaaggggg agaggcggaa

841 gagagtgcca cagacacgac cagtgaaact aacaagagat ttagctcatt gtcatacaag

901 tctcgggaag aagaccccac tctcacagaa gaagagatct cagctatgta ctcatcagta

961 aataaacctg gacagttagt gaataaatcg gggcagtcgc ttacagttcc ggagtccacc

1021 tacacctcca ttcaagggga cccacagagg tcaccctcct cctgtaatga tctctatgct

1081 actgttaaag acttcgaaaa aactccaaac agcacacttc caccagcagg gaggcccagc

1141 gaggagccag agcctgatta tgaagcgata cagactctca acagagagga agaaaaggcc

1201 accctgggga ccaatggcca ccacggtctc gtcccaaagg agaacgacta cgagagcata

1261 agtgacttgc agcaaggcag agatattacc aggctctag (SEQ ID NO: 50) .

[0070] In some embodiments, the sequence encoding the at least one PAG1 domain is codon optimized. For example the sequence encoding the at least one PAG1 domain can be codon optimized for expression in a mammal, or a human. Inhibitory Receptors

[0071] The disclosure provides inhibitory receptors comprising a PAG1 intracellular domain, or a functional fragment or variant thereof. Inhibitory receptors can specifically bind target ligands, e.g. antigens or inhibitor ligands. In various embodiments, the inhibitory receptor comprises an engineered receptor, comprising at least a first polypeptide comprising on one or more of: a phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1) intracellular domain or a functional variant thereof; a transmembrane domain, and an extracellular ligand binding domain or a fragment thereof. In some embodiments, the inhibitory receptor comprises a hinge domain. In some embodiments, the ligand binding domain comprises an antigen binding domain. In some embodiments, the inhibitory receptor comprises a second polypeptide. In some embodiments, the second polypeptide comprises an extracellular ligand binding domain or a portion thereof.

Intracellular domain

[0072] The disclosure provides an inhibitory receptor comprising a polypeptide comprising an intracellular domain isolated or derived from PAG1.

[0073] As used herein, “intracellular domain” refers to the cytoplasmic or intracellular domain of a protein, such as a receptor, that interacts with the interior of the cell, and carries out a cytosolic function. As used herein, “cytosolic function” refers to a function of a protein or protein complex that is carried out in the cytosol of a cell. For example, intracellular signal transduction cascades are cytosolic functions.

[0074] Any functional variant or fragment of the PAG1 intracellular domain capable of inhibiting immune cell activation is envisaged within the scope of the disclosure. Fragments and functional variants of the PAG1, include, inter alia, truncations of the PAG1 intracellular domain, including N-terminal truncations of the PAG1 intracellular domain, C-terminal truncations of the PAG1 intracellular domain, and PAG1 intracellular domains comprising both N- and C-terminal truncations of the intracellular domain. In some embodiments, functional variants of the PAG1 intracellular domain include mutations in the PAG1 intracellular domain, including, but not limited to, point mutations, insertions, internal deletions, and inversions. In some embodiments, functional variants of the PAG1 intracellular domain include both truncations and mutations.

[0075] In some embodiments, the PAG1 intracellular domain comprises amino acid residues 37-432 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to amino acid residues 37-432 SEQ ID NO: 1. In some embodiments, the PAG1 intracellular domain comprises a sequence of:

1 CSSCDREKKP RQHSGDHENL MNVPSDKEMF SRSVTSLATD APASSEQNGA LTNGDILSED

61 STLTCMQHYE EVQTSASDLL DSQDSTGKPK CHQSRELPRI PPESAVDTML TARSVDGDQG

121 LGMEGPYEVL KDSSSQENMV EDCLYETVKE IKEVAAAAHL EKGHSGKAKS TSASKELPGP

181 QTEGKAEFAE YASVDRNKKC RQSVNVESIL GNSCDPEEEA PPPVPVKLLD ENENLQEKEG

241 GEAEESATDT TSETNKRFSS LSYKSREEDP TLTEEEI SAM YSSVNKPGQL VNKSGQSLTV

301 PESTYTSIQG DPQRSPSSCN DLYATVKDFE KTPNSTLPPA GRPSEEPEPD YEAIQTLNRE

361 EEKATLGTNG HHGLVPKEND YESI SDLQQG RDITRL (SEQ ID NO: 2).

[0076] In some embodiments, the PAG1 intracellular domain consists essentially of

SEQ ID NO: 2.

[0077] In some embodiments, the PAG1 intracellular domain comprises a truncated PAG1 intracellular domain. In some embodiments, the truncation is an N-terminal truncation. In some embodiments, the truncation is a C-terminal truncation. In some embodiments, the truncation comprises both N- and C-terminal truncations. In some embodiments, the truncation does not include amino acid residues 217-326 of SEQ ID NO: 1. Both short truncations, e.g., about 1, 2, 3, 4 or 5 amino acid residues, and longer truncations, are included with the scope of the disclosure. In some embodiments, the truncation is between about 1-150 amino acids, 1-100 amino acids, 1-80 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 5-10 amino acids, 5-20 amino acids, 5-30 amino acids, 5-50 amino acids, 5-100 amino acids, 10-20 amino acids, 10-30 amino acids, 10-40 amino acids, 10-50 amino acids, 10-80 amino acids, 10-90 amino acids, 20-30 amino acids, 20-40 amino acids, 20-50 amino acids, 20-80 amino acids, 20-90 amino acids, 20-100 amino acids, 20-150 amino acids, 30-50 amino acids, 30-80 amino acids, 30-100 amino acids, 30-20 amino acids, 30-150 amino acids, 50-100 amino acids, or 50-150 amino acids.

[0078] In some embodiments, the truncated PAG1 intracellular domain comprises amino acid residues 41-432 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 41-432 SEQ ID NO: 1. In some embodiments, the truncated PAG1 intracellular domain comprises a sequence of:

1 DREKKPRQHS GDHENLMNVP SDKEMFSRSV T SLAT DAP AS SEQNGALTNG DILSEDSTLT 61 CMQHYEEVQT SASDLLDSQD STGKPKCHQS RELPRI PPES AVDTMLTARS VDGDQGLGME 121 GPYEVLKDSS SQENMVEDCL YETVKEIKEV AAAAHLEKGH SGKAKSTSAS KELPGPQTEG 181 KAEFAEYASV DRNKKCRQSV NVESILGNSC DPEEEAPPPV PVKLLDENEN LQEKEGGEAE 241 ESATDTTSET NKRFSSLSYK SREEDPTLTE EEI SAMYSSV NKPGQLVNKS GQSLTVPEST 301 YTSIQGDPQR SPSSCNDLYA TVKDFEKTPN STLPPAGRPS EEPEPDYEAI QTLN REEEKA 361 TLGTNGHHGL VPKENDYESI SDLQQGRDIT RL (SEQ ID NO: 3).

[0079] In some embodiments, the truncated PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 3. In some embodiments, the truncated PAG1 intracellular domain consists essentially of SEQ ID NO: 3.

[0080] In some embodiments, the truncated PAG1 intracellular domain comprises amino acid residues 93-432 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 93-432 SEQ ID NO: 1. In some embodiments, the PAG1 intracellular domain comprises a sequence of:

1 LSEDSTLTCM QHYEEVQTSA SDLLDSQDST GKPKCHQSRE LPRI PPESAV DTMLTARSVD 61 GDQGLGMEGP YEVLKDSSSQ ENMVEDCLYE TVKEIKEVAA AAHLEKGHSG KAKSTSASKE 121 LPGPQTEGKA EFAEYASVDR NKKCRQSVNV ESILGNSCDP EEEAPPPVPV KLLDENENLQ 181 EKEGGEAEES ATDTTSETNK RFSSLSYKSR EEDPTLTEEE I SAMYSSVNK PGQLVNKSGQ 241 SLTVPESTYT SIQGDPQRSP SSCNDLYATV KDFEKTPNST LPPAGRPSEE PEPDYEAIQT 301 LNREEEKATL GTNGHHGLVP KENDYESISD LQQGRDITRL (SEQ ID NO: 4).

[0081] In some embodiments, the truncated PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the truncated PAG1 intracellular domain consists essentially of SEQ ID

NO: 4.

[0082] In some embodiments, the truncated PAG1 intracellular domain comprises amino acid residues 112-432 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 112-432 SEQ ID NO: 1. In some embodiments, the PAG1 intracellular domain comprises a sequence of:

1 ASDLLDSQDS TGKPKCHQSR ELPRI PPESA VDTMLTARSV DGDQGLGMEG PYEVLKDSSS

61 QENMVEDCLY ETVKEIKEVA AAAHLEKGHS GKAKSTSASK ELPGPQTEGK AEFAEYASVD

121 RNKKCRQSVN VESILGNSCD PEEEAPPPVP VKLLDENENL QEKEGGEAEE SATDTTSETN

181 KRFSSLSYKS REEDPTLTEE EISAMYSSVN KPGQLVNKSG QSLTVPESTY TSIQGDPQRS

241 PSSCNDLYAT VKDFEKTPNS TLPPAGRPSE EPEPDYEAIQ TLNREEEKAT LGTNGHHGLV

301 PKENDYESIS DLQQGRDITR L (SEQ ID NO: 5).

[0083] In some embodiments, the truncated PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 5. In some embodiments, the truncated PAG1 intracellular domain consists essentially of SEQ ID NO: 5.

[0084] In some embodiments, the truncated PAG1 intracellular domain comprises amino acid residues 41-326 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 41-326 SEQ ID NO: 1. In some embodiments, the PAG1 intracellular domain comprises a sequence of:

1 DREKKPRQHS GDHENLMNVP SDKEMFSRSV T SLAT DAP AS SEQNGALTNG DILSEDSTLT 61 CMQHYEEVQT SASDLLDSQD STGKPKCHQS RELPRI PPES AVDTMLTARS VDGDQGLGME 121 GPYEVLKDSS SQENMVEDCL YETVKEIKEV AAAAHLEKGH SGKAKSTSAS KELPGPQTEG 181 KAEFAEYASV DRNKKCRQSV NVESILGNSC DPEEEAPPPV PVKLLDENEN LQEKEGGEAE 241 ESATDTTSET NKRFSSLSYK SREEDPTLTE EEI SAMYSSV NKPGQL (SEQ ID NO: 6). [0085] In some embodiments, the truncated PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 6. In some embodiments, the truncated PAG1 intracellular domain consists essentially of SEQ ID

NO: 6.

[0086] In some embodiments, the truncated PAG1 intracellular domain comprises amino acid residues 41-371 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 41-371 SEQ ID NO: 1. In some embodiments, the PAG1 intracellular domain comprises a sequence of:

1 DREKKPRQHS GDHENLMNVP SDKEMFSRSV T SLAT DAP AS SEQNGALTNG DILSEDSTLT 61 CMQHYEEVQT SASDLLDSQD STGKPKCHQS RELPRI PPES AVDTMLTARS VDGDQGLGME 121 GPYEVLKDSS SQENMVEDCL YETVKEIKEV AAAAHLEKGH SGKAKSTSAS KELPGPQTEG 181 KAEFAEYASV DRNKKCRQSV NVESILGNSC DPEEEAPPPV PVKLLDENEN LQEKEGGEAE 241 ESATDTTSET NKRFSSLSYK SREEDPTLTE EEI SAMYSSV NKPGQLVNKS GQSLTVPEST 301 YTSIQGDPQR SPSSCNDLYA TVKDFEKTPN S (SEQ ID NO: 7).

[0087] In some embodiments, the truncated PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 7. In some embodiments, the truncated PAG1 intracellular domain consists essentially of SEQ ID

NO: 7.

[0088] In some embodiments, the truncated PAG1 intracellular domain comprises amino acid residues 41-400 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 41-400 SEQ ID NO: 1. In some embodiments, the PAG1 intracellular domain comprises a sequence of:

1 DREKKPRQHS GDHENLMNVP SDKEMFSRSV T SLAT DAP AS SEQNGALTNG DILSEDSTLT 61 CMQHYEEVQT SASDLLDSQD STGKPKCHQS RELPRI PPES AVDTMLTARS VDGDQGLGME 121 GPYEVLKDSS SQENMVEDCL YETVKEIKEV AAAAHLEKGH SGKAKSTSAS KELPGPQTEG 181 KAEFAEYASV DRNKKCRQSV NVESILGNSC DPEEEAPPPV PVKLLDENEN LQEKEGGEAE 241 ESATDTTSET NKRFSSLSYK SREEDPTLTE EEI SAMYSSV NKPGQLVNKS GQSLTVPEST 301 YTSIQGDPQR SPSSCNDLYA TVKDFEKTPN STLPPAGRPS EEPEPDYEAI QTLN REEEKA

(SEQ ID NO: 8).

[0089] In some embodiments, the truncated PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the truncated PAG1 intracellular domain consists essentially of SEQ ID NO: 8.

[0090] In some embodiments, the truncated PAG1 intracellular domain comprises amino acid residues 112-326 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 112-326 SEQ ID NO: 1. In some embodiments, the PAG1 intracellular domain comprises a sequence of:

1 ASDLLDSQDS TGKPKCHQSR ELPRI PPESA VDTMLTARSV DGDQGLGMEG PYEVLKDSSS 61 QENMVEDCLY ETVKEIKEVA AAAHLEKGHS GKAKSTSASK ELPGPQTEGK AEFAEYASVD 121 RNKKCRQSVN VESILGNSCD PEEEAPPPVP VKLLDENENL QEKEGGEAEE SATDTTSETN 181 KRFSSLSYKS REEDPTLTEE EISAMYSSVN KPGQL (SEQ ID NO: 9).

[0091] In some embodiments, the truncated PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 9. In some embodiments, the truncated PAG1 intracellular domain consists essentially of SEQ ID NO: 9.

[0092] In some embodiments, the truncated PAG1 intracellular domain comprises amino acid residues 217-326 of SEQ ID NO: 1, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 217-326 SEQ ID NO: 1. In some embodiments, the PAG1 intracellular domain comprises a sequence of:

1 QTEGKAEFAE YASVDRNKKC RQSVNVESIL GNSCDPEEEA PPPVPVKLLD ENENLQEKEG 61 GEAEESATDT TSETNKRFSS LSYKSREEDP TLTEEEI SAM YSSVNKPGQL (SEQ ID NO:

10).

[0093] In some embodiments, the truncated PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 10. In some embodiments, the truncated PAG1 intracellular domain consists essentially of SEQ ID NO: 10.

[0094] In some embodiments, the PAG1 intracellular domain comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to:

(SEQ ID NO: 90). [0095] In some embodiments, the PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 90. In some embodiments, the PAG1 intracellular domain consists essentially of SEQ ID NO: 90.

[0096] In some embodiments, the PAG1 intracellular domain comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to:

1 DREKKPRQHS GDHENLMNVP SDKEMFSRSV TSLATDAPAS SEQNGALTNG DILSEDSTLT 61 CMQHYEEVQT SASDLLDSQD STGKPKCHQS RELPRIPPES AVDTMLTARS VDGDQGLGME 121 GPYEVLKDSS SQENMVEDCL YETVKEIKEV AAAAHLEKGH SGKAKSTSAS KELPGPQTEG 181 KAEFAEYASV DRNKKCRQSV NVESILGNSC DPEEEAPPPV PVKLLDENEN LQEKEGGEAE 241 ESATDTTSET NKRFSSLSYK SREEDPTLTE EEISAMYSSV NKPGQLVNKS GQSLTVPEST 301 YTSIQGDPQR SPSSCNDLYA TVKDFEKTPN S (SEQ ID NO: 91).

[0097] In some embodiments, the PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 91. In some embodiments, the PAG1 intracellular domain consists essentially of SEQ ID NO: 91.

[0098] In some embodiments, the PAG1 intracellular domain comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to:

1 DREKKPRQHS GDHENLMNVP SDKEMFSRSV TSLATDAPAS SEQNGALTNG DILSEDSTLT 61 CMQHYEEVQT SASDLLDSQD STGKPKCHQS RELPRIPPES AVDTMLTARS VDGDQGLGME 121 GPYEVLKDSS SQENMVEDCL YETVKEIKEV AAAAHLEKGH SGKAKSTSAS KELPGPQTEG 181 KAEFAEYASV DRNKKCRQSV NVESILGNSC DPEEEAPPPV PVKLLDENEN LQEKEGGEAE 241 ESATDTTSET NKRFSSLSYK SREEDPTLTE EEISAMYSSV NKPGQL (SEQ ID NO:

92).

[0099] In some embodiments, the PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 92. In some embodiments, the PAG1 intracellular domain consists essentially of SEQ ID NO: 92.

[0100] In some embodiments, the PAG1 intracellular domain comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to:

1 DREKKPRLSE DSTLTCMQHY EEVQTSASDL LDSQDSTGKP KCHQSRELPR IPPESAVDTM 61 LTARSVDGDQ GLGMEGPYEV LKDSSSQENM VEDCLYETVK EIKEVAAAAH LEKGHSGKAK 121 STSASKELPG PQTEGKAEFA EYASVDRNKK CRQSVNVESI LGNSCDPEEE APPPVPVKLL 181 DENENLQEKE GGEAEESATD TTSETNKRFS SLSYKSREED PTLTEEEISA MYSSVNKPGQ 241 LVNKSGQSLT VPESTYTSIQ GDPQRSPSSC NDLYATVKDF EKTPNSTLPP AGRPSEEPEP 301 DYEAIQTLNR EEEKATLGTN GHHGLVPKEN DYESISDLQQ GRDITRL (SEQ ID NO:

93).

[0101] In some embodiments, the PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 93. In some embodiments, the PAG1 intracellular domain consists essentially of SEQ ID NO: 93. [0102] In some embodiments, the PAG1 intracellular domain comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to:

1 DREKKPRASD LLDSQDSTGK PKCHQSRELP RIPPESAVDT MLTARSVDGD QGLGMEGPYE 61 VLKDSSSQEN MVEDCLYETV KEIKEVAAAA HLEKGHSGKA KSTSASKELP GPQTEGKAEF 121 AEYASVDRNK KCRQSVNVES ILGNSCDPEE EAPPPVPVKL LDENENLQEK EGGEAEESAT 181 DTTSETNKRF SSLSYKSREE DPTLTEEEIS AMYSSVNKPG QLVNKSGQSL TVPESTYTSI

241 QGDPQRSPSS CNDLYATVKD FEKTPNSTLP PAGRPSEEPE PDYEAIQTLN REEEKATLGT

301 NGHHGLVPKE NDYESISDLQ QGRDITRL (SEQ ID NO: 94).

[0103] In some embodiments, the PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 94. In some embodiments, the PAG1 intracellular domain consists essentially of SEQ ID NO: 94.

[0104] In some embodiments, the PAG1 intracellular domain comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to:

1 DREKKPRASD LLDSQDSTGK PKCHQSRELP RIPPESAVDT MLTARSVDGD QGLGMEGPYE 61 VLKDSSSQEN MVEDCLYETV KEIKEVAAAA HLEKGHSGKA KSTSASKELP GPQTEGKAEF 121 AEYASVDRNK KCRQSVNVES ILGNSCDPEE EAPPPVPVKL LDENENLQEK EGGEAEESAT

181 DTTSETNKRF SSLSYKSREE DPTLTEEEIS AMYSSVNKPG QL (SEQ ID NO: 95).

[0105] In some embodiments, the PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 95. In some embodiments, the PAG1 intracellular domain consists essentially of SEQ ID NO: 95.

[0106] In some embodiments, the PAG1 intracellular domain comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to:

1 DREKKPRQTE GKAEFAEYAS VDRNKKCRQS VNVESILGNS CDPEEEAPPP VPVKLLDENE 61 NLQEKEGGEA EESATDTTSE TNKRFSSLSY KSREEDPTLT EEEISAMYSS VNKPGQL

(SEQ ID NO: 96).

[0107] In some embodiments, the PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 96. In some embodiments, the PAG1 intracellular domain consists essentially of SEQ ID NO: 96.

[0108] In some embodiments, the PAG1 intracellular domain comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to:

1 DREKKPRASD LLDSQDSTGK PKCHQSRELP RIPPESAVDT MLTARSVDGD QGLGMEGPYE 61 VLKDSSSQEN MVEDCLYETV KEIKEV (SEQ ID NO: 97).

[0109] In some embodiments, the PAG1 intracellular domain consists essentially of a sequence having at least 95% identity to SEQ ID NO: 97. In some embodiments, the PAG1 intracellular domain consists essentially of SEQ ID NO: 97. [0110] In some embodiments, the truncated PAG1 intracellular domain comprises or consists essentially of a sequence selected from the group consisting of amino acid residues 41-432 of SEQ ID NO: 1, residues 93-432 of SEQ ID NO: 1, residues 112-432 of SEQ ID NO: 1, residues 41-326 of SEQ ID NO: 1, residues 41-371 of SEQ ID NO: 1, residues 41-400 of SEQ ID NO: 1, residues 112-326 of SEQ ID NO: 1, and residues 217-326 of SEQ ID NO: 1.

Transmembrane domain

[0111] The disclosure provides an inhibitory receptor comprising a polypeptide comprising a transmembrane domain. In some embodiments, the transmembrane domain is isolated or derived from PAG1, LILRB1 or CD8. However, any suitable transmembrane may be used with the inhibitory receptors described herein.

[0112] A “transmembrane domain”, as used herein, refers to a domain of a protein that spans a membrane of the cell. Transmembrane domains typically consist predominantly of non-polar amino acids, and may traverse the lipid bilayer once or several times. Transmembrane domains usually comprise alpha helices, a configuration which maximizes internal hydrogen bonding.

[0113] Transmembrane domains isolated or derived from any source are envisaged as within the scope of the inhibitory receptors of the disclosure.

[0114] The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD 8 a, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0115] In some embodiments, the transmembrane domain can be attached directly to the antigen binding domain or ligand binding domain.

[0116] In some embodiments, the transmembrane domain can be attached to the antigen binding domain or ligand binding domain via a hinge, e.g., a hinge from a human protein. For example, in some embodiments, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, a CD8a hinge or a PAG1 hinge. [0117] In some embodiments, the inhibitory receptor comprises polypeptide comprising a PAG1 transmembrane domain or a functional variant thereof. In some embodiments, the PAG1 transmembrane domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to LWGSLAAVAIFFVITFLIFL (SEQ ID NO: 11). In some embodiments, the PAG1 transmembrane comprises SEQ ID NO: 11. In some embodiments, the PAG1 transmembrane domain consists essentially of SEQ ID NO: 11

[0118] In some embodiments, the inhibitory receptor comprises a polypeptide comprising a LILRB 1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB 1 transmembrane comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to VVIGILVAVILLLLLLLLLFLIL (SEQ ID NO: 12). In some embodiments, the LILRB 1 transmembrane domain comprises SEQ ID NO: 12. In some embodiments, the LILRB 1 transmembrane domain consists essentially SEQ ID NO: 12. In some embodiments, the LILRB 1 transmembrane comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to V VIGIL V A VILLLLLLLLLFLI (SEQ ID NO: 88). In some embodiments, the LILRBl transmembrane domain comprises SEQ ID NO: 88. In some embodiments, the LILRBl transmembrane domain consists essentially SEQ ID NO: 88

[0119] In some embodiments, the inhibitory receptor comprises a polypeptide comprising a CD8a transmembrane domain or a functional variant thereof. In some embodiments, the CD8a transmembrane domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to IYIWAPLAGTCGVLLLSLVITLY CNH (SEQ ID NO: 13). In some embodiments, the CD8a transmembrane domain comprises a sequence identical to SEQ ID NO: 13. In some embodiments, the CD8a transmembrane domain consists essentially of SEQ ID NO: 13.

Hinge Domain

[0120] The disclosure provides an inhibitory receptor comprising a polypeptide comprising a hinge domain. In some embodiments, the hinge domain is isolated or derived from PAG1, LILRBl, CD8 or a combination thereof. However, any suitable hinge domain may be used with the inhibitory receptors of the disclosure. [0121] As used herein, a “hinge domain” refers to an extracellular domain between the ligand binding domain and the transmembrane domain.

[0122] In some embodiments, the polypeptide comprises a PAG1 hinge domain or a functional variant thereof. In some embodiments, the PAG1 hinge domain or functional variant thereof comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to GPAGSLLGSGQMQIT (SEQ ID NO: 14). In some embodiments, the PAG1 hinge or functional variant thereof comprises SEQ ID NO: 14. In some embodiments, the PAG1 hinge domain consists essentially of SEQ ID NO: 14.

[0123] In some embodiments, the polypeptide comprises a LILRB1 hinge domain or a functional variant thereof. The LILRB1 protein has four immunoglobulin (Ig) like domains termed Dl, D2, D3 and D4. In some embodiments, the LILRB1 hinge domain comprises an LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1 D3D4 domain comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to YGSQSSKPYLLTHPSDPLEL (SEQ ID NO: 15). In some embodiments, the LILRB1 D3D4 domain comprises SEQ ID NO: 15. In some embodiments, the LILRB1 D3D4 domain consists essentially of SEQ ID NO: 15. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or is identical to YGSQSSKPYLLTHPSDPLEL VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQ SGLGRHLG (SEQ ID NO: 16). In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or is identical to SEQ ID NO: 16. In some embodiments, the LILRB1 hinge domain comprises SEQ ID NO: 16. In some embodiments, the LILRB1 hinge domain consists essentially of SEQ ID NO: 16. In some embodiments, the LILRB1 hinge domain comprises or functional variant thereof comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLG (SEQ ID NO: 17). In some embodiments, the LILRB1 hinge domain comprises SEQ ID NO: 17. In some embodiments, the LILRB1 hinge domain consists essentially of SEQ ID NO: 17. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or is identical to YGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQ SGLGRHLGV (SEQ ID NO: 87). In some embodiments, the LILRB1 hinge domain comprises SEQ ID NO: 87. In some embodiments, the LILRB1 hinge domain consists essentially of SEQ ID NO: 87. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or is identical to VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 178). In some embodiments, the LILRB1 hinge domain comprises SEQ ID NO: 178. In some embodiments, the LILRB1 hinge domain consists essentially of SEQ ID NO: 178. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or is identical to

AGSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSGGSGNSSGSGGSPVPSTPPTPSPS TPPTPSPS (SEQ ID NO: 179). In some embodiments, the LILRB1 hinge domain comprises SEQ ID NO: 179. In some embodiments, the LILRB1 hinge domain consists essentially of SEQ ID NO: 179. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or is identical to AGSGGSGGSGGSPVPSTPPTNSSSTPPTPSPSPVPSTPPTNSSSTPPTPSPSPVPST PPTNSSSTPPTPSPSASV (SEQ ID NO: 180). In some embodiments, the LILRB1 hinge domain comprises SEQ ID NO: 180. In some embodiments, the LILRB1 hinge domain consists essentially of SEQ ID NO: 180. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or is identical to VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHVVSGPSGGPSSPT TGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 181). In some embodiments, the LILRB1 hinge domain comprises SEQ ID NO: 181. In some embodiments, the LILRB1 hinge domain consists essentially of SEQ ID NO: 181. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or is identical to TTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 182). In some embodiments, the LILRB1 hinge domain comprises SEQ ID NO: 182. In some embodiments, the LILRB1 hinge domain consists essentially of SEQ ID NO: 182. [0124] In embodiments, the LILRB1 hinge domain or functional fragment or 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: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 87. In some embodiments, the LILRB1 hinge domain comprises a sequence at least 95% identical to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 87. In some embodiments, the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 87. In some embodiments, the LILRB1 hinge domain consists essentially of a sequence identical to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 87.

[0125] In some embodiments, the hinge domain is isolated or derived from CD8a. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or is identical to a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18). In some embodiments, the CD8a hinge domain comprises SEQ ID NO: 18. In some embodiments, the CD8a hinge domain consists essentially of SEQ ID NO: 18. In some embodiments, the CD8a hinge 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: accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagccc ctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagg gggctggacttcgcctgtgat (SEQ ID NO: 19).

[0126] In some embodiments, the hinge domain is isolated or derived from CD28. In some embodiments, the CD28 hinge 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

CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 20). In some embodiments, the CD28 hinge domain comprises or consists essentially of SEQ ID NO: 20. In some embodiments, the CD28 hinge 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: tgtaccattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccat tatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc

(SEQ ID NO: 21). [0127] In some embodiments, the hinge domain comprises both PAG1 and LILRB1 sequences. In some embodiments, the hinge 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: YGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQ SGLGRHLGVGPAGSLLGSGQMQIT SEQ ID NO: 86.

[0128] In some embodiments, the hinge comprises SEQ ID NO: 86. In some embodiments, the hinge consists essentially of SEQ ID NO: 86.

Polypeptides

[0129] The disclosure provides an inhibitory receptor comprising at least a first polypeptide, the first polypeptide comprising one or more of: (a) a PAG1 intracellular domain or a functional fragment or variant thereof, (b) a transmembrane domain, and (c) an extracellular ligand binding domain. In some embodiments, the polypeptide further comprises a hinge domain.

[0130] In some embodiments of the inhibitory receptors provided herein, the polypeptide comprises a PAG1 intracellular domain or functional fragment thereof and a transmembrane domain. In some embodiments, the polypeptide comprises an extracellular ligand binding domain or a portion thereof, a transmembrane domain, and a PAG1 intracellular domain or functional fragment or variant thereof. In some embodiments, the polypeptide comprises an extracellular ligand binding domain or a portion thereof, a hinge domain, a transmembrane domain, and a PAG1 intracellular domain or functional fragment thereof.

[0131] In some embodiments, the polypeptide comprises an extracellular ligand binding domain or portion thereof, a PAG1 hinge domain, a PAG1 transmembrane domain, and a PAG1 intracellular domain. In some embodiments, the polypeptide comprises a full length PAG1 protein, e.g., a PAG1 protein with an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 1, which is fused at the N-terminus to an extracellular ligand binding domain. In some embodiments, the polypeptide comprises or consists essentially of SEQ ID NO: 1 fused to an extracellular ligand binding domain at the N- terminus of SEQ ID NO: 1.

[0132] In some embodiments, the polypeptide comprises a PAG1 transmembrane domain and a PAG1 intracellular domain. For example, the polypeptide can comprise an extracellular ligand binding domain or a portion thereof, a PAG1 transmembrane domain, and a PAG1 intracellular domain. As a further non-limiting example, the polypeptide can comprise n extracellular ligand binding domain or a portion thereof, a hinge isolated or derived from any of PAG1, LILRB 1 or CD8a, a PAG1 transmembrane domain, and a PAG1 intracellular domain. In some embodiments, the PAG1 transmembrane domain and PAG1 intracellular domain comprise a sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to:

1 LWGSLAAVAI FFVITFLI FL CSSCDREKKP RQHSGDHENL MNVPSDKEMF SRSVTSLATD

61 APASSEQNGA LTNGDILSED STLTCMQHYE EVQTSASDLL DSQDSTGKPK CHQSRELPRI

121 PPESAVDTML TARSVDGDQG LGMEGPYEVL KDSSSQENMV EDCLYETVKE IKEVAAAAHL

181 EKGHSGKAKS TSASKELPGP QTEGKAEFAE YASVDRNKKC RQSVNVESIL GNSCDPEEEA

241 PPPVPVKLLD ENENLQEKEG GEAEESATDT TSETNKRFSS LSYKSREEDP TLTEEEI SAM

301 YSSVNKPGQL VNKSGQSLTV PESTYTSIQG DPQRSPSSCN DLYATVKDFE KTPN STLPPA

361 GRPSEEPEPD YEAIQTLNRE EEKATLGTNG HHGLVPKEND YESI SDLQQG RDITRL (SEQ

ID NO: 47).

[0133] In some embodiments, the PAG1 transmembrane domain and PAG1 intracellular domain comprise SEQ ID NO: 47. In some embodiments, the PAG1 intracellular domain and PAG1 transmembrane domain consist essentially of SEQ ID NO: 47.

[0134] In some embodiments, the polypeptide comprises a LILRB 1 hinge domain and a LILRB 1 transmembrane domain, or functional variants thereof. Lor example, the polypeptide comprises an extracellular ligand binding domain or a portion thereof, a LILRB 1 hinge domain, a LILRB 1 transmembrane domain, and a PAG1 intracellular domain or functional fragment or variant thereof. In some embodiments, the LILRB 1 hinge domain and a LILRB 1 transmembrane domain comprise a sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to:

YGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQ SGLGRHLGVVIGILVAVILLLLLLLLLLLI (SEQ ID NO: 48).

[0135] In some embodiments, the LILR1B hinge domain and LILRB 1 transmembrane domain comprise SEQ ID NO: 48. In some embodiments, the LILRB 1 hinge domain and LILRBl transmembrane domain consist essentially of SEQ ID NO: 48. In some embodiments, the LILR1B hinge domain and LILRBl transmembrane domain comprise a sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to: YGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQ SGLGRHLGVVIGILVAVILLLLLLLLLFLIL (SEQ ID NO: 89).

[0136] In some embodiments, the LILRB1 hinge domain and LILRB1 transmembrane domain consist essentially of SEQ ID NO: 89. In some embodiments, the polypeptide comprises SEQ ID NO: 48 and a truncated PAG1 intracellular domain or functional variant thereof, for example SEQ ID NO: 2. In some embodiments, the polypeptide comprises a LILRB 1 hinge domain, a LILRB 1 transmembrane domain, and a truncated PAG1 intracellular domain selected from the group consisting of amino acid residues 41-432 of SEQ ID NO: 1, residues 93-432 of SEQ ID NO: 1, residues 112-432 of SEQ ID NO: 1, residues 41-326 of SEQ ID NO: 1, residues 41-371 of SEQ ID NO: 1, residues 41-400 of SEQ ID NO: 1, residues 112-326 of SEQ ID NO: 1, and residues 217-326 of SEQ ID NO: 1.

[0137] In some embodiments, the polypeptide comprises a LILRB 1 hinge domain, a PAG1 transmembrane domain, and a PAG1 intracellular domain. For example, the polypeptide comprises an extracellular ligand binding domain or a portion thereof, a LILRB 1 hinge domain, a PAG1 transmembrane domain, and a PAG1 intracellular domain or functional fragment or variant thereof.

[0138] In some embodiments, the polypeptide comprises a hinge domain comprising both LILRB 1 and PAG1 sequences, a PAG1 transmembrane domain, and a PAG1 intracellular domain. For example, the polypeptide comprises an extracellular ligand binding domain or a portion thereof, a hinge domain comprising both LILRB 1 and PAG1 sequences, a PAG1 transmembrane domain, and a PAG1 intracellular domain. In some embodiments, the PAG1 intracellular domain comprises SEQ ID NO: 2. In some embodiments, the PAG1 intracellular domain is a truncated PAG1 intracellular domain as described herein.

[0139] In some embodiments, the polypeptide comprises a CD8a hinge domain and a CD8a transmembrane domain, or functional variants thereof. For example, the polypeptide comprises an extracellular ligand binding domain or a portion thereof, a CD8a hinge domain, a CD8a transmembrane domain, and a PAG1 intracellular domain or functional fragment or variant thereof. In some embodiments, the CD8a hinge domain and CD8a transmembrane domain comprise a sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to:

[0140] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLY CNH (SEQ ID NO: 49). In some embodiments, the CD8a hinge domain and CD8a transmembrane domain comprise SEQ ID NO: 49. In some embodiments, the CD8a hinge domain and CD8a transmembrane domain consist essentially of SEQ ID NO: 49. In some embodiments, the polypeptide comprises a CD8a hinge domain and CD8a transmembrane domain, for example SEQ ID NO: 49, and a truncated PAG1 intracellular domain or functional variant thereof, for example SEQ ID NO: 2. In some embodiments, the polypeptide comprises a CD8a hinge domain, a CD 8 a transmembrane domain, and a truncated PAG1 intracellular domain is selected from the group consisting of amino acid residues 41-432 of SEQ ID NO: 1, residues 93-432 of SEQ ID NO: 1, residues 112-432 of SEQ ID NO: 1, residues 41-326 of SEQ ID NO: 1, residues 41-371 of SEQ ID NO: 1, residues 41-400 of SEQ ID NO: 1, residues 112-326 of SEQ ID NO: 1, and residues 217-326 of SEQ ID NO: 1.

[0141] In some embodiments, the polypeptide comprises a CD8a hinge domain, and a PAG1 transmembrane domain. For example, the polypeptide comprises an extracellular ligand binding domain or a portion thereof, a CD8a hinge domain, a PAG1 transmembrane domain, and a PAG1 intracellular domain or functional fragment or variant thereof. In some embodiments, the CD 8a hinge domain comprises a sequence having at least 95% identity to SEQ ID NO: 18 and the PAG1 transmembrane domain comprises a sequence having at least 95% identity to SEQ ID NO: 11. In some embodiments, the CD8a hinge domain comprises SEQ ID NO: 18 and the PAG1 transmembrane domain comprises SEQ ID NO: 11. In some embodiments, the CD8a hinge domain consists essentially of SEQ ID NO: 18 and the PAG1 transmembrane domain consists essentially of SEQ ID NO: 11. In some embodiments, the PAG1 intracellular domain comprises SEQ ID NO: 2. In some embodiments, the PAG1 intracellular domain is a truncated PAT1 intracellular domain selected from the group consisting of amino acid residues 41-432 of SEQ ID NO: 1, residues 93-432 of SEQ ID NO: 1, residues 112-432 of SEQ ID NO: 1, residues 41-326 of SEQ ID NO: 1, residues 41-371 of SEQ ID NO: 1, residues 41-400 of SEQ ID NO: 1, residues 112-326 of SEQ ID NO: 1, or residues 217-326 of SEQ ID NO: 1.

[0142] In some embodiments, the inhibitory receptor is a single chain receptor. For example, the extracellular ligand binding domain, optionally the hinge domain, the transmembrane domain and PAG1 intracellular domain are encoded by a single polypeptide.

[0143] In some embodiments, the inhibitory receptor is a multi-chain receptor. Any suitable multi-chain receptor architectures are envisaged as within the scope of the instant disclosure and include, inter alia, TCR architectures and inhibitory receptors wherein at least the extracellular ligand binding domain is encoded by more than one polypeptide. In some embodiments, the inhibitory receptor comprises a first and a second polypeptide.

Extracellular Domains

[0144] The disclosure provides an inhibitory receptor comprising a polypeptide comprising an extracellular ligand binding domain, or a portion thereof. In some embodiments, the receptor comprises at least a first polypeptide comprising the extracellular ligand binding domain or a portion thereof, and the PAG1 intracellular domain or a functional fragment or variant thereof. In some embodiments, the receptor comprises at least a first polypeptide comprising the extracellular ligand binding domain or a portion thereof, optionally a hinge domain, a transmembrane domain and the PAG1 intracellular domain or a functional fragment or variant thereof.

[0145] In some embodiments, the extracellular ligand binding domain comprises an antigen binding domain. Suitable antigen binding domains include, but are not limited to, antigen binding domains from antibodies, antibody fragments, scFv, antigen binding domains derived from T cell receptors, and the like. All forms of antigen binding domains known in the art are envisaged as within the scope of the disclosure.

[0146] In some embodiments, the extracellular ligand binding domain comprises a nb- only domain. The term “l domain”, “nb-only domain”, “b chain variable domain” or “single variable domain TCR (svd-TCR)” refers to an antigen binding domain that consists essentially of a single T Cell Receptor (TCR) beta variable domain that specifically binds to an antigen in the absence of a second TCR variable domain nb- only domains are described in U.S. Patent Pub. No. US 2019/0255186 Al, the contents of which are incorporated by reference herein in their entirety.

[0147] An “extracellular domain”, as used herein, refers to the extracellular portion of a protein. The “extracellular domain” can also comprise a fusion domain, for example of fusions between additional domains capable of binding to and targeting a specific antigen and the endogenous extracellular domain of PAG 1.

[0148] 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. [0149] 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 VF or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.

[0150] 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.

[0151] “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.

[0152] Unless specified, as used herein a scFv may have the VF and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VF-linker-VH or may comprise VH-linker-VF. [0153] 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 (“l”) light chains refer to the two major antibody light chain isotypes.

[0154] 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.

[0155] In some embodiments, for example those embodiments wherein the receptor comprises a first and a second polypeptide, the antigen binding domain is isolated or derived from a T cell receptor (TCR) extracellular domain or an antibody.

[0156] In some embodiments, the inhibitory receptor comprises an antigen binding domain. The antigen-binding domain or domains of the inhibitory receptor may be provided on the same or a different polypeptide as the intracellular PAG1 domain. [0157] In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv). In some embodiments, a single polypeptide comprises the scFv and the PAG1 intracellular domain.

[0158] In some embodiments, the inhibitory receptor comprises a second polypeptide. The disclosure provides receptors having two polypeptides each having a part of a ligand-binding domain (e.g. cognates of a heterodimeric LDB, such as a TCRa/b- or Fab-based LBD). The disclosure further provides receptors having two polypeptides, each having a part of a ligand-binding domain (e.g. cognates of a heterodimeric LDB, such as a TCRa/b- or Fab-based LBD) and one part of the ligand binding domain is fused to a hinge or transmembrane domain, while the other part of the ligand binding domain has no intracellular domain. Further variations include receptors where each polypeptide has a hinge domain, and where each polypeptide has a hinge and transmembrane domain. In some embodiments, the hinge domain is absent. In other embodiments, the hinge domain is a membrane proximal extracellular region (MPER), such as the LILRBl D3D4 domain.

[0159] In any of the embodiments disclosed herein, the domains may be fused adjacent to one another with linkers between them. Suitable linkers will be known to persons of ordinary skill in the art and include, inter alia, polyGlycine and polySerine linkers, and Glycine-Serine linkers. In some embodiments, the first polypeptide comprises a first chain of an antibody and the second polypeptide comprise a second chain of said antibody.

[0160] In some embodiments, the receptor comprises a Fab fragment of an antibody. In embodiments, a first polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody and a PAG1 intracellular domain, and a second polypeptide comprises an antigen-binding fragment of the light chain of the antibody. In some embodiments, the first polypeptide comprises an antigen-binding fragment of the light chain of the antibody and the PAG1 intracellular domain, and the second polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody.

[0161] In some embodiments, the inhibitory receptor comprises an extracellular fragment of a T cell receptor (TCR). In some embodiments, a first polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR and the PAG1 intracellular domain, and a second polypeptide comprises an antigen-binding fragment of the beta chain of the TCR. In some embodiments, a first polypeptide comprises an antigen-binding fragment of the beta chain of the TCR and the PAG1 intracellular domain, and the second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR.

Illustrative Antigen-Binding Domains

[0162] Various single variable domains known in the art or disclosed herein are suitable for use in embodiments of the PAG 1 -based inhibitory receptors and/or activatory receptors described herein.

[0163] PAGl-based inhibitory receptors comprise, inter alia, ligand binding domains for inhibitor ligands. Exemplary inhibitor ligands that are recognized by the ligand binding domain of the PAGl-based inhibitor receptors comprise alleles lost due to loss of heterozygosity, minor histocompatibility antigens, and loss of Y antigens as described herein.

[0164] Various single variable domains known in the art or disclosed herein are suitable for use in embodiments. Such scFv’s include, for example and without limitation the following mouse and humanized scFv antibodies that bind HFA-A*02 in a peptide- independent way (complementarity determining regions underlined):

C-001765

MMTOTPFSFPVSFGDOASISCRSSOSIVHSNGNTYFEWYFOKPGOSPK FFIYKVSNRFSGVPDRFSGSGSGTDFTFKISRVEAEDFGVYYCFOGSHVPRTS GGGTKFEIKGGGGSGGGGSGGGGSGGOVOFOOSGPEFVKPGASVRISCKASG YTFTSYHIHWVKORPGOGFEWIGWIYPGNVNTEYNEKFKGKATFTADKSSST AYMHFSSFTSEDSAVYFCAREEITYAMDYWGOGTSVTVSSYG (SEQ ID NO: 35); or

DVLMTOTPLSLPVSLGDOASISCRSSOSIVHSNGNTYLEWYLOKPGOS PKLLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYY CFOGSHVPR

TSGGGTKFEIKGGGGSGGGGSGGGGSGGQVQFQQSGPEFVKPGASVRISCKA SGYTFTSYHIHWVKORPGOGLEWIGWIYPGNVNTEYNEKFKGKATLTADKS S STAYMHLSSLTSEDSAVYFCAREEITYAMDYWGQGTSVTVSS (SEQ ID NO: 125, the corresponding polynucleotide sequence is provided as SEQ ID NO: 241)

C-002159

QLV Q SGAEVKKPGS S VKV S CKASGYTFTSYHIHWVRQ APGQGLEWM GWIYPGNVNTEYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCAREE ITYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGEIVLTQSPGTLSLSPGE RATLSCRSSOSIVHSNGNTYLEWYOOKPGOAPRLLIYKVSNRFSGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCFOGSHVPRTFGGGTKVEIK (SEQ ID NO: 36) C-002160

OLVOSGAEVKKPGSSVKVSCKASGYTFTSYHIHWVRO APGQGLEWM GWIYPGNVNTEYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCAREE ITYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGDIVMTQTPLSLPVTPG EPASISCRSSOSIVHSNGNTYLEWYLOKPGOSPOLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCFOGSHVPRTFGGGTKVEIK (SEQ ID NO:

37)

C-002161

OLVESGGGLVKPGGSLRLSCAASGYTFTSYHIHWVROAPGKGLEWVG WIYPGNVNTEYNEKFKGRFTISRDDSKNTLYLOMNSLKTEDTAVYYCAREEI TYAMDYWGOGTTVTVSSGGGGSGGGGSGGGGSGGDIOMTOSPSSLSASVGD RVTITCRSSOSIVHSNGNTYLEWY OOKPGKAPKLLIYKV SNRFSGVPSRFSGS GSGTDFTLTISSLOPEDFATYYCFOGSHVPRTFGGGTKVEIK (SEQ ID NO: 38) C-002162

QLV Q SGAEVKKPGS S VKV S CKASGYTFTSYHIHWVRQ APGQGLEWIG WIYPGNVNTEYNEKFKGKATITADESTNTAYMELS SLRSEDTAVYY CAREEIT YAMDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGDIOMTOSPSTLSASVGD RVTITCRSSOSIVHSNGNTYLEWY OOKPGKAPKLLIYKV SNRFSGVPARFSGS GSGTEFTLTISSLOPDDFATYYCFOGSHVPRTFGOGTKVEVK (SEQ ID NO: 39) C-002163

QLV Q SGAEVKKPGS S VKV S CKASGYTFTSYHMHWVRQ APGQGLEWI GYIYPGNVNTEYNEKFKGKATLTADKSTNTAYMELSSLRSEDTAVYFCAREE ITYAMDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGDVOMTOSPSTLSASVG DRVTITCS S S 0 SIVHSNGNTYMEWY OOKPGKAPKLLIYKV SNRF SGVPDRF SG SGSGTEFTLTISSLOPDDFATYYCHOGSHVPRTFGOGTKVEVK (SEQ ID NO:

40)

C-002164

OVOFOOSGPEFVKPGASVKMSCKASGYTFTSYHIOWVKORPGOGFE WIGWIYPGDGSTOYNEKFKGKTTFTADKSSSTAYMFFSSFTSEDSAIYFCARE GTYYAMDYWGOGTSVTVSSGGGGSGGGGSGGGGSGGDVFMTOTPFSFPVS FGDOVSISCRSSOSIVHSNGNTYFEWYFOKPGOSPKFFIYKVSNRFSGVPDRFS GSGSGTDFTFKISRVEAEDFGVYY CF OGSHVPRTFGGGTKFEIK (SEQ ID NO:

41)

C-002165

OFOFOESGPGFVKPSETFSFTCTVSGYTFTSYHIOWIROPPGKGFEWIG WIYPGDGSTOYNEKFKGRATISVDTSKNOFSFNFDSV SAADTAIYY CAREGT YYAMDYWGKGSTVTVSSGGGGSGGGGSGGGGSGGDIOMTOSPSSFSASVGD RVTITCRSSOSIVHSNGNTYFEWY OOKPGKAPKFFIYKV SNRFSGVPSRFSGS GSGTDFTFTISSFOPEDIATYYCFOGSHVPRTFGPGTKVDIK (SEQ ID NO: 42) C-002166

EVOLVOSGAELKKPGSSVKVSCKASGYTFTSYHIOWVKOAPGOGLEW IGWIYPGDGSTOYNEKFKGKATLTVDKSTNTAYMELSSLRSEDTAVYYCARE GTYYAMDYWGOGTFVTVSSGGGGSGGGGSGGGGSGGDIOMTOSPSTFSASV GDRVTITCRSSOSIVHSNGNTYFEWY OOKPGKAPKFFIYKV SNRFSGVPSRFS GSGSGTDFTFTISSFOPDDFATYYCFOGSHVPRTFGOGTKVEVK (SEQ ID NO:

43)

C-002167

QV QFV Q SGAEVKKPGS S VKV S CKASGYTFTSYHIQWVRQ APGQGFE WMGWIYPGDGSTOYNEKFKGRVTITADKSTSTAYMEFSSFRSEDTAVYYCA REGTYYAMDYWGOGTTVTVSSGGGGSGGGGSGGGGSGGEIVFTOSPGTFSF SPGERATFSCRSSQSIVHSNGNTYFEWY QQKPGQAPRFFIYKVSNRFSGIPDRF SGSGSGTDFTFTISRFEPEDFAVYYCFOGSHVPRTFGGGTKVEIK (SEQ ID NO:

44)

C-002168

OVTLKOSGAEVKKPGSSVKV SCTASGYTFTSYHV SWVROAPGOGLE WLGRIYPGDGSTOYNEKFKGKVTITADKSMDTSFMELTSLTSEDTAVYYCAR EGTYYAMDFWGOGTFVTVSSGGGGSGGGGSGGGGSGGEIVFTOSPGTFSFSP GERATFSCRSSOSIVHSNGNTYFAWYOOKPGOAPRFFISKVSNRFSGVPDRFS GSGSGTDFTLTISRLEPEDFAVYYCOOGSHVPRTFGGGTKVEIK (SEQ ID NO: 45)

C-002169

QV QLVQSGAEVKKPGAS VKV SCKASGYTFTSYHMHWVRQAPGQRLE WMGWIYPGDGSTOYNEKFKGKVTITRDTSASTAYMELSSLRSEDTAVYYCA REGTYYAMDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGDIVMTOTPLSLP VTPGEP ASIS CRS S O SIVHSNGNTYLDWYLOKPGO SPOLLIYKV SNRF SGVPDR FSGSGSGTDFTLKISRVEAEDVGVYYCMOGSHVPRTFGGGTKVEIK (SEQ ID NO: 46)

[0165] In some embodiments, the scFv comprises the complementarity determined regions (CDRs) of any one of SEQ ID NOS: 22-33 (Table 1). In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOS: 22- 33. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOS: 22-33. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs of any one of SEQ ID NOS: 25-27 or 31-33, and the light chain of the antibody comprises the light chain CDRs of any one of SEQ ID NOS: 22-24 or 28- 30. In some embodiments, the heavy chain of the antibody comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOS: 35-46 or 125, and wherein the light chain of the antibody comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOS: 35-46 or 125. In some embodiments, the heavy chain comprises all of SEQ ID NOS: 25-27, and the light chain comprises all of SEQ ID NOS: 22-24. In some embodiments, the heavy chain comprises all of SEQ ID NOS: 31-33, and the light chain comprises all of SEQ ID NOS: 28-30. [0166] Table 1. Exemplary Complementarity Determining Regions (CDRs) of Antibodies that bind HLA-A*02

[0167] In some embodiments, the heavy chain of the antibody comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOS: 35-46 or 125, and wherein the light chain of the antibody comprises a sequence identical to the light chain portion of any one of SEQ ID NOS: 35-46 or 125.

[0168] In some embodiments, the 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: 35-46 or 125.

[0169] Additional antigen binding domains used with the activator and/or inhibitor receptors of the disclosure can include amino acid sequences selected from any one of SEQ ID NOs: 253, 260-264, or can be encoded by the nucleic acid sequences selected from any one of SEQ ID NOs: 254, 265-269.

[0170] Additional antigen binding domains used with the activator and/or inhibitor receptors of the disclosure are described in Table 2 below. In table 2, the name of the construct is described as scFv Inhibitor name [B]/scFv Activator name [A] .

[0171] Table 2. Additional antigen binding domain sequences

Signal Peptides

[0172] In some embodiments, the polypeptide comprises a signal peptide. For example, the polypeptide comprises a VK1 signal peptide. In some embodiments, the signal peptide is an N-terminal signal peptide. In some embodiments, the signal peptide comprises a sequence at least 95% identical to a sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 128). In some embodiments, the signal peptide comprises a sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 128). In some embodiments, the signal peptide is encoded by a sequence at least 95% identical to a sequence of

ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTC CGAGGTGCCAGATGT (SEQ ID NO: 129), or a sequence identical thereto.

Exemplary PAG1 Receptors

[0173] Sequences of exemplary, but non-limiting, PAG1 inhibitory receptors described herein are shown in Table 3 below.

[0174] Table 3. Exemplary PAG1 inhibitory receptors

[0175] In Table 3, NY-ESO-1 scFv sequences are underlined, hinge sequences are in bold, transmembrane sequences are in bold and underlined, and PAG1 intracellular domain sequences are in regular text.

Antigens

[0176] The disclosure provides inhibitory receptors comprising extracellular ligand binding domains. In some embodiments, the ligand for the extracellular ligand binding domain is an antigen binding domain.

[0177] The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen for the receptors described herein. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components. [0178] In some embodiments, the antigen-binding domain specifically binds to a target selected from etiolate receptor, anbb integrin, TNF receptor superfamily member 17 (TNFRSF17, or BCMA), CD276 molecule (CD276, or B7-H3), natural killer cell cytotoxicity receptor 3 ligand 1( NCRL3LR1, or B7-H6), carbonic anhydrase 9 (CA9, or CAIX), CD 19 molecule (CD 19), CD20 molecule (CD20), CD22 molecule (CD22), CD30 molecule (CD30), CD33 molecule (CD33), CD37 molecule (CD37), CD44 molecule (CD44), CD44v6, CD44v7/8, CD70 molecule (CD70), CD 123 molecule (CD123), CD138 molecule (CD138), CD171 molecule (CD171), CEA cell adhesion molecule (CEA), delta like canonical Notch ligand 4 (DLL4), epithelial cell adhesion molecule (EGP-2, EGP-40 or EPCAM), chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor receptor (EGFR), erb-b2 receptor tyrosine kinase 2 (ErbB2, or HER2), erb-b2 receptor tyrosine kinase 3 (ErbB3, or Her3), erb-b2 receptor tyrosine kinase 4 (ErbB4, or Her4), epidermal growth factor receptor vIII (EGFRvIII), EPH receptor A2 (EphA2), fibroblast activation protein alpha (FAP), folate receptor alpha (FBP), fetal acetylcholine receptor, frizzled class receptor 7 (Fzd7), Disialoganglioside GD2 (GD2), ganglioside GD3 (GD3), Glypican-3 (GPC3), trophoblast glycoprotein (h5T4), interleukin 11 receptor (IL-11R), interleukin 13 receptor subunit alpha 2 (IL13R-a2), kinase insert domain receptor (KDR), k light chain, l light chain, LeY,Ll cell adhesion molecule (LI CAM), MAGE-A1, mesothelin (MSLN), MHC presented peptides, mucin 1, cell surface associated (MUC1), mucin 16, cell surface associated (MUC16), neural cell adhesion molecule 1 (NCAM), NKG2D ligands, Notchl, Notch2/3, NY-ESO-1, preferentially expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Survivin, tumor associated glycoprotein-72 antigen (TAG-72), TEMs, telomerase reverse transcriptase (TERT), kinase insert domain receptor (KDR, or VEGFR2), and receptor tyrosine kinase like orphan receptor 1 (ROR1).

[0179] In some embodiments, the antigen-binding domain specifically binds peptide MHC (pMHC) as the antigen. Exemplary pMHC antigens include, but are not limited to, MAGE-A3 pMHC (e.g., FLWGPRALV (SEQ ID NO: 270) and MPKVAELVHFL (SEQ ID NO: 271) peptides), HPV E6 pMHC (e.g, TIHDIILECV (SEQ ID NO: 272) peptide), HPV E7 pMHC (e.g, YMLDLQPET (SEQ ID NO: 276) peptide) and NY- ESO-1 pMHC (e.g, LLEFYLAMPFA (SEQ ID NO: 274) or SLLMWITQV (SEQ ID NO: 275) peptides).

[0180] In some embodiments, the antigen-binding domain specifically binds to a target selected from CD33, CD38, a human leukocyte antigen (HLA), an organ specific antigen, a blood-brain barrier specific antigen, an Epithelial-mesenchymal transition (EMT) antigen, E-cadherin, cytokeratin, Opioid-binding protein/cell adhesion molecule (OPCML), HYLA2, Deleted in Colorectal Carcinoma (DCC), Scaffold/Matrix attachment region-binding protein 1 (SMAR1), cell surface carbohydrate and mucin type O-glycan.

[0181] In some embodiments, the antigen-binding domain of the inhibitory receptor specifically binds to an antigen from a gene with high, homogeneous surface expression across tissues. High, homogeneous surface expression across tissues allows the inhibitor ligand to deliver a large, even inhibitory signal.

[0182] In some embodiment, the antigen is encoded by a gene that is absent or polymorphic in in many tumors.

[0183] Methods of distinguishing the differential expression of inhibitor ligands (e.g., antigens) between target and non-target cells will be readily apparent to the person or ordinary skill in the art. For example, the presence or absence of inhibitor ligands in non-target and target cells can be assayed by immunohistochemistry with an antibody that binds to the inhibitor ligand, followed by microscopy or FACS, RNA expression profiling of target cells and non-target cells, or DNA sequencing of non-target and target cells to determine if the genomic locus of the inhibitor ligand comprises mutations in either the target or non-target cells.

Alleles lost due to Loss of Heterozygosity (LOH)

[0184] Homozygous deletions in primary tumors are rare and small, and therefore unlikely to yield target B candidates. For example, in an analysis of 2218 primary tumors across 21 human cancer types, the top 4 candidates were CDKN2A, RB 1 , PTEN and N3PB2. However, CDKN2A (P16) was deleted in only 5% homozygous deletion across all cancers. Homozygous HLA-A deletions were found in less than 0.2% of cancers (Cheng et al., Nature Comm. 8:1221 (2017)). In contrast, deletion of a single copy of gene in cancer cells due to loss of hemizygosity occurs far more frequently. [0185] In some embodiments, the inhibitor ligand comprises an allele of a gene that is lost in target cells due to loss of heterozygosity. In some embodiments, the target cells comprises cancer cells. Cancer cells undergo frequent genome rearrangements, including duplication and deletions. These deletions can lead to the deletion of one copy of one or more genes in the cancer cells.

[0186] As used herein, “loss of heterozygosity (LOH)” refers to a genetic change, whereby one of the two alleles in the genome of a cell or cells is deleted, leaving a single mono-allelic (hemizygous) locus.

HLA class I alleles

[0187] In some embodiments, the inhibitor ligand comprises an HLA class I allele. The major histocompatibility complex (MHC) class I is a protein complex that displays antigens to cells of the immune system, triggering immune response. The Human Leukocyte Antigens (HLAs) corresponding to MHC class I are HLA-A, HLA-B, HLA- C, HLA-E, HLA-L, and HLA-G. HLAs are highly polymorphic, and HLA alleles are frequently lost in cancers through loss of heterozygosity.

[0188] In some embodiments, the inhibitor ligand comprises HLA class I allele. In some embodiments, the inhibitor ligand comprises an allele of HLA class I that is lost in a target cell through LOH. HLA-A is a group of human leukocyte antigens (HLA) of the major histocompatibility complex (MHC) that are encoded by the HLA-A locus. Receptors comprising an a chain encoded by HLA-A are one of three major types of human MHC class I cell surface receptors. The receptor is a heterodimer comprising a heavy a chain and smaller b chain. The a chain is encoded by a variant of HLA-A, while the b chain ^2-microglobulin) is an invariant. There are several hundred different HLA-A genes, and several thousand variant alleles, all of which fall within the scope of the instant disclosure.

[0189] In some embodiments, the inhibitor ligand comprises an HLA-B allele. The HLA-B gene has many possible variations (alleles). Hundreds of versions (alleles) of the HLA-B gene are known, each of which is given a particular number (such as HLA-B27).

[0190] In some embodiments, the inhibitor ligand comprises an HLA-C allele. 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 have been described.

[0191] In some embodiments, the inhibitor ligand comprises an HLA-E allele. HLA-E has several alleles that can be inhibitor ligands including, without limitation, HLA- E*01:01 and HLA-E* 01:03.

[0192] In some embodiments, the inhibitor ligand comprises an HLA-F allele. HLA-F is expressed as a heavy chain noncovalently complexed to 2-microglobulin (B2M), and at least 22 HLA-F alleles are known. In some embodiments, the inhibitor ligand comprises an HLA-G allele. The HLA-G locus encodes at least 16 distinct functional proteins from at least 50 alleles.

[0193] In some embodiments, the HLA class I allele has broad or ubiquitous RNA expression.

[0194] In some embodiments, the HLA class I allele has a known, or generally high minor allele frequency.

[0195] In some embodiments, the HLA class I allele does not require a peptide-MHC antigen, for example when the HLA class I allele is recognized by a pan-HLA ligand binding domain.

[0196] In some embodiments, the inhibitor ligand comprises an HLA-A allele. In some embodiments the HLA-A allele comprises HLA-A*02. Various single variable domains known in the art or disclosed herein that bind to and recognize HLA-A* 02 are suitable for use in embodiments. Such scFvs include, for example and without limitation the following mouse and humanized scFv antibodies that bind HLA-A *02 in a peptide-independent way shown in Table 1.

Minor histocompatibility antigens

[0197] In some embodiments, the second, inhibitor ligand comprises a minor histocompatibility antigen (MiHA). In some embodiments, the second, inhibitor ligand comprises an allele of a MiHA that is lost in a target cell through LOH.

[0198] MiHAs are peptides derived from proteins that contain nonsynonymous differences between alleles and are displayed by common HLA alleles. The non- synonymous differences can arise from SNPs, deletions, frameshift mutations or insertions in the coding sequence of the gene encoding the MiHA. Exemplary MiHAs can be about 9-12 amino acids in length and can bind to MHC class I and MHC class II proteins. Binding of the TCRto the MHC complex displaying the MiHA can activate T cells. The genetic and immunological properties of MiHAs will be known to the person of ordinary skill in the art. Candidate MiHAs are known peptides presented by known HLA class I alleles, are known to elicit T cell responses in the clinic (for example, in graft versus host disease, or transplant rejection, and allow for patient selection by simple SNP genotyping.

[0199] In some embodiments, the MiHA has broad or ubiquitous RNA expression. [0200] In some embodiments, the MiHA has high minor allele frequency.

[0201] In some embodiments, the MiHA comprises a peptide derived from a Y chromosome gene.

[0202] In some embodiments, the second inhibitor ligand comprises a MiHA selected from the group of MiHAs disclosed in Tables 4 and 5.

[0203] Exemplary, but non-limiting, examples of MiHAs that are envisaged as within the scope of the instant invention are disclosed in Table 4 below. Columns in Table 4 indicate, from left to right, the name of the MiHA, the gene which from which it is derived, MHC class I variant which can display the MiHA and the sequences of the peptide variants [A/B variants indicated in brackets).

Table 4. HLA Class I Autosomal MiHAs.

[0204] Exemplary, but non-limiting, examples of MiHAs that are envisaged as within the scope of the instant invention are disclosed in Table 5 below. Columns in Table 5 indicate, from left to right, the name of the MiHA, the gene which from which it is derived, MHC class I variant which can display the MiHA and the sequences of the peptide variants [A/B variants indicated in brackets).

Table 5. HLA Class I Y linked MiHAs

[0205] In some embodiments, the MiHA comprises HA-1. HA-1 is a peptide antigen having a sequence of VL[H/R]DDLLEA (SEQ ID NO: 34), and is derived from the Rho GTPase activating protein 45 (HA-1) gene.

Exemplary ligand binding domains that selectively bind to HA-1 variant H peptide (VLHDDLLEA (SEQ ID NO: 277)) are shown in Table 6 below. TCR alpha and TCR beta sequences in SEQ ID NO: 279 are separated by a P2A self-cleaving polypeptide of sequence ATNF SLLKQ AGD VEENPGP (SEQ ID NO: 278) with an N terminal GSG linker. Table 6. Ftcr HA-l(H) Inhibitory Receptor Sequences

[0206] In some embodiments, the second, inhibitory ligand comprises HA- 1(H). In some embodiments, the second, inhibitory ligand binding is isolated or derived from a TCR. In some embodiments, the second, inhibitory ligand binding domain comprises TCR alpha and TCR beta variable domains. In some embodiments, the TCR alpha and TCR beta variable domains are separated by a self-cleaving polypeptide sequence. In some embodiments, the TCR alpha and TCR beta variable domains separated by a self cleaving polypeptide sequence comprise SEQ ID NO: 279. In some embodiments, the TCR alpha and TCR beta variable domains separated by a self-cleaving polypeptide sequence comprise SEQ ID NO: 279, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR alpha and TCR beta variable domains are encoded by a sequence of SEQ ID NO: 377, or a sequence having at least 80% identity, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR alpha variable domain comprises SEQ ID NO: 280 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR beta variable domain comprises SEQ ID NO: 281 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto.

Loss of Y chromosome

[0207] In some embodiments, the inhibitor ligand comprises a Y chromosome gene, i.e. peptide encoded by a gene on the Y chromosome. In some embodiments, the inhibitor ligand comprises a peptide encoded by a Y chromosome gene that is lost in target cells through loss of Y chromosome (LoY). For example, about a third of the characterized MiHAs come from the Y chromosome. The Y chromosome contains over 200 protein coding genes, all of which are envisaged as within the scope of the instant disclosure.

[0208] As used herein, “loss of Y”, or “LoY” refers a genetic change that occurs at high frequency in tumors whereby one copy of part or all of the Y chromosome is deleted, leading to a loss of Y chromosome encoded gene(s).

[0209] Loss of Y chromosome is known to occur in certain cancers. For example, there is a reported 40% somatic loss of Y chromosome in renal clear cell cancers (Arseneault et al, Sci. Rep. 7: 44876 (2017)). Similarly, clonal loss of the Y chromosome was reported in 5 out of 31 in male breast cancer subjects(Wong et al, Oncotarget 6(42):44927-40 (2015)). Loss of the Y chromosome in tumors from male patients has been described as a “consistent feature” of head and neck cancer patients (el-Naggar et al., Am J Clin Pathol 105(1): 102-8 (1996)). Further, Y chromosome loss was associated with X chromosome disomy in four of seven male patients with gastric cancer (Saal et al., Virchows Arch B Cell Pathol (1993)). Thus, Y chromosome genes can be lost in a variety of cancers, and can be used as inhibitor ligands with the engineered receptors of the instant disclosure targeting cancer cells.

Polynucleotides and Vectors

[0210] In other aspects, the disclosure provides polynucleotides comprising a nucleic acid sequence encoding the inhibitory receptor polypeptides of the disclosure. In some embodiments, the polynucleotides encode at least a first polypeptide comprising one or more of a PAG1 intracellular domain or a functional fragment or variant thereof, a transmembrane and an extracellular ligand binding domain. In some embodiments, the polynucleotides comprise a nucleic acid sequence encoding a polypeptide comprising an extracellular ligand bind domain or a portion thereof, a transmembrane domain and a PAG1 intracellular domain or a functional fragment or variant thereof. In some embodiments, the polypeptides, and the polynucleotides encoding same, comprise a hinge domain.

[0211] In some embodiments, the polynucleotides comprise a sequence encoding a PAG1 domain. In some embodiments, the PAG1 domain is a truncated PAG1 intracellular domain as described herein. In some embodiments, the truncation is a N- terminal truncation, a C-terminal truncation or both an N- and C-terminal truncation. [0212] In some embodiments, the polynucleotides comprise a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a nucleic acid sequence that encodes a polypeptide of SEQ ID NO: 1.

[0213] In some embodiments, the polynucleotides comprise a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2. In some embodiments, the polynucleotide comprises a nucleic acid sequence that encodes a polypeptide of SEQ ID NO: 2.

[0214] In some embodiments, the polynucleotides comprise a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of amino acid residues 41-432 of SEQ ID NO: 1, residues 93-432 of SEQ ID NO: 1, residues 112-432 of SEQ ID NO: 1, residues 41-326 of SEQ ID NO: 1, residues 41-371 of SEQ ID NO: 1, residues 41-400 of SEQ ID NO: 1, residues 112-326 of SEQ ID NO: 1, and residues 217-326 of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a nucleic acid sequence that encodes a polypeptide selected from the group consisting of amino acid residues 41-432 of SEQ ID NO: 1, residues 93-432 of SEQ ID NO: 1, residues 112-432 of SEQ ID NO: 1, residues 41-326 of SEQ ID NO: 1, residues 41-371 of SEQ ID NO: 1, residues 41-400 of SEQ ID NO: 1, residues 112-326 of SEQ ID NO: 1, and residues 217-326 of SEQ ID NO: 1. [0215] The disclosure provides polynucleotides encoding a polypeptide comprising a transmembrane domain of an inhibitory receptor described herein. In some embodiments, the transmembrane domain is isolated or derived from PAG1, LILRB1 or CD8a. In some embodiments, the polynucleotides comprise a sequence encoding a polypeptide at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to any one of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 88 or SEQ ID NO: 13. In some embodiments, the polynucleotides comprise a sequence encoding any one of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 88 or SEQ ID NO: 13.

[0216] The disclosure provides polynucleotides encoding a polypeptide sequence of a hinge domain of an inhibitory receptor described herein. In some embodiments, the hinge domain is isolated or derived from PAG1, LILRB1, CD8a or a combination thereof. In some embodiments, the polynucleotides comprise a sequence encoding a polypeptide at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 86, SEQ ID NO: 87 or SEQ ID NO: 18. In some embodiments, the polynucleotides comprise a sequence encoding any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 86, SEQ ID NO: 87 or SEQ ID NO: 18.

[0217] In some embodiments, the polynucleotides comprise a sequence encoding a polypeptide at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to any one SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. In some embodiments, the polynucleotides comprise a sequence encoding any one of SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49.

[0218] The disclosure provides polynucleotides encoding a polypeptide sequence of an extracellular ligand binding domain of an inhibitory receptor as described herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence that encodes a polypeptide that is at least 95% identical to the heavy chain portion or the light chain portion of any one of SEQ ID NOS: 35-46. In some embodiments, the polynucleotide comprises a nucleic acid sequence that encodes a CDR sequence of any one of SEQ ID NOS: 22-33.

[0219] In another aspect, the disclosure provides vectors comprising the polynucleotides encoding the inhibitory receptors of the disclosure. [0220] 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.

[0221] The expression of natural or synthetic nucleic acids encoding receptors is typically achieved by operably linking a nucleic acid encoding 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. [0222] The polynucleotides encoding the inhibitory receptor polypeptides 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.

[0223] Further, the expression vector may be provided to cells, such as immune cells, in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

[0224] 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, lenti virus vectors are used.

[0225] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 basepairs (bp) upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0226] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor- la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, 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. Inducible promoters are also contemplated. 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.

[0227] In order to assess the expression of receptor the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). [0232] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitory receptor, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or other assays. Engineered Cells

[0233] In other aspects, the disclosure provides immune cells comprising a nucleic acid sequence or vector encoding the inhibitory receptor polypeptides of the disclosure and/or expressing inhibitory receptors of the disclosure.

[0234] In some embodiments, immune cell activation is reduced when the cell is contacted with the antigen or a cell expressing the antigen on its surface. In some embodiments, immune cell activation comprises expression of a gene operatively linked to an NFAT promoter. Immune cell activation and/or inhibition of activation can be measured by various other methods.

[0235] As used herein, the term “immune cell” refers to a cell involved in the innate or adaptive (acquired) immune systems. Exemplary innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, Natural Killer (NK) cells, polymophonuclear leukocytes such as neutrophils eosinophils and basophils and mononuclear cells such as monocytes, macrophages and mast cells. Immune cells with roles in acquired immunity include lymphocytes such as T-cells and B-cells.

[0236] In some embodiments, the immune cell is a T cell, an NK cell, a B cell or a macrophage. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is autologous. In some embodiments, the immune cell is allogeneic. In some embodiments, the immune cell is non-natural.

[0237] 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.

[0238] The disclosure provides immune cells comprising the inhibitory receptors comprising a PAG1 intracellular domain described herein. In some embodiments, the immune cells further comprise an activatory receptor.

[0239] In some embodiments, the activatory receptor is a chimeric antigen receptor (CAR) or T Cell Receptor (TCR).

[0240] In some embodiments, the activatory receptor is a single chain receptor.

[0241] In some embodiments, the activatory receptor is a multi-chain receptor. Chimeric Antigen Receptors (CARs)

[0242] In some embodiments, the activatory receptor is a chimeric antigen receptor (CAR). All CAR architectures are envisaged as within the scope of the instant disclosure. An exemplary CAR comprises a polypeptide comprising an extracellular antigen binding domain, optionally a hinge domain, a transmembrane domain and one or more intracellular domains, such as a stimulatory domain and co-stimulatory domain.

[0243] In some embodiments, the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo. Exemplary hinges can be isolated or derived from IgD and CD8 domains, for example IgGl.

[0244] In some embodiments, the hinge is isolated or derived from CD8a or CD28. In some embodiments, the CD8a hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18).

[0245] In some embodiments, the CD8a hinge comprises or consists essentially of SEQ ID NO: 18. In some embodiments, the CD8a hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of: accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgc ccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat

(SEQ ID NO: 19).

[0246] In some embodiments, the CD8a hinge is encoded by SEQ ID NO: 20.

[0247] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of:

CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 20). [0248] In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 20. 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: tgtaccattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaaca cctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 21).

[0249] In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 21.

[0250] 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.

[0251] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions may be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively the transmembrane domain may 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 may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0252] 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: FWVLWVGGVLACY SLLVTVAFIIFWV (SEQ ID NO: 55).

[0253] In some embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 55. 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: ttctgggtgctggtcgttgtgggcggcgtgctggcctgctacagcctgctggtgacagtggccttcatc atcttttgggtg (SEQ ID NO: 56).

[0254] In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 56.

[0255] 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: 57).

[0256] In some embodiments, the IL-2Rbeta transmembrane domain comprises or consists essentially of SEQ ID NO: 7. In some embodiments, the IL-2Rbeta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of: attccgtggc tcggccacct cctcgtgggc ctcagcgggg cttttggctt catcatctta gtgtacttgc tgatc (SEQ ID NO: 58).

[0257] In some embodiments, the IL-2Rbeta transmembrane domain is encoded by SEQ ID NO: 58.

[0258] 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. Effector functions of a regulatory T cell, for example, include the suppression or downregulation of induction or proliferation of effector T cells. 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 may be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired functions of the CAR-T cells of the instant disclosure. The term intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal. [0259] 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.

[0260] 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 domain is a CD247 molecule (0O3z) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DN AX-activating protein of 12 kDa (DAP 12) activation domain. In some embodiments, the CD3z 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:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 59).

[0261] In some embodiments, the 0O3z activation domain comprises or consists essentially of SEQ ID NO: 59. In some embodiments, the 0O3z 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: agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgag ctcaatctaggacgaagagaggagtacgatgttttggacaagcgtagaggccgggaccctgagatgggg ggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggag gcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccaggga ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID

NO: 60).

[0262] In some embodiments, the 0O3z activation domain is encoded by SEQ ID NO: 60.

[0263] 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).

[0264] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. In some embodiments, the ITAM contains a tyrosine separated from a leucine or an isoleucine by any two other amino acids (YxxL) (SEQ ID NO: 61).

[0265] In some embodiments, the cytoplasmic domain contains 1, 2, or 3 ITAMs. In some embodiments, the cytoplasmic domain contains 1 ITAM. In some embodiments, the cytoplasmic domain contains 2 ITAMs. In some embodiments, the cytoplasmic domain contains 3 ITAMs. In some embodiments, the cytoplasmic domain contains 4 ITAMs. In some embodiments, the cytoplasmic domain contains 5 ITAMs.

[0266] In some embodiments, the cytoplasmic domain is a Oϋ3z activation domain. In some embodiments, Oϋ3z activation domain comprises a single ITAM. In some embodiments, 6Ό3z activation domain comprises two ITAMs. In some embodiments, Oϋ3z activation domain comprises three ITAMs.

[0267] In some embodiments, the Oϋ3z activation domain comprising a single ITAM comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 62).

[0268] In some embodiments, the 6Ό3z activation domain comprises or consists essentially of SEQ ID NO: 62. In some embodiments, the 6Ό3z activation domain comprising a single ITAM is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of: agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttgcacat gcaggccctg ccccctcgc (SEQ ID NO: 63).

[0269] In some embodiments, the 6Ό3z activation domain is encoded by SEQ ID NO: 63. [0270] Further examples of ITAM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from TCR FcRy, FcR , CD3y, CD35, CD3s, Oϋ3z, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the instant invention comprises a cytoplasmic signaling sequence derived from Oϋ3z.

[0271] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise the CD3z signaling domain by itself or combined with any other desired cytoplasmic domain(s). For example, the cytoplasmic domain of the CAR can comprise a CD3z 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-2R , Fc Receptor gamma (FcRy), Fc Receptor beta (FcR ), CD3g molecule gamma (CD3y), CD35, CD3s, 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 fragments thereof.

[0272] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker.

[0273] In some embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In some embodiments, the co stimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 64).

[0274] In some embodiments, the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 64. 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: aggagcaagcggagcagactgctgcacagcgactacatgaacatgaccccccggaggcctggccccacc cggaagcactaccagccctacgcccctcccagggatttcgccgcctaccggagc (SEQ ID NO: 65).

[0275] In some embodiments, the CD28 co-stimulatory domain is encoded by SEQ ID NO: 65.

[0276] In some embodiments, the intracellular domain of the CARs of the instant disclosure comprises an interleukin-2 receptor beta-chain (IL-2Rbeta or IL-2R-beta) cytoplasmic domain. In some embodiments, the IL-2Rbeta domain is truncated. In some embodiments, the IL-2Rbeta cytoplasmic domain comprises one or more STAT5- recruitment motifs. In some embodiments, the CAR comprises one or more STAT5- recruitment motifs outside the IL-2Rbeta cytoplasmic domain.

[0277] In some embodiments, the IL-2Rbeta intracellular 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:

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLS S PFPS S S FS PGGLAPEI S PLEVLERDKVT QLLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 66).

[0278] In some embodiments, the IL2Rbeta intracellular domain comprises or consists essentially of SEQ ID NO: 66. In some embodiments, the IL-2R-beta intracellular 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:

1 aactgcagga acaccgggcc atggctgaag aaggtcctga agtgtaacac cccagacccc

61 tcgaagttct tttcccagct gagctcagag catggaggcg acgtccagaa gtggctctct

121 tcgcccttcc cctcatcgtc cttcagccct ggcggcctgg cacctgagat ctcgccacta

181 gaagtgctgg agagggacaa ggtgacgcag ctgctccccc tgaacactga tgcctacttg

241 tctctccaag aactccaggg tcaggaccca actcacttgg tg (SEQ ID NO: 67). [0279] In some embodiments, the IL-2Rbeta intracellular domain is encoded by SEQ ID NO: 67.

[0280] In an embodiment, the IL-2R-beta cytoplasmic domain comprises one or more STAT5-recruitment motifs. Exemplary STAT5-recruitment motifs are provided by Passerini et al. (2008) STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25+ effector T cells. International Immunology, Vol. 20, No. 3, pp. 421-431, and by Kagoya et al. (2018) A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects. Nature Medicine doi:10.1038/nm.4478.

[0281] In some embodiments, the STAT5-recruitmentmotif(s) consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 68).

T Cell Receptors (TCRs)

[0282] As used herein, a “TCR”, sometimes also called a “TCR complex” or “TCR/CD3 complex” refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more of the invariant CD3 chains (zeta, gamma, delta and epsilon), sometimes referred to as subunits. The TCR alpha and beta chains can be disulfide-linked to function as a heterodimer to bind to peptide-MHC complexes. Once the TCR alpha/beta heterodimer engages peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal. In an exemplary TCR complex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3 epsilon and CD3 delta form a heterodimer, CD3 epsilon and CD3 gamma for a heterodimer, and two CD3 zeta form a homodimer. [0283] The disclosure provides a second activatory receptor that may be an engineered TCR. Any suitable ligand binding domain may be fused to an extracellular domain, hinge domain or transmembrane of the engineered TCRs described herein. For example, the ligand binding domain can be an antigen binding domain of an antibody or TCR, or comprise an antibody fragment, a l only domain, a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb).

[0284] In some embodiments, the ligand binding domain is fused to an extracellular domain of a TCR subunit. The TCR subunit can be TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. For example, the ligand binding domain can be fused to TCR alpha, or TCR beta, or portions of the ligand binding can be fused to two subunits, for example portions of the ligand binding domain can be fused to both TCR alpha and TCR beta.

[0285] In some embodiments, the activatory receptor comprises an extracellular fragment of a T cell receptor (TCR). In some embodiments, a first polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR and the intracellular domain, and a second polypeptide comprises an antigen-binding fragment of the beta chain of the TCR. In some embodiments, a first polypeptide comprises an antigen-binding fragment of the beta chain of the TCR and the intracellular domain, and the second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR.

[0286] TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma and CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta or CD3 epsilon, or fragments or derivative thereof, can be fused to one or more domains capable of providing a stimulatory signal of the disclosure, thereby enhancing TCR function and activity.

[0287] The disclosure provides an activatory engineered TCR comprising a transmembrane domain. The disclosure provides polypeptides comprising a transmembrane domain, and an intracellular domain capable of providing a stimulatory signal or an inhibitory signal. In some embodiments, the engineered TCR comprises multiple intracellular domains capable of providing a stimulatory signal.

[0288] Transmembrane domains isolated or derived from any source are envisaged as within the scope of the fusion proteins of the disclosure. The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane -bound or transmembrane protein.

[0289] In some embodiments, the transmembrane domain is one that is associated with one of the other domains of the fusion protein, or isolated or derived from the same protein as one of the other domains of the fusion protein. In some embodiments, the transmembrane domain and the second intracellular domain are from the same protein, for example a TCR complex subunit such as TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. In some embodiments, the extracellular domain (svd-TCR), the transmembrane domain and the second intracellular domain are from the same protein, for example a TCR complex subunit such as TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. In other embodiments, the extracellular domain (comprising one or more ligand binding domains, such as nb-only domain and scFv domains), the transmembrane domain and the intracellular domain(s) are from different proteins. For example, in some embodiments the engineered svd-TCR comprises a CD28 transmembrane domain with a CD28, 4-1BB and 0Ό3z intracellular domain. [0290] In some embodiments, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex has bound to a target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0291] In some embodiments, the transmembrane domain can be attached to the extracellular region of a polypeptide of the engineered TCR, e.g., the antigen binding domain of the TCR alpha or beta chain, via a hinge, e.g. , a hinge from a human protein. For example, the hinge can be a human immunoglobulin (Ig) hinge, e.g. , an IgG4 hinge, or a CD 8 a hinge.

[0292] In some embodiments, the hinge is isolated or derived from CD8a or CD28. In some embodiments, the CD8a hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18). In some embodiments, the CD8a hinge comprises or consists essentially of SEQ ID NO: 18. In some embodiments, the CD8a hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 20. In some embodiments, the CD8a hinge is encoded by SEQ ID NO: 20.

[0293] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 20). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 20. 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: 21. In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 21. [0294] In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain. In some embodiments, the TCR alpha transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 69). In some embodiments, the TCR alpha transmembrane domain comprises, or consists essentially of, SEQ ID NO: 69. In some embodiments, the TCR alpha transmembrane domain is encoded by a sequence of:

GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTC ATGACGCTGCGGCTGTGG (SEQ ID NO: 70).

[0295] In some embodiments, the transmembrane domain comprises a TCR beta transmembrane domain. In some embodiments, the TCR beta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of:

TILYEILLGKATLY A VL V S AL VL (SEQ ID NO: 71). In some embodiments, the TCR beta transmembrane domain comprises, or consists essentially of, SEQ ID NO: 71. In some embodiments, the TCR beta transmembrane domain is encoded by a sequence of [0296] ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCG TGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO: 72).

[0297] In some embodiments, the transmembrane comprises a CD3 zeta transmembrane domain. In some embodiments, the CD3 zeta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: LCYLLDGILFIYGVILTALFL (SEQ ID NO: 73). In some embodiments, the CD3 zeta transmembrane domain comprises, or consists essentially of, SEQ ID NO: 73.

[0298] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region).

[0299] In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0300] When present, the transmembrane domain may be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain may be a membrane anchor domain. Without limitation, a natural or artificial transmembrane domain may comprise a hydrophobic a-helix of about 20 amino acids, often with positive charges flanking the transmembrane segment. The transmembrane domain may have one transmembrane segment or more than one transmembrane segment. Prediction of transmembrane domains/segments may be made using publicly available prediction tools (e.g. TMHMM, Krogh et al. Journal of Molecular Biology 2001; 305(3):567-580; or TMpred, Hofmann & Stoffel Biol. Chem. Hoppe-Seyler 1993; 347: 166). Non-limiting examples of membrane anchor systems include platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (added post- translationally to a signal sequence) and the like.

[0301] The disclosure provides engineered TCRs comprising one or more intracellular domains.

[0302] In some embodiments, the intracellular domain comprises one or more domains capable of providing a stimulatory signal to a transmembrane domain. In some embodiments, the intracellular domain comprises a first intracellular domain capable of providing a stimulatory signal and a second intracellular domain capable of providing a stimulatory signal. In other embodiments, the intracellular domain comprises a first, second and third intracellular domain capable of providing a stimulatory signal. The intracellular domains capable of providing a stimulatory signal are selected from the group consisting of a CD28 molecule (CD28) domain, a LCK proto-oncogene, Src family tyrosine kinase (Lck) domain, a TNF receptor superfamily member 9 (4- IBB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto oncogene, Src family tyrosine kinase (Fyn) domain, a zeta chain of T cell receptor associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) alpha, TCRbeta, CD3 delta, CD3 gamma and CD3 epsilon intracellular domains.

[0303] In some embodiments, an intracellular domain comprises at least one intracellular signaling domain. An intracellular signaling domain generates a signal that promotes a function a cell, for example an immune effector function of a TCR containing cell, e.g., a TCR-expressing T-cell. In some embodiments, the intracellular domain of the fusion proteins of the disclosure includes at least one intracellular signaling domain. For example, the intracellular domains of CD3 gamma, delta or epsilon comprise signaling domains.

[0304] In some embodiments, the extracellular domain, transmembrane domain and intracellular domain are isolated or derived from the same protein, for example T-cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon.

[0305] Examples of intracellular domains for use in activatory receptors of the disclosure include the cytoplasmic sequences of the TCR alpha, TCR beta, CD3 zeta, and 4- IBB, and the intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0306] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the proteins responsible for primary stimulation, or antigen dependent stimulation.

[0307] In some embodiments, the intracellular domain comprises a CD3 delta intracellular domain. In some embodiments, the CD3 delta intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of:

GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKGGSRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 75).

[0308] In some embodiments, the CD3 delta intracellular domain comprises or consists essentially of, SEQ ID NO: 75. [0309] In some embodiments, the CD3 delta intracellular domain is encoded by a sequence of:

1 ggacatgaga ctggaaggct gtctggggct gccgacacac aagctctgtt gaggaatgac

61 caggtctatc agcccctccg agatcgagat gatgctcagt acagccacct tggaggaaac

121 tgggctcgga acaagggcgg aagcaggagc aagcggagca gactgctgca cagcgactac

181 atgaacatga ccccccggag gcctggcccc acccggaagc actaccagcc ctacgcccct

241 cccagggatt tcgccgccta ccggagcta (SEQ ID NO: 76).

[0310] In some embodiments, the intracellular domain comprises a CD3 epsilon intracellular domain. In some embodiments, the CD3 epsilon intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLN QRRIGGSRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 98).

[0311] In some embodiments, the CD3 epsilon intracellular domain comprises or consists essentially of, SEQ ID NO: 98. In some embodiments, the CD3 epsilon intracellular domain is encoded by a sequence of:

1 aagaatagaa aggccaaggc caagcctgtg acacgaggag cgggtgctgg cggcaggcaa

61 aggggacaaa acaaggagag gccaccacct gttcccaacc cagactatga gcccatccgg

121 aaaggccagc gggacctgta ttctggcctg aatcagcgca gaatcggcgg aagcaggagc

181 aagcggagca gactgctgca cagcgactac atgaacatga ccccccggag gcctggcccc

241 acccggaagc actaccagcc ctacgcccct cccagggatt tcgccgccta ccggagctag

(SEQ ID NO: 77).

[0312] In some embodiments, the intracellular domain comprises a CD3 gamma intracellular domain. In some embodiments, the CD3 gamma intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNGGSRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 78).

[0313] In some embodiments, the CD3 gamma intracellular domain comprises, or consists essentially of, SEQ ID NO: 78. In some embodiments, the CD3 gamma intracellular domain is encoded by a sequence of:

1 ggacaggatg gagttcgcca gtcgagagct tcagacaagc agactctgtt gcccaatgac

61 cagctctacc agcccctcaa ggatcgagaa gatgaccagt acagccacct tcaaggaaac

121 cagttgagga ggaatggcgg aagcaggagc aagcggagca gactgctgca cagcgactac

181 atgaacatga ccccccggag gcctggcccc acccggaagc actaccagcc ctacgcccct

241 cccagggatt tcgccgccta ccggagctag (SEQ ID NO: 79).

[0314] In some embodiments, the intracellular domain comprises a CD3 zeta intracellular domain. In some embodiments, the CD3 zeta intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of:

1 rvkfsrsada payqqgqnql ynelnlgrre eydvldkrrg rdpemggkpq rrknpqegly 61 nelqkdkmae ayseigmkge rrrgkghdgl yqglstatkd tydalhmqal ppr (SEQ ID

NO: 85) or a portion thereof.

[0315] In some embodiments, the CD3 zeta intracellular domain comprises, or consists essentially of, SEQ ID NO: 85. In some embodiments, the CD3 zeta intracellular domain comprises a mutation of Q44K.

[0316] In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain. In some embodiments, a TCR alpha intracellular domain comprises Ser-Ser. In some embodiments, a TCR alpha intracellular domain is encoded by a sequence of TCCAGC.

[0317] In some embodiments, the intracellular domain comprises a TCR beta intracellular domain. In some embodiments, the TCR beta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, or is identical to a sequence of: MAMVKRKDSR (SEQ ID NO: 81). In some embodiments, the TCR beta intracellular domain comprises, or consists essentially of SEQ ID NO: 81. In some embodiments, the TCR beta intracellular domain is encoded by a sequence of:

ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 82). [0318] In some embodiments, the intracellular signaling domain comprises at least one stimulatory intracellular domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and one additional stimulatory intracellular domain, for example a co-stimulatory domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.

[0319] Exemplary co-stimulatory intracellular signaling domains include those derived from proteins responsible for co-stimulatory signals, or antigen independent stimulation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA, a Toll ligand receptor, as well as DAP 10, DAP 12, CD30, LIGHT, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1 (CDl la/CD18) 4-1BB (CD137, TNF receptor superfamily member 9), and CD28 molecule (CD28). A co-stimulatory protein can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, B7-H3, a ligand that specifically binds with CD83, CD4, and the like. The co-stimulatory domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

[0320] In some embodiments, the stimulatory domain comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises a CD28 or 4-1BB co-stimulatory domain. CD28 and 4- IBB are well characterized co-stimulatory molecules required for full T cell activation and known to enhance T cell effector function. For example, CD28 and 4- IBB have been utilized in chimeric antigen receptors (CARs) to boost cytokine release, cytolytic function, and persistence over the first-generation CAR containing only the CD3 zeta signaling domain. Likewise, inclusion of co-stimulatory domains, for example CD28 and 4-1BB domains, in engineered TCR can increase T cell effector function and specifically allow co stimulation in the absence of co-stimulatory ligand, which is typically down-regulated on the surface of tumor cells. [0321] In some embodiments, the stimulatory domain comprises a CD28 intracellular domain. In some embodiments, the CD28 intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 64). [0322] In some embodiments, the CD28 intracellular domain comprises, or consists essentially of, SEQ ID NO: 64. In some embodiments, a CD28 intracellular domain is encoded by a nucleotide sequence comprising SEQ ID NO: 65.

[0323] In some embodiments, the stimulatory domain comprises a 4-1BB intracellular domain. In some embodiments, the 4- IBB intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 80). [0324] In some embodiments, the 4- IBB intracellular domain comprises, or consists essentially of, KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 80).

[0325] In some embodiments, a 4- IBB intracellular domain is encoded by a nucleotide sequence comprising:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAG GCCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAG AAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 83).

Activator Ligand Binding Domains and Ligands

[0326] Any suitable ligand binding domain is envisaged as within the scope of the activatory receptors described herein.

[0327] In some embodiments, the activatory receptor comprises a ligand binding domain that is an antigen binding domain. In some embodiments, the antigen binding domain comprises an antibody fragment, a nb only domain, a linear antibody, a single chain variable fragment (scFv), or a single domain antibody (sdAb).

[0328] The disclosure provides activatory receptors having two polypeptides each having a part of a ligand-binding domain (e.g. cognates of a heterodimeric LDB, such as a TCRa/b- or Fab-based LBD). The disclosure further provides activatory receptors having two polypeptides, each having a part of a ligand-binding domain (e.g. cognates of a heterodimeric LDB, such as a TCRa/b- or Fab-based LBD) and one part of the ligand binding domain is fused to a hinge or transmembrane domain, while the other part of the ligand binding domain has no intracellular domain. Further variations include receptors where each polypeptide has a hinge domain, and where each polypeptide has a hinge and transmembrane domain. In some embodiments, the hinge domain is absent.

[0329] In some embodiments, the activatory receptor comprises a Fab fragment of an antibody.

[0330] In embodiments, a first polypeptide of the activatory receptor comprises an antigen-binding fragment of the heavy chain of the antibody and an intracellular domain, and a second polypeptide of the activatory receptor comprises an antigen binding fragment of the light chain of the antibody. In some embodiments, the first polypeptide comprises an antigen-binding fragment of the light chain of the antibody and the intracellular domain, and the second polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody.

[0331] In some embodiments, the activatory receptor comprises an extracellular fragment of a T cell receptor (TCR). In some embodiments, a first polypeptide of the activatory receptor comprises an antigen-binding fragment of the alpha chain of the TCR and an intracellular domain, and a second polypeptide of the activatory receptor comprises an antigen-binding fragment of the beta chain of the TCR. In some embodiments, a first polypeptide comprises an antigen-binding fragment of the beta chain of the TCR and an intracellular domain, and the second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR.

[0332] In some embodiments, the activator ligand is transferrin receptor (TFRC). Human transferrin receptor is described inNCBI record No. AAA61153.1, the contents of which are incorporated herein by reference. In some embodiments, TFRC is encoded by SEQ ID NO: 84.

[0333] In some embodiments, the activator ligand is a tumor specific antigen (TSA). In some embodiments, the tumor specific antigen is mesothelin (MSLN), CEACAM5 or EGFR. Table 7 shows illustrative scFv antigen binding domains that target tumor specific TSAs. In some embodiments, the TSA is MSLN, CEA, EGFR, DLL4, CA125, GD2, ROR1 or HER2/NEU. In some embodiments, the antigen binding domains targeting TSAs comprise an amino acid sequence selected from any one or more of SEQ ID NOs: 137-153. In some embodiments, the antigen binding domains are encoded by a nucleic sequence that shares about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity selected from any one or more of SEQ ID NOs: 154-170.

[0334] Table 7. Exemplary scFv antigen binding domains target tumor specific antigens (TSAs)

[0335] In some embodiments, the activator ligand is EGFR, and the activator ligand binding domain is an EGFR binding domain. In some embodiments, the EGFR binding domain comprises a VH and/or a VL domain comprising an amino acid sequence selected from any one or more of SEQ ID NOs: 172-192, or a sequence having at least 90% identity thereto.

[0336] In some embodiments, the activator ligand is EGFR, and the activator ligand binding domain is an EGFR binding domain. In some embodiments, the EGFR binding domain comprises CDRs from Table 8. In some embodiments, the EGFR binding domain comprises CDRs having at least 95% sequence identity to CDRs disclosed in Table 8. In some embodiments, the EGFR binding domain comprises a sequence at least 95% identical to any one of SEQ ID NOS: 193-226. In some embodiments, the EGFR binding domain comprises a sequence identical to any one of SEQ ID NOS: 193- 226. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs of any one of SEQ ID NOS: 193-210, and the light chain of the antibody comprises the light chain CDRs of any one of SEQ ID NOS: 211-226.

[0337] Table 8. EGFR antigen binding domain CDRs.

[0338] In some embodiments, the activator ligand is a pan-HLA ligand, and the activator binding domain is a pan-HLA binding domain, i.e. a binding domain that binds to and recognizes an antigenic determinant shared among HLA I products, such as the HLA A, B and C loci. Various single variable domains known in the art or disclosed herein are suitable for use in embodiments. Such scFvs include, for example and without limitation the mouse and humanized pan-HLA scFv antibodies comprising amino acid sequences selected from SEQ ID NO: 227-232. Nucleic acid sequences encoding mouse and humanized pan-HLA scFvs include, without limitation, nucleic acid sequences selected from 233-238. An exemplary pan-HLA ligand is W6/32, which recognizes a conformational epitope, reacting with HLA class I alpha3 and alpha2 domains. Further exemplary antibodies with broad HLA binding are known in the art and include HC-10 and TFL-006.

Method of Making Engineered Cells

[0339] In other aspects, the disclosure provides methods comprising introducing polynucleotides of the disclosure into cells, optionally using vectors of the disclosure. In embodiments, the resulting cell expresses the polynucleotide. In embodiments, the resulting cell expresses the inhibitory receptor encoded by the polynucleotide. In embodiments, the cell is an immune cell. In embodiments, the immune cell is a T cell. [0340] Methods of transforming populations of immune cells, such as T cells, with the vectors of the instant disclosure will be readily apparent to the person of ordinary skill in the art. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi), according to manufacturer’s instructions. T cells can be cultured at a density of 1 x 10^L6 cells/mL in X-Vivo 15 media supplemented with 5% human A/B serum and 1% Pen/strep in the presence of CD3/28 Dynabeads (1:1 cell to bead ratio) and 300 Units/mL of IL-2 (Miltenyi). After 2 days, T cells can be transduced with viral vectors, such as lentiviral vectors using methods known in the art. In 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 peripheral blood mononuclear cells can be achieved after 5 days of growth to generate a >99% CD3+ highly transduced cell population.

[0341] Methods of activating and culturing populations of T cells comprising the receptors, polynucleotides or vectors of the disclosure will be readily apparent to the person of ordinary skill in the art.

[0342] Whether prior to or after genetic modification, 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.

[0343] In some embodiments, T cells of the instant disclosure are expanded and activated in vitro. Generally, the T cells of the instant disclosure are expanded in vitro by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti- CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Bcsancon. France) can be used as can other methods commonly known in the art (Berg et ak, Transplant Proc. 30(8):3975-3977, 1998; Haanen et ak, J. Exp. Med. 190(9): 13191328, 1999; Garland et ak, J. Immunol Meth. 227(l-2):53-63, 1999).

[0344] In some embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (/. e. , in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In some embodiments, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells.

[0345] In further embodiments of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent- coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0346] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached to contact the T cells. In one embodiment, the cells (for example, CD4+ T cells) and beads (for example, DYNABEADS CD3/CD28 T paramagnetic beads at a ratio of 1:1) are combined in a buffer. Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (/. e. , increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. In some embodiments, cells that are cultured at a density of lxlO⁶ cells/mL are used.

[0347] In some embodiments, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the beads and T cells are cultured together for 2-3 days. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF , and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. In some embodiments, the media comprises X-VIVO- 15 media supplemented with 5% human A/B serum, 1% penicillin/streptomycin (pen/strep) and 300 Units/ml of IL-2 (Miltenyi).

[0348] The T cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% C02).

[0349] In some embodiments, the T cells comprising receptors of the disclosure are autologous. Prior to expansion and genetic modification, a source of T cells is obtained from a subject. Immune cells such as T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. [0350] In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0351] In some embodiments, immune cells such as T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD4 - conjugated beads, for a time period sufficient for positive selection of the desired T cells.

[0352] Enrichment of an immune cell population, such as a T cell population, by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immune-adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD l ib, CD 16, HLA-DR, and CD8.

[0353] For isolation of a desired population of immune cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e.. increase the concentration of cells), to ensure maximum contact of cells and beads.

[0354] In some embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10° C or at room temperature.

[0355] T cells for stimulation, or PBMCs from which immune cells such as T cells are isolated, can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C. or in liquid nitrogen.

Assaying Signaling

[0356] In some embodiments, activation of an immune cell comprising the inhibitory receptors described herein is reduced when the cell is contacted with the antigen or a cell expressing the antigen on its surface. In some embodiments, immune cell activation comprises expression of a gene operatively linked to an NFAT promoter. Nuclear factor of activated T-cells (NFAT) is a family of transcription factors shown to be important in immune response. The NFAT transcription factor family consists of five members NFATcl, NFATc2, NFATc3, NFATc4, and NFAT5. NFAT plays a role in regulating inflammation.

[0357] As used herein, an NFAT promoter is a promoter that is regulated (/. e. , activated or repressed) when NFAT is expressed in a cell. NFAT target promoters are described in Badran, B. M. et al.(2002) J. Biological Chemistry Vol. 277: 47136-47148, and contain NFAT consensus sequences such as GGAAA.

[0358] Methods of assessing the effects of receptor activation on gene expression are known in the art, and include the use of reporter genes, whose expression can be quantified. Reporter genes are used for identifying potentially transfected or transduced cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription. In exemplary embodiments, an NFAT promoter operably linked to a reporter gene is used to evaluate the expression of the receptors of the disclosure on NFAT signaling.

Pharmaceutical Compositions

[0359] The disclosure provides pharmaceutical compositions comprising immune cells comprising the PAG1 -based inhibitory receptors of the disclosure and a pharmaceutically acceptable diluent, carrier or excipient.

[0360] Such compositions may 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.

Methods of Use

[0361] The disclosure provides methods of using the PAGl-based inhibitory receptors described herein, immune cells comprising PAGl-based inhibitory receptors, and pharmaceutical compositions comprising same.

[0362] In some embodiments, the methods comprise administering to a subject a plurality of immune cells comprising a chimeric antigen receptor or T cell receptor (TCR) and an inhibitory receptor as described herein. For example, the subject can have a disease or disorder, such as cancer, that would benefit from, or be treatable with, immunotherapy comprising CAR T or TCR T cells. CAR T therapies using the inhibitory receptors described herein may be used for the treatment of glioblastoma, metastatic breast cancer, leukemia, lymphoma, sarcoma, multiple myeloma and pediatric acute lymphoblastic leukemia, among other malignancies.

[0363] In some embodiments, administration of CAR T or TCR T cells further comprising the inhibitory receptors of the disclosure can increase specificity of the immune cells for a target cell or tissue compare to immune cells that express the CAR or TCR but do not express the inhibitory receptor administration of CAR T or TCR T cells further comprising the inhibitory receptors of the disclosure can reduce side effects in a subject when compared to immune at express the CAR or TCR but do not express the inhibitory receptor.

Kits and Articles of Manufacture

[0364] The disclosure provides kits and articles of manufacture comprising the polynucleotides and vectors encoding the inhibitory receptors described herein. In some embodiments, the kit comprises articles such as vials, syringes and/or instructions for use.

[0365] In some embodiments, the kit comprises a polynucleotide or vector comprising a sequence encoding one or more inhibitory receptor polypeptides of the disclosure. For example, the polynucleotide or vector comprises a sequence of a PAG1 intracellular domain as described herein.

[0366] In some embodiments, the kit comprises a plurality of immune cells comprising an inhibitory receptor as described herein. In some embodiments, the plurality of immune cells comprises a plurality of T cells.

[0367] The present description sets forth numerous exemplary configurations, methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments.

EXAMPLES

Example 1: Materials and Methods [0368] Cell Culture

[0369] Jurkat cells encoding an NFAT Luciferase reporter were maintained in RPMI media supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Pen/Strep) and 0.4mg/mL G418/Geneticin. T2 cells (ATCC CLR-1992) were maintained in IMDM media + 20% FBS and 1% Pen/Strep.

[0370] Plasmid Construction

[0371] Phosphoprotein associated with glycosphingolipid-enriched microdomains 1, (PAG1) was rendered responsive to ligand stimulation via fusion to an NY-ESO- l/HLA-A*02-selective ligand binding scFv domain (C-266). Fusions were made to either the N-terminus of the native full-length sequence of PAG1, truncated PAG1, e.g. a PAG1 sequence including the intracellular domain or a portion of the intracellular domain, or fused via hinge and/or hinge/transmembrane regions of leukocyte immunoglobulin-like receptor subfamily B member 1, LILRB1 (LIR1) or CD 8. Gene segments were combined using Golden Gate cloning and inserted downstream of an eFla promoter contained in a lentiviral expression plasmid (pLentil).

[0372] NFAT Luciferase Assay

[0373] Jurkat cells were transfected via 100 pL format Neon electroporation system (Thermo Fisher) according to the manufacturer's protocol. The following settings were used: 3 pulses, 1500 V, 10 milliseconds. Co-transfection was performed with 3 pg of activating TCR construct (CT-139) or CAR construct (C-1511) and 3 pg of either inactivating CAR construct or empty vector (pLentiO) per 1 million cells and recovered in RPMI media supplemented with 20% heat-inactivated FBS and 0.1% Pen/Strep. [0374] Peptides, e.g. MAGE-A3 (FLWGPRALV; SEQ ID NO: 270) and modified NY- ESO-1 (SLLMWITQV; SEQ ID NO: 275), were synthesized by Genscript. Activating peptide, MAGE-A3, was serially diluted 5 -fold starting at 50 pM. Inactivating peptide, NY-ESO-1, was diluted to 50 pM, 5 pM, 0.5 pM, or 0.05 uM and these constant amounts were added to the MAGE-A3 serial dilutions and subsequently loaded onto 10,000 T2 cells in 15 pL of RPMI supplemented with 1% BSA and 0.1 % Pen/Strep and incubated in Coming® 384-well Low Flange White Flat Bottom Polystyrene TC- treated Microplates. The following day, 10,000 Jurkat cells were resuspended in 15 pL of RPMI supplemented with 10% heat-inactivated FBS and 0.1% Pen/Strep, added to the peptide -loaded T2 cells and co-cultured for 6 hours. The ONE-Step Luciferase Assay System (BPS Bioscience) was used to evaluate Jurkat luminescence. Assays were performed in technical duplicates.

Example 2: Inhibition of chimeric antigen receptor (CAR) signaling [0375] PAG1 constructs were assayed for their ability to inhibit CAR signaling in a ligand dependent manner, and for the effect of the LILRB 1 hinge and transmembrane domains on PAG1- mediated inhibition of CAR signaling.

[0376] Jurkat-NFAT luciferase reporter cells were transfected with an activating CAR construct recognizing MAGE-A3 pMHC and an inhibitory PAG1 construct recognizing NY-ESO-1 pMHC, or an empty vector. The effect on activation of NFAT-luciferase was measured by co-culturing transfected cells using the methods described in Example 1. In brief, Jurkat cells expressing the indicated constructs in Table 9 were co-cultured with T2 cells loaded with varying amounts of MAGE-A3 peptide plus 10 uM inhibitory NY-ESO-1 peptide. The results are shown in FIG. 1.

[0377] The data shown in FIG. 1 demonstrate that PAG1 can be made ligand responsive by the addition of an extracellular ligand binding domain (LBD). PAG1 fusion constructs inhibit CAR mediated activation. A LILRB1 hinge or LILRB1 hinge and transmembrane domain (TM) improves blocking potency of PAG 1 constructs.

[0378] Table 9. Activator CAR and inhibitory PAG1 constructs

Example 3: Inhibition of T cell receptor (TCR) signaling

[0379] PAG1 inhibitory receptor constructs were assayed for their ability to inhibit TCR signaling in a ligand dependent manner, and for the effect of the LILRB 1 hinge and transmembrane domains on PAG 1 -mediated inhibition of T cell receptor (TCR) signaling.

[0380] FIG. 2 shows that the FIFRB 1 hinge region improves the inhibitory effect of the PAG1 intracellular domain on MAGE-A3 TCRs. In FIG. 2, Jurkat-NFAT luciferase reporter cells were transfected with an activating TCR construct recognizing MAGE- A3 pMHC and an inhibitory construct recognizing NY-ESO-1 pMHC, or an empty vector. The effect on activation of NFAT-luciferase was measured by co-culturing transfected Jurkat cells with T2 cells loaded with varying amounts of MAGE-A3 peptide plus 5 uM inhibitory NY-ESO-1 peptide. The constructs assayed in FIG. 2 are shown in Table 10. [0381] The data shown in FIG. 2 demonstrate that PAG1 fusion inhibits TCR mediated activation. Furthermore, truncation of the PAG1 intracellular domain (ICD), which included a potential palmitoylation site, did not have any effect on inhibition.

[0382] Table 10. Activator TCR and inhibitory PAG1 constructs

Example 4: ScFv-PAGl inhibition of TCR activation is observed with conventional CD8-based hinge and TM domains and improved by LILRB1 hinge and TM domains

[0383] The effect of a CD8 hinge, CD8 hinge and transmembrane domains, or LILRB 1 hinge and transmembrane domains on ScFv-PAGl inhibition of MAGE-A3 pMHC TCR activation was assayed in Jurkat cells as described in Example 1.

[0384] Jurkat-NFAT luciferase reporter cells were transfected with an activating TCR construct recognizing MAGE-A3 pMHC and an inhibitory construct recognizing NY - ESO-1 pMHC, or an empty vector as described in Example 1. The effect on activation of NFAT-luciferase was measured by co-culturing transfected Jurkat cells with T2 cells loaded with varying amounts of MAGE-A3 peptide plus 5 mM inhibitory NY-ESO-1 peptide. The data are shown in FIG. 3, and the constructs are described in Table 11. [0385] As shown by the data in FIG. 3, scFv-PAGl construct-mediated inhibition of MAGE-A3 TCR activation is improved by using the LILRB 1 hinge and transmembrane domain regions compared to the CD8 hinge and transmembrane domains of other CARs. PAG1 constructs show better inhibitory activity when fused to LILRB 1 hinge/TM than when fused to CD8 hinge or CD8 hinge/TM.

[0386] Table 11. Activator TCR and inhibitory PAG1 constructs with CD8 and LILRB1 hinge domains

Example 5: Effect of truncations at either end of the PAG1 intracellular domain on ScFv-PAGl inhibition of CAR signaling

[0387] The effect of N- and C-terminal truncations of the PAG1 intracellular domain (I CD) on ScFv-PAGl inhibition of MAGE-A3 pMHC CAR activation was assayed in Jurkat cells as described in Example 1.

[0388] Jurkat-NFAT luciferase reporter cells were transfected with an activating CAR construct recognizing MAGE-A3 pMHC and an inhibitory PAG1 construct recognizing NY-ESO-1 pMHC, or an empty vector. The effect on activation of NFAT-luciferase was measured by co-culturing transfected Jurkat cells with T2 cells loaded with varying amounts of MAGE-A3 peptide plus 10 uM inhibitory NY-ESO-1 peptide as described in Example 1. The data are shown in FIG. 4, and the constructs are described in Table 12

[0389] As shown in FIG. 4, receptors having truncations to the membrane-proximal end or C-terminal end of the PAG1 ICD retain the ability of the PAG1 ICD to block CAR signaling.

[0390] Table 12. Activator CAR and inhibitory PAG1 constructs with N- and C- terminal truncations of the ICD

[0391] An scFv-PAGl construct with truncations at both the N-and C-termini of the ICD was also assayed for its ability to inhibit MAGE-A3 pMHC CAR signaling.

[0392] Jurkat-NFAT luciferase reporter cells were transfected with an activating CAR construct recognizing MAGE-A3 pMHC and an inhibitory construct recognizing NY - ESO-1 pMHC, or an empty vector. The effect on activation of NFAT-luciferase was measured by co-culturing transfected Jurkat cells with T2 cells loaded with varying amounts of MAGE-A3 peptide plus 10 mM inhibitory NY-ESO-1 peptide, as described in Example 1. The data is shown in FIG. 5, and the constructs are described in Table 13

[0393] As shown in FIG. 5, simultaneous truncations to both the membrane-proximal end and C-terminal end of the PAG1 ICD do not have a large effect on the ability of the PAG1 ICD to block CAR signaling.

[0394] Table 13. Activator CAR and inhibitory PAG1 construct with N- and C- terminal truncation of the ICD

Example 6: Effect of truncation or modification of the ICD at amino acid residues 217-326 on scFv-PAGl inhibitory receptor function

[0395] The effect of truncations or modifications of the PAG1 ICD at residues 217-326 on CAR signaling were assayed in Jurkat cells as described in Example 1.

[0396] Jurkat-NFAT luciferase reporter cells were transfected with an activating CAR construct recognizing MAGE-A3 pMHC and an inhibitory construct recognizing NY -

ESO-1 pMHC, or an empty vector. The effect on activation of NFAT-luciferase was measured by co-culturing transfected Jurkat cells with T2 cells loaded with varying amounts of MAGE-A3 peptide plus 10 pM inhibitory NY-ESO-1 peptide. The data are shown in FIG. 6, and constructs are described in Table 14. [0397] As shown in FIG. 6, scFv-PAGl inhibition of MAGE-A3 CAR higher inhibition is observed when the PAG1[112-326] domain is used and when Y227 Y299 or Y317 residues are conserved. Truncations and mutations within residues 217-326 of the PAG1 ICD affect the ability of the PAG1 ICD to block CAR signaling.

[0398] Table 14. Activator CAR and inhibitory PAG1 constructs with truncations and modifications to residues 217-326 of the PAG1 intracellular domain

[0399] Table 15. Summary of results from examples 2-6 (effects of various PAG1 inhibitors and CAR and TCR activators

Claims

CLAIMS What is claimed is:

1. An inhibitory receptor comprising at least a first polypeptide, the first polypeptide comprising one or more of: a. a phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1) intracellular domain or a functional fragment or variant thereof, b. a transmembrane domain, and c. an extracellular ligand binding domain or a portion thereof.

2. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 37-432 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

3. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises a sequence identical to amino acid residues 37-432 of SEQ ID NO:

1

4. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises a truncated PAG1 intracellular domain.

5. The method of claim 5, wherein the truncated PAG1 intracellular domain comprises an N-terminal truncation, a C-terminal truncation, or both.

6. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 41-432 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

7. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 93-432 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

8. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 112-432 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

9. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 41-326 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

10. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 41-371 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

11. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 41-400 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

12. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 112-326 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

13. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 217-326 of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

14. The inhibitory receptor of claim 1, wherein the PAG1 intracellular domain comprises amino acid residues 41-432 of SEQ ID NO: 1, residues 93-432 of SEQ ID NO: 1, residues 112-432 of SEQ ID NO: 1, residues 41-326 of SEQ ID NO: 1, residues 41-371 of SEQ ID NO: 1, residues 41-400 of SEQ ID NO: 1, residues 112-326 of SEQ ID NO: 1, or residues 217-326 of SEQ ID NO: 1.

15. The inhibitory receptor of any one of claims 1-14, wherein the transmembrane domain comprises a PAG1 transmembrane domain, a LILRB1 transmembrane domain, a CD8a transmembrane domain, or a functional fragment or variant of any of these.

16. The inhibitory receptor of claim 15, wherein the PAG1 transmembrane domain comprises a sequence of SEQ ID NO: 11, or a sequence having at least 95% identical thereto.

17. The inhibitory receptor of claim 15, wherein the PAG1 transmembrane domain comprises a sequence identical to SEQ ID NO: 11.

18. The inhibitor receptor of claim 15, wherein the LILRB1 transmembrane domain comprises a sequence of SEQ ID NO: 88, or a sequence having at least 95% identity thereto.

19. The inhibitor receptor of claim 15, wherein the LILRB 1 transmembrane domain comprises a sequence identical to SEQ ID NO: 88.

20. The inhibitory receptor of claim 15, wherein the CD8a transmembrane domain comprises a sequence of SEQ ID NO: 13, or a sequence having at least 95% identity thereto.

21. The inhibitory receptor of claim 15, wherein the CD8a transmembrane domain comprises a sequence identical to SEQ ID NO: 13.

22. The inhibitory receptor of any one of claims 1-21, wherein the polypeptide comprises a hinge domain.

23. The inhibitory receptor of claim 22, wherein the hinge domain comprises a CD 8 hinge domain, a LILRB1 hinge domain, a PAG1 hinge domain or a combination, functional fragment or variant of any of these.

24. The inhibitory receptor of claim 23, wherein the LILRB1 hinge domain comprises a sequence of SEQ ID NO: 16, SEQ ID NO: 87, or a sequence having at least 95% identity thereto.

25. The inhibitory receptor of claim 23, wherein the LILRB1 hinge domain comprises a sequence of SEQ ID NO: 17, or a sequence having at least 95% identity thereto.

26. The inhibitory receptor of claim 23, wherein the LILRB1 hinge domain comprises a sequence of SEQ ID NO: 15 or a sequence having at least 95% identity thereto.

27. The inhibitory receptor of claim 23, wherein the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO: 16, SEQ ID NO: 87, SEQ ID NO: 17, or SEQ ID NO: 15.

28. The inhibitory receptor of claim 23, wherein the PAG1 hinge domain comprises a sequence of SEQ ID NO: 14 or a sequence having at least 95% identity thereto.

29. The inhibitory receptor of claim 23, wherein the PAG1 hinge domain comprises a sequence identical to SEQ ID NO: 14.

30. The inhibitory receptor of claim 23, wherein the CD8a hinge domain comprises a sequence of SEQ ID NO: 18, or a sequence having at least 95% identity thereto.

31. The inhibitory receptor of claim 23, wherein the hinge domain comprises both PAG1 and LILRB1 hinge sequences.

32. The inhibitory receptor of claim 31, wherein the hinge domain comprises a sequence of SEQ ID NO: 86, or a sequence having at least 95% identity thereto.

33. The inhibitory receptor of any one of claims 1-14, wherein the first polypeptide comprises a LILRB 1 hinge domain, a LILRB 1 transmembrane domain and the PAG1 intracellular domain or a functional fragment or variant of any of these.

34. The inhibitory receptor of claim 33, wherein the LILRB 1 hinge domain and the LILRB1 transmembrane domain comprise a sequence of SEQ ID NO: 48, or a sequence having at least 95% identity thereto.

35. The inhibitory receptor of any one of claims 1-14, wherein the first polypeptide comprises a PAG1 transmembrane domain and a PAG1 intracellular domain, or a functional fragment or variant of either of these.

36. The inhibitory receptor of claim 35, wherein the PAG1 transmembrane domain and PAG1 intracellular domain comprises a sequence with at least 95% identity to SEQ ID NO: 47.

37. The inhibitory receptor of any one of claims 1-14, wherein the first polypeptide comprises a CD8a hinge domain, a CD8a transmembrane domain, and a PAG1 intracellular domain, or functional fragment or variant of any of these.

38. The inhibitory receptor of claim 37, wherein the CD8a hinge domain and the CD8a transmembrane domain comprise a sequence of SEQ ID NO: 49, or a sequence having at least 95% identity thereto.

39. The inhibitory receptor of any one of claims 1-14, wherein the first polypeptide comprises a CD8a hinge domain, a PAG1 transmembrane domain and the PAG1 intracellular domain, or a functional fragment or variant of any of these.

40. The inhibitory receptor of claim 39, wherein the first polypeptide comprises a sequence of SEQ ID NO: 86, or a sequence having at least 95% identity thereto.

41. The inhibitory receptor of any one of claims 1-14, wherein the first polypeptide comprises a PAG1 hinge domain, a PAG1 transmembrane domain and a PAG1 intracellular domain or a functional fragment or variant of any of these.

42. The inhibitory receptor of claim 41, wherein the first polypeptide comprises a sequence of SEQ ID NO: 1, or a sequence having at least 95% identity thereto.

43. The inhibitory receptor of claim 41, wherein the first polypeptide comprises a sequence of SEQ ID NO: 1.

44. The inhibitory receptor of any one of claims 1-43, wherein the extracellular ligand binding domain comprises an antigen binding domain.

45. The inhibitory receptor of claim 44, wherein the antigen binding domain is specific to an antigen that is lost in a cancer cell through loss of heterozygosity.

46. The inhibitory receptor of claim 44 or 45, wherein the antigen binding domain is specific to a minor histocompatibility antigen (MiHA).

47. The inhibitory receptor of claim 44 or 45, wherein the antigen binding domain is specific to an antigen that is lost in a cancer cell through loss of Y chromosome.

48. The inhibitory receptor of claim 44 or 45, wherein the antigen binding domain is specific to a major histocompatibility class I allele.

49. The inhibitory receptor of claim 48, wherein the major histocompatibility class I allele comprises an HLA-A, an HLA-B, an HLA-C allele, an HLA-E allele, and HLA-F allele or an HLA-G allele.

50. The inhibitory receptor of claim 49, wherein the HLA-A allele comprises an HLA-A* 02 allele.

51. The inhibitor receptor of any one of claims 44-50, wherein the antigen binding domain comprises an antibody fragment, a nb only domain, a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb).

52. The inhibitory receptor of claim 50, wherein the antigen binding domain comprises a heavy chain and a light chain of an antibody, wherein the heavy chain comprises heavy chain complementarity determining regions (CDRs) of any one of SEQ ID NOs: 25-27 or 31-33, and wherein the light chain comprises light chain CDRs of any one of SEQ ID NOs: 22-24 or 28-30.

53. The inhibitory receptor of claim 50, wherein the antigen binding domain comprises a heavy chain and a light chain of an antibody, wherein the heavy chain comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 35-46, and wherein the light chain comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOs: 35-46 or 125.

54. The inhibitory receptor of claim 51, wherein the scFv comprises CDRs of any one of SEQ ID NOs: 22-33.

55. The inhibitory receptor of claim 51, wherein the ScFv comprises a sequence at least 95% identical to any one of SEQ ID NOs: 35-46 or 125.

56. The inhibitory receptor of claim 51, wherein the ScFv comprises a sequence identical to any one of SEQ ID NOs: 35-46 or 125.

57. The inhibitory receptor of any one of claims 1-50, wherein the receptor comprises a second polypeptide.

58. The inhibitory receptor of claim 57, wherein the second polypeptide comprises an extracellular ligand binding domain or a portion thereof.

59. The inhibitory receptor of claim 58, wherein the extracellular ligand binding domain of the first polypeptide comprises a first chain of an antibody and the extracellular ligand binding domain of the second polypeptide comprise a second chain of said antibody.

60. The inhibitory receptor of any one of claims 57-59, wherein the antibody comprises a Fab fragment of an antibody.

61. The inhibitory receptor of claim 59 or 60, wherein: a. the first polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody, and b. the second polypeptide comprises an antigen-binding fragment of the light chain of the antibody.

62. The inhibitory receptor of claim 59 or 60, wherein: a. the first polypeptide comprises an antigen-binding fragment of the light chain of the antibody, and b. the second polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody.

63. The inhibitory receptor of claim 61 or 62, wherein the heavy chain of the antibody comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 35-46 or 125, and wherein the light chain of the antibody comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 35-46 or 125.

64. The inhibitory receptor of claim 57, wherein the receptor comprises an extracellular fragment of a T cell receptor (TCR).

65. The inhibitory receptor of claim 64, wherein: a. the first polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR, and b. the second polypeptide comprises an antigen-binding fragment of the beta chain of the TCR.

66. The inhibitory receptor of claim 64, wherein: a. the first polypeptide comprises an antigen-binding fragment of the beta chain on the TCR, and b. the second polypeptide comprises an antigen-binding fragment of the alpha chain of the TCR.

67. The inhibitory receptor of any one of claims 44-66, wherein the extracellular ligand binding domain binds to an antigen that is present on a normal cell but is not present on a cancer cell.

68. A polynucleotide comprising a nucleic acid sequence encoding the inhibitory receptor of any one of claim 1-67.

69. A vector comprising the polynucleotide of claim 68.

70. An immune cell comprising the inhibitory receptor of any one of claims 1-67, the polynucleotide of claim 68, or the vector of claim 69.

71. The immune cell of claim 70, wherein immune cell activation is reduced when the cell is contacted with the extracellular ligand or a cell expressing the extracellular ligand on its surface.

72. The immune cell of claim 70 or 71, wherein the immune cell is a T cell, a B cell, an NK cell or a macrophage.

73. The immune cell of 64 or 65, wherein the extracellular ligand is an antigen.

74. The immune cell of any one of claims 70-73, wherein the immune cell comprises a second receptor, wherein the second receptor is an activatory receptor.

75. The immune cell of claim 74, wherein the activatory receptor activates the immune cell or promotes activation of the immune cell.

76. The immune cell of claim 74 or 75, wherein the activatory receptor is a chimeric antigen receptor (CAR) or an engineered T Cell Receptor (TCR).

77. The immune cell of any one of claims 70-76, wherein the activatory receptor comprises an antigen binding domain.

78. The immune cell of claim 77, wherein the antigen binding domain binds to a tumor specific antigen (TSA).

79. The immune cell of claim 78, wherein the TSA is selected from the group consisting of EGFR, mesothelin (MSLN) and cancer embryonic antigen (CEA or CEACAM5).

80. The immune cell of claim 77, wherein the antigen binding domain binds to a transferrin receptor (TFRC) antigen.

81. The immune cell of claim 77, wherein the activatory receptor comprises an antigen binding domain that binds to a pan-HLA antigen.

82. The immune cell of any one of claims 70-81, wherein the immune cell is isolated.

83. The immune cell of any one of claims 70-82, wherein the immune cell is non natural.

84. A method, comprising introducing the polynucleotide of claim 68, or the vector of claim 69 into a cell.

85. The method of claim 84, wherein the cell expresses the polynucleotide.

86. The method of claim 84 or claim 85, wherein the cell is an immune cell.

87. The method of claim 81, wherein the immune cell is a T cell, a B cell, an NK cell or a macrophage.

88. The method of any one of claims 82-87, wherein immune cell activation is reduced when the cell is contacted with the extracellular ligand or a cell expressing the extracellular ligand on its surface.

89. The method of claim 88, wherein the extracellular ligand is an antigen.

90. The method of claim 88 or 89, wherein the cell expressing the extracellular ligand on its surface is not a cancer cell.

91. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a plurality of immune cells comprising a chimeric antigen receptor (CAR) or engineered T-cell receptor (TCR), and comprising the inhibitory receptor of any one of claims 1-67.

92. The method of claim 91, wherein the immune cells are T cells.

93. The method of claim 91 or 92, wherein the inhibitory receptor increases the specificity of the immune cells for a target cell or tissue compared to immune cells that express the CAR or TCR but do not express the inhibitory receptor.

94. The method of any one of claims 91-93, wherein the immune cells comprise a second activatory receptor.

95. The method of claim 94, wherein the activatory receptor activates the immune cell or promotes activation of the immune cell.

96. The method of claim 94 or 95, wherein the activatory receptor is a chimeric antigen receptor (CAR) or an engineered T Cell Receptor (TCR).

97. The method of any one of claims 94-96, wherein the activatory receptor comprises an antigen binding domain.

98. The method of claim 97, wherein the antigen binding domain binds to a tumor specific antigen (TSA).

99. The method of claim 98, wherein the TSA is selected from the group consisting of EGFR, mesothelin (MSLN) and cancer embryonic antigen (CEA or CEACAM5).

100. The method of 97, wherein the antigen binding domain binds to a transferrin receptor (TFRC) antigen.

101. The method of 97, wherein the antigen binding domain binds to a pan-HLA antigen.

102. The method of any one of claims 91-101, wherein the plurality of immune cells are isolated.

103. The method of any one of claims 91-102, wherein the plurality of immune cells are non-natural.

104. The method of any one of claims 91-103, wherein the immune cells comprising the inhibitory receptor have reduced side effects when compared to immune cells that express the CAR or TCR but do not express the inhibitory receptor.

105. The method of any one of claims 91-104, wherein the disease is cancer.

106. A pharmaceutical composition, comprising a therapeutically effective amount of the immune cell of any one of 70-83 and a pharmaceutically acceptable carrier or diluent.

107. The immune cell of any one of 70-83 for use as a medicament.

108. The immune cell of any one of 70-83 for use in a method of treatment of a subject in need thereof.

109. A kit, comprising the polynucleotide of claim 68 or the vector of claim 69, or the immune cell of any one of 70-83.

110. The kit of claim 109, further comprising instructions for use.