Compositions and methods for treating HER2-positive cancer

Abstract

The present disclosure relates to immune cells, and methods of making and using same, that contain a dual receptor system that responds to loss of heterozygosity in target cells, where the first receptor comprises an activating receptor specific for the HER2 antigen and the second receptor comprises an inhibitory receptor specific for an antigen lost in cancer but not in wild-type cells that suppresses activation of the immune cell by the first receptor.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 149,952, filed February 16, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application has been filed with a Sequence Listing in electronic format. The Sequence Listing is provided as the file A2BI_035_01WO_Seq_List_ST25.txt, created on February 16, 2022, and is 5,413,164 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. [Background technology]

[0003] Cell therapy is a powerful tool for the treatment of various diseases, especially cancer. In conventional adoptive cell therapy, immune cells are engineered to express specific receptors, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which induce the activation of immune cells against cellular targets through receptor interaction with ligands expressed by the target cells. Identification of suitable target molecules remains difficult, as many targets are expressed in normal tissues. One example of such a target is erb-b2 receptor tyrosine kinase 2 (ERBB2, or HER2), which is expressed on the surface of many solid tumors, as well as normal epithelial cells. This expression can result in toxicity when transplanted cells target normal tissues that express the HER2 target molecule. Clinical trials with HER2 CARs have shown serious side effects, and the only current antibody therapy targeting HER2 is only effective in breast cancer, where HER2 levels are very high. Thus, there is a need in the art for compositions and methods useful for the treatment of diseases, especially cancer, by adoptive cell therapy. Summary of the Invention

[0004] The present disclosure provides an immune cell responsive to loss of heterozygosity in a cancer cell, the immune cell comprising: (a) an activator receptor comprising an extracellular ligand binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen; and (b) an inhibitory receptor comprising an extracellular ligand binding domain specific for a non-target antigen that is not expressed by the cancer cell due to the loss of heterozygosity in the cancer cell.

[0005] In some embodiments of the immune cells of the present disclosure, the non-target antigen is an HLA class I allele or a minor histocompatibility antigen (MiHA). In some embodiments, the HLA class I allele comprises HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the HLA class I allele is HLA-A*02.

[0006] In some embodiments of the immune cells of the present disclosure, the target antigen is HER2 or a peptide antigen of HER2 in complex with the major histocompatibility complex class I complex (MHC I).

[0007] In some embodiments of the immune cells of the present disclosure, the cancer cells express HER2. In some embodiments, the cancer cells are breast cancer cells, bladder cancer cells, ovarian cancer cells, gastric cancer cells, salivary duct cancer cells, non-small cell lung cancer cells, pancreatic cancer cells, or colon cancer cells. In some embodiments, the cancer cells are breast cancer cells or gastric cancer cells.

[0008] In some embodiments of the immune cells of the present disclosure, the HLA-A*02 non-target antigen is expressed by healthy cells of the subject. In some embodiments, the healthy cells of the subject express both the target antigen and the HLA-A*02 non-target antigen. In some embodiments, both the activator receptor and the inhibitory receptor specifically activate the immune cell in the presence of cancer cells.

[0009] In some embodiments of the immune cells of the present disclosure, the immune cells are T cells. In some embodiments, the T cells are CD8+CD4- T cells.

[0010] In some embodiments of the immune cells of the present disclosure, the HER2 antigen comprises a sequence or subsequence that is at least 95% identical to the sequence or subsequence of any one of SEQ ID NOs: 2-29.

[0011] In some embodiments of the immune cells of the present disclosure, the activator receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the extracellular ligand-binding domain of the activator receptor comprises an antibody fragment, a single chain Fv antibody fragment (scFv), a beta chain variable domain (Vβ), or a TCR alpha chain variable domain and a TCR beta chain variable domain. In some embodiments, the extracellular ligand-binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complementarity determining regions (CDRs) selected from the group consisting of SEQ ID NOs: 30-42. In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of GFNIKDTYIH (SEQ ID NO: 36), ARIYPTNGYTRYADSVKG (SEQ ID NO: 38), and SRWGGDGFYAMD[Y / V] (SEQ ID NO: 40) or (SEQ ID NO: 41), and the VL region comprises one or more CDR sequences selected from the group consisting of RASQDVNTAVA (SEQ ID NO: 30), SASFLY (SEQ ID NO: 32), and QQHYTTPP (SEQ ID NO: 34). In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of NYGMN (SEQ ID NO: 37), WINTSTGESTFADDFKG (SEQ ID NO: 39), and WEVYHGYVPY (SEQ ID NO: 42), and the VL region comprises one or more CDR sequences selected from the group consisting of KASQDVYNAVA (SEQ ID NO: 31), SASSRYT (SEQ ID NO: 33), and QQHFRTPFT (SEQ ID NO: 35). In some embodiments, the VH region comprises the CDR sequences of GFNIKDTYIH (SEQ ID NO: 36), ARIYPTNGYTRYADSVKG (SEQ ID NO: 38), and SRWGGDGFYAMD[Y / V] (SEQ ID NO: 40) or (SEQ ID NO: 41), and the VL region comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO: 30), SASFLY (SEQ ID NO: 32), and QQHYTTPP (SEQ ID NO: 34).In some embodiments, the VH region comprises the CDR sequences of NYGMN (SEQ ID NO:37), WINTSTGESTFADDFKG (SEQ ID NO:39), and WEVYHGYVPY (SEQ ID NO:42), and the VL region comprises the CDR sequences of KASQDVYNAVA (SEQ ID NO:31), SASSRYT (SEQ ID NO:33), and QQHFRTPFT (SEQ ID NO:35). In some embodiments, the VH region comprises the CDR sequences of DTYIH (SEQ ID NO:313), RIYPTNGYTRYADSVKG (SEQ ID NO:314), and WGGDGFYAMDV (SEQ ID NO:315), and the VL region comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO:30), SASFLYS (SEQ ID NO:311), QQHYTTPPT (SEQ ID NO:312), and DTYIH (SEQ ID NO:313). In some embodiments, the scFv comprises a sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to or is identical to a sequence selected from the group consisting of SEQ ID NOs: 51, 53, 55, 57.59 and 61.

[0012] In some embodiments of the immune cells of the present disclosure, the CAR comprises a hinge sequence isolated or derived from CD8, CD28, IgG1, or IgG4, or a synthetic hinge. In some embodiments, the CAR comprises a transmembrane domain isolated or derived from CD8 or CD28. In some embodiments, the CAR comprises an intracellular domain isolated or derived from CD28, 4-1BB, or CD3z, or a combination thereof.

[0013] In some embodiments of the immune cells of the present disclosure, the inhibitory receptor comprises a TCR or a CAR. In some embodiments, the extracellular ligand-binding domain of the inhibitory receptor comprises an antibody fragment, a single chain Fv antibody fragment (scFv), a beta chain variable domain (Vβ), or a TCR alpha chain variable domain and a TCR beta chain variable domain. In some embodiments, the extracellular ligand-binding domain of the inhibitory receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise CDRs selected from the group consisting of SEQ ID NOs: 87-102. In some embodiments, the VH region comprises one or more CDRs selected from the group consisting of ASGYTFTSYHIH (SEQ ID NO: 95), WIYPGNVNTEYNEKFKGK (SEQ ID NO: 98) and EEITYAMDY (SEQ ID NO: 101), and the VL region comprises one or more CDRs selected from the group consisting of RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFSGVP[D / A]R (SEQ ID NO: 90) or (SEQ ID NO: 91), and FQGSHVPRT (SEQ ID NO: 92). In some embodiments, the VH region comprises one or more CDRs selected from the group consisting of SGYTFTSYHMH (SEQ ID NO: 97), WIYPGDGSTQYNEKFKG (SEQ ID NO: 100) and EGTYYAMDY (SEQ ID NO: 102), and the VL region comprises one or more CDRs selected from the group consisting of RSSQSIVHSNGNTYLD (SEQ ID NO: 89), KVSNRFSGVP[D / A]R (SEQ ID NO: 90) or (SEQ ID NO: 91) and MQGSHVPRT (SEQ ID NO: 94). In some embodiments, the scFv comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to or identical to a sequence selected from the group consisting of SEQ ID NOs: 63-74, 207, 209 and 211. In some embodiments, the VH region comprises the CDR sequences of SYHIH (SEQ ID NO: 566), WIYPGNVNTEYNEKFKG (SEQ ID NO: 567), and EEITYAMDY (SEQ ID NO: 101), and the VL region comprises the CDR sequences of RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFS (SEQ ID NO: 565), and FQGSHVPRT (SEQ ID NO: 92).

[0014] In some embodiments of the immune cells of the present disclosure, the inhibitory receptor comprises the LILRB1 intracellular domain or a functional variant thereof. In some embodiments, the inhibitory receptor comprises the LILRB1 hinge domain and transmembrane domain, or a functional variant thereof. In some embodiments, the inhibitory receptor comprises a sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to or is identical to YGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 254).

[0015] The present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the immune cells of the present disclosure. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient.

[0016] The present disclosure provides a pharmaceutical composition comprising the immune cells of the present disclosure for use as a medicament in the treatment of cancer.

[0017] The present disclosure provides a polynucleotide system comprising one or more polynucleotides comprising polynucleotide sequences encoding (a) an activator receptor comprising an extracellular ligand binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen, and (b) an inhibitory receptor comprising an extracellular ligand binding domain specific for a non-target antigen that is not expressed by a cancer cell due to loss of heterozygosity in the cancer cell.

[0018] The present disclosure provides a vector comprising the polynucleotide system of the present disclosure.

[0019] The present disclosure provides a method of treating a HER2+ cancer in a subject identified as having, or suspected of having, a loss of heterozygosity in an allele encoding a non-target antigen in the HER2+ cancer, comprising administering to the subject an immune cell or pharmaceutical composition of the present disclosure.

[0020] The present disclosure provides a method of producing an immune cell therapy comprising transforming an immune cell with a polynucleotide system or vector of the present disclosure.

[0021] The present disclosure provides a kit comprising the immune cells or pharmaceutical compositions of the present disclosure. In some embodiments, the kit further comprises instructions for use.

[0022] The present disclosure also provides a method of selectively killing HER2-positive tumor cells having loss of heterozygosity at alleles encoding non-target antigens in HER2-positive cancer, comprising contacting the HER2-positive tumor cells with a composition or immune cells, wherein the tumor cells are in mixed culture. In some embodiments, the non-target antigen is selected from the group consisting of HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-B*07, and HLA-C*07.

[0023] The disclosure also provides (a) an activator receptor comprising an extracellular ligand binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen, wherein the extracellular ligand binding domain of the activator receptor comprises: (i) a VH region comprising the CDR sequences of DTYIH (SEQ ID NO: 313), RIYPTNGYTRYADSVKG (SEQ ID NO: 314), and WGGDGFYAMDV (SEQ ID NO: 315); and (ii) a VL region comprising the CDR sequences of RASQDVNTAVA (SEQ ID NO: 30), SASFLYS (SEQ ID NO: 311), QQHYTTPPT (SEQ ID NO: 312), and DTYIH (SEQ ID NO: 313), and a light chain variable (VL) region. and (b) an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen not expressed by the cancer cell due to loss of heterozygosity in the cancer cell, wherein the extracellular ligand-binding domain of the inhibitory receptor comprises (i) a heavy chain variable (VH) region comprising CDR sequences of SYHIH (SEQ ID NO: 566), WIYPGNVNTEYNEKFKG (SEQ ID NO: 567), and EEITYAMDY (SEQ ID NO: 101), and (ii) a light chain variable (VL) region comprising RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFS (SEQ ID NO: 565), and FQGSHVPRT (SEQ ID NO: 92).

[0024] The present disclosure also provides an immune cell comprising: (a) an activator receptor comprising an extracellular ligand binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen, wherein the extracellular ligand binding domain of the activator receptor is an scFv comprising the sequence of SEQ ID NO: 51; and (b) an inhibitory receptor comprising an extracellular ligand binding domain specific for a non-target antigen that is not expressed by a cancer cell due to loss of heterozygosity in the cancer cell, wherein the extracellular ligand binding domain of the inhibitory receptor is an scFv comprising the sequence of SEQ ID NO: 209.

[0025] The disclosure also provides a method of treating HER2+ cancer in a subject identified as having, or suspected of having, a loss of heterozygosity at an allele encoding HLA-A*02, comprising the steps of: (a) an activator receptor comprising an extracellular ligand binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen, the extracellular ligand binding domain of the activator receptor comprising: (i) a VH region comprising the CDR sequences of DTYIH (SEQ ID NO: 313), RIYPTNGYTRYADSVKG (SEQ ID NO: 314), and WGGDGFYAMDV (SEQ ID NO: 315); (ii) a VH region comprising the CDR sequences of RASQDVNTAVA (SEQ ID NO: 30), SASFLYS (SEQ ID NO: 311), QQHYTTPPT (SEQ ID NO: 312), and DTYIH (SEQ ID NO: 313); and (b) an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen not expressed by a cancer cell due to loss of heterozygosity in the cancer cell, wherein the extracellular ligand-binding domain of the inhibitory receptor comprises (i) a heavy chain variable (VH) region comprising the CDR sequences of SYHIH (SEQ ID NO: 566), WIYPGNVNTEYNEKFKG (SEQ ID NO: 567), and EEITYAMDY (SEQ ID NO: 101), and (ii) a light chain variable (VL) region comprising RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFS (SEQ ID NO: 565), and FQGSHVPRT (SEQ ID NO: 92).

[0026] The disclosure also provides a method of treating HER2+ cancer in a subject identified as having, or suspected of having, a loss of heterozygosity at an allele encoding HLA-A*02, comprising administering to the subject an immune cell comprising: (a) an activator receptor comprising an extracellular ligand binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen, wherein the extracellular ligand binding domain of the activator receptor is an scFv comprising the sequence of SEQ ID NO: 51; and (b) an inhibitory receptor comprising an extracellular ligand binding domain specific for a non-target antigen that is not expressed by the cancer cell due to the loss of heterozygosity in the cancer cell, wherein the extracellular ligand binding domain of the inhibitory receptor is an scFv comprising the sequence of SEQ ID NO: 209.

[0027] The disclosure also provides a method of selectively killing HER2-positive tumor cells having a loss of heterozygosity at an allele encoding HLA-A*02, comprising: (a) an activator receptor comprising an extracellular ligand binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen, the extracellular ligand binding domain of the activator receptor comprising: (i) a VH region comprising the CDR sequences of DTYIH (SEQ ID NO: 313), RIYPTNGYTRYADSVKG (SEQ ID NO: 314), and WGGDGFYAMDV (SEQ ID NO: 315); and (ii) a VH region comprising the CDR sequences of RASQDVNTAVA (SEQ ID NO: 30), SASFLYS (SEQ ID NO: 311), QQHYTTPPT (SEQ ID NO: 312), and DTYIH (SEQ ID NO: 313), and a light chain variable (VL) region comprising the CDR sequences of and (b) an inhibitory receptor comprising an extracellular ligand binding domain specific for a non-target antigen not expressed by the cancer cell due to loss of heterozygosity in the cancer cell, wherein the extracellular ligand binding domain of the inhibitory receptor comprises (i) a heavy chain variable (VH) region comprising the CDR sequences of SYHIH (SEQ ID NO: 566), WIYPGNVNTEYNEKFKG (SEQ ID NO: 567), and EEITYAMDY (SEQ ID NO: 101), and (ii) a light chain variable (VL) region comprising the CDR sequences of RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFS (SEQ ID NO: 565), and FQGSHVPRT (SEQ ID NO: 92).

[0028] The disclosure also provides a method of selectively killing HER2-positive tumor cells having a loss of heterozygosity at an allele encoding HLA-A*02, comprising contacting the HER2-positive tumor cells with an immune cell comprising: (a) an activator receptor comprising an extracellular ligand binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen, wherein the extracellular ligand binding domain of the activator receptor is an scFv comprising the sequence of SEQ ID NO: 51; and (b) an inhibitory receptor comprising an extracellular ligand binding domain specific for a non-target antigen that is not expressed by the cancer cell due to the loss of heterozygosity in the cancer cell, wherein the extracellular ligand binding domain of the inhibitory receptor is an scFv comprising the sequence of SEQ ID NO: 209. [Brief description of the drawings]

[0029] 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 disclosure are utilized, and the accompanying drawings of which:

[0030] [Figure 1] Table comparing the activity of different chimeric antigen receptors with their target HER2. FRP5* and 4D5* each have a single amino acid change in the VH domain compared to FRP5 and 4D5, respectively. IgG4(EQ) has a mutated CH2 region to avoid Fc receptor binding. Linker sequence: GGGSSGGGSG (SEQ ID NO: 1). [Figure 2-1] 1 is a plot showing HER2 activator EC50 in Jurkat cells as measured by antigen titration on a solid substrate. HER2 CAR2 showed no activity. [Figure 2-2] 1 is a plot showing HER2 activator EC50 in Jurkat cells as measured by HCT.116 target cell number titration. HER2 CAR2 and HER2 CAR3 showed no activity. [Figure 2-3]2A and 2B are plots and tables showing HER2 activator EC50 in Jurkat cells as measured by titration of target HER2 mRNA. HER2 CAR2 (C297) showed no activity in antigen titration on solid substrates (FIG. 2A) or target cell number titrations (FIG. 2B) and was further engineered with different hinges (CT1094, CT1095). [Diagram 3] 1 is a pair of plots showing that activation of Jurkat cells co-expressing HER2 (CT292) CAR1 activator and HLA-A*02 specific inhibitory receptor (HLA-A*02 blocker) is blocked by target cells expressing ligands for both the activator and blocker receptors. Target A: activator ligand (HER2), target B: inhibitory ligand (HLA-A*02). Left panel shows HCT.116 target cells, right panel: HeLa target cells. [Figure 4] 1 is a pair of plots showing that activation of Jurkat cells co-expressing the HER2 (CT299) CAR1 activator and the HLA-A*02 specific inhibitory receptor (HLA-A*02 blocker) is blocked by HCT.116 target cells expressing ligands for both the activator and blocker receptors. Target A: activator ligand (HER2), target B: inhibitory ligand (HLA-A*02). [Diagram 5] A series of fluorescence-activated cell sorting (FACS) plots showing expression of HER2 CAR1 or HER2 CAR4 alone or in combination with HLA-A*02 blocking agent in Jurkat cells. [Figure 6] 1 is a series of FACS plots showing expression of HER2 CAR1 (CT292) in primary T cells alone and in combination with HLA-A*02 blocker constructs mPA2.1 (C1765), humanized (hu) PA2.1.14 (C2162), and humanized PA2.1.18 (C2163). HER2 CAR1 and PA2.1 HLA-A*02 blockers can be expressed and enriched in primary T cells. [Figure 7]1 is a set of plots and images showing that HER2 CAR1 (CT292)-mediated primary T cell killing of HeLa target cells is inhibited when T cells express HLA-A*02 inhibitory receptors (as indicated) and HeLa target cells co-express HER2 activator ligand and HLA-A*02 blocker target ligand at a ratio of 1:40. Target A: activator ligand (HER2), target B: inhibitor ligand (HLA-A*02), hu: humanized, SW: selectivity window. Images were taken at 40 hours and show HeLa cell expression of activator (left image of each panel) or a combination of activator and inhibitor ligands (right panel of each image). Effector and target cells were at a ratio of 1:1. [Figure 8] 1 is a set of plots and images showing that HER2 CAR1 (CT292)-mediated primary T cell killing of HCT.116 target cells is inhibited when T cells express HLA-A*02 inhibitory receptors (as indicated) and HCT.116 target cells co-express a HER2 activator ligand and an HLA-A*02 blocker target ligand at a 1:4 ratio. Target A: activator ligand (HER2), target B: inhibitor ligand (HLA-A*02), hu: humanized, SW: selectivity window. Images were taken at 40 hours and show HCT.116 cell expression of activator (left image of each panel) or a combination of activator and inhibitor ligands (right panel of each image). Effector and target cells were at a 1:1 ratio. [Figure 9]FIG. 1 is a series of plots showing that inhibition of primary T cell activation by HER2 CAR1 (CT292) through the HLA-A*02 inhibitory receptor is reversible. Primary T cell activation was assayed using HCT.116 target cells. Target AB: HCT.116 cells expressing both activator (HER2) and inhibitory (HLA-A*02) ligands, target A: HCT.116 cells expressing HER2, RD: round, E: T stands for effector:target. Primary T cells expressing only the HER2 CAR and not the HLA-A*02 inhibitory receptor were depleted by round 3, whereas primary T cells expressing both the HER2 CAR and the HLA-A*02 inhibitory receptor were not depleted. [Figure 10] FIG. 1 shows an experiment assaying the ability of primary T cells expressing a HER2 CAR and an HLA-A*02 inhibitory receptor to kill target cells in a mouse xenograft model of HER2-positive cancer that has lost expression of HLA-A*02. [Figure 11] A pair of plots showing that primary T cells expressing a HER2 CAR and an HLA-A*02 inhibitory receptor selectively kill tumor cells but not normal cells in a mouse xenograft model of HER2-positive cancer that has lost expression of HLA-A*02. [Figure 12]

[0023] Figure 1 is a series of FACS plots showing expression of activator and repressor ligands by HeLa, HCT.116 and MS751 target cells. Cell surface antigens were quantified using the Agilent QIFI kit. [Figure 13] 1 is a table summarizing antigen density and activator to blocker antigen ratios on HeLa, HCT.116 and MS751 target cells. Cell surface antigens were quantified using the Agilent QIFI kit. [Figure 14]A series of plots showing the sensitivity of nine receptor pairs, including scFv CT292 (left column), CT299 (middle column), or CT1094 (right column) paired with no module (CAR only), mouse blocker (C1765), humanized blocker 1 (C2162), or humanized blocker 2 (C2163), to activators (top) and blockers (bottom). [Figure 15] 1 is a series of plots showing killing of HeLa target cells by primary T cells transduced with six candidate activator-blocker pairs at an E:T ratio of 1:1. The activator-blocker pairs include scFvs of CT292 (left column), CT299 (middle column), or CT1094 (right column) with either the murine (C1765, top) or humanized (C2162, bottom) form of the blocker. [Figure 16] Paired plots showing cell killing of tumor (solid circles) or normal (open circles) A375 target cells by CT1118 or CT1121 constructs. This analysis was performed on two HLA-A*02- T cell donors (left vs. right). [Figure 17] A pair of plots showing cell killing of HeLa (left panel) or A375 (right panel) target cells by primary T cells transfected with HER2 CAR1 (CT292) and CAR / human blocker (CT2162) at a 1:1 E:T ratio. [Figure 18] A pair of plots showing cell killing of HER2+ / HLA-A*02- HeLa target cells (left panel) or normal HeLa target cells (right panel) by primary T cells transfected with HER2 CAR1 or CT2162 in three 48-h rounds of tumor re-challenge at an E:T ratio of 1:1. [Figure 19] A series of plots showing the reversibility of the ability of HER2 CAR1 and CT2162 to kill tumor (target A, HER2+ / HLA-A*02-) or normal (target AB, HER2+ / HLA-A*02+) HeLa cells in a 48 hour round at an E:T ratio of 1:1. Arrows indicate the order of exposure: tumor→normal→tumor (top panel) vs. normal→top→normal tumor (bottom panel). [Figure 20]1 is a series of images showing the reversibility of the ability of untransduced T cells (UTD), HER2 CAR1 T cells, or CT2162 cells to kill HER2+ / HLA-A*02- HeLa cells ("tumor") or HER2+ / HLA-A*02+ HeLa cells ("normal") at a 1:1 E:T ratio. Images of target cells at t=48 hours are shown for each sequence: left panel: tumor→normal→tumor vs. right panel: normal→tumor→normal tumor. Scale bar=50 μm. [Figure 21] Plot showing selective killing of tumor (target A, solid circles) and normal (target AB, open circles) HeLa target cells by HER2 CAR1 and CT2162 cells at a 1:1 ratio over three 48 hour rounds. [Figure 22] A series of images showing selective killing of tumor but not normal HeLa target cells in mixed cultures after 20 h of co-culture with untransduced (UTD, left), CT292 (center), or CT118 (right) cells. [Diagram 23] A series of images showing selective killing of tumor but not normal HeLa target cells upon re-challenge of the mixed cultures (i.e., round 2) after 24 h of co-culture with untransduced (UTD, left), CT292 (center), or CT118 (right) cells. [Figure 24] Quantitative plots of HER2 and HLA-A2 expression in normal, tumor (assuming LOH of HLA-A2), and cell lines (stars) are shown. The EC50 and IC50 of CT1118 are marked with dashed lines. The lighter dashed lines indicate the range of EC50 and IC50 measured. A375, the cell line used in the in vivo studies described in Example 12, has similar HER2 and HLA-A2 expression as the EC50 and IC50 of HER2 CT1118, respectively. [Diagram 25] Tumor volumes in mice bearing tumor (left panel) and normal (right panel) A375 xenografts following treatment with untransduced ("UTD") cells, CT292 cells, and CT1118 cells are shown. [Figure 26-1]1 shows Jurkat NFAT-luciferase (JNL) cell suppression by CT292 paired with either HLA-A*03, HLA-A*11, or HLA-B*07 blocking agents as measured by mRNA titration assay. [Figure 26-2] Same as above. [Figure 26-3] Same as above. [Figure 27] Figure 1 shows titrated HLA-A*11:01 or HLA-B*07:02 protein on the surface of HeLa cells upon mRNA transfection. HeLa cells were transfected with two-fold serial dilutions of mRNA encoding either FLAG-tagged HLA-A*11:01 or FLAG-tagged HLA-B*07:02, with the highest dose being 2000 ng / 200,000 HeLa cells. The bottom panel contains 0 ng of mRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present disclosure describes engineered receptors, such as chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), adoptive cell therapy, and methods of their use. The engineered receptors can target the HER2 antigen and activate immune cells that have been genetically modified to express the HER2-targeted receptor. Immune cells expressing a HER2-targeted activator receptor can be used in compositions, for example, as part of adoptive cell therapy in the treatment of cancers that are HER2 positive. It is also contemplated that the immune cells expressing a HER2-targeted activator receptor further comprise a second inhibitory receptor. The second inhibitory receptor can prevent or inhibit the activation of the immune cell mediated by the activator receptor. For example, an immune cell expressing an activating receptor that targets HER2 and an inhibitory receptor that specifically binds to a separate antigen, for example, an MHC class I antigen, is activated in the presence of cells expressing HER2, but not in the presence of cells expressing both HER2 and the HLA class I antigen that is the target of the inhibitory receptor. Selective activation and inhibition of immune cells expressing both activating and inhibitory receptors is useful to reduce toxicity in adoptive cell therapy.The inventors have found that this strategy can be used using the HER2-targeting activating receptor and inhibitory receptor described herein.Both activating and inhibitory receptors can be, for example, chimeric antigen receptors (CARs) or T cell receptors (TCRs).

[0032] The term "chimeric antigen receptor" or "CAR" as used herein may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and includes engineered receptors that graft artificial specificity onto specific immune effector cells, such as helper T cells (CD4+), cytotoxic T cells (CD8+), or NK cells. CARs may be utilized to confer the specificity of a monoclonal antibody to T cells, thereby allowing large numbers of specific T cells to be generated, for example, for use in adoptive cell therapy. In certain embodiments, CARs direct the specificity of cells to a tumor-associated antigen, such as HER2. In some embodiments, CARs include an extracellular domain that includes an intracellular signaling domain, a transmembrane domain, and an antigen-binding region. In some embodiments, CARs include a fusion of a single chain variable fragment (scFv) or scFab derived from a monoclonal antibody fused to a transmembrane domain and an intracellular signaling domain. The fusion may also include a hinge. Either heavy-light (HL) and light-heavy (LH) scFvs may be used. The specificity of the CAR design may come from the ligand (e.g., peptide) of the receptor. Depending on the type of intracellular domain, the CAR may be an activator receptor or an inhibitory receptor. In some embodiments, for example, when the CAR is an activator receptor, the CAR contains domains for additional costimulatory signaling, such as CD3, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some embodiments, molecules may be co-expressed with the CAR, including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally eliminate T cells upon addition of a prodrug, homing receptors, cytokines, and cytokine receptors. As used herein, the properties attributed to a chimeric antigen receptor may be understood to refer to the receptor itself or to a host cell that contains the receptor.

[0033] As used herein, "TCR," sometimes referred to as "TCR complex" or "TCR / CD3 complex," refers to a protein complex that includes one or more of the TCR alpha chain, TCR beta chain, and invariant CD3 chain (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 that binds to peptide-MHC complexes. When the TCR alpha / beta heterodimer engages peptide-MHC, a conformational change in the TCR complex within the associated invariant CD3 subunit is 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, and CD3 epsilon and CD3 gamma for the heterodimer, as well as two CD3 zetas form homodimers.

[0034] The term "stimulation" refers to a primary response induced by the binding of a stimulatory domain or molecule (e.g., the TCR / CD3 complex) to 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, for example, altered expression of certain molecules and / or rearrangements of cytoskeletal structures.

[0035] The term "stimulatory molecule" or "stimulatory domain" refers to a molecule or portion thereof that, when naturally expressed by a T cell, provides a primary cytoplasmic signaling sequence that stimulatorily regulates activation of the TCR complex for at least some aspects of the T cell signaling pathway. The TCR alpha and / or TCR beta chains of a wild-type TCR complex do not contain a stimulatory domain and require association with a CD3 subunit, such as CD3 zeta, to initiate signaling. In one embodiment, the primary stimulatory signal is initiated by binding of the TCR / CD3 complex with, for example, a peptide-bound major histocompatibility complex (MHC), which results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. One or more stimulatory domains 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.

[0036] As used herein, a "domain capable of providing a stimulatory signal" refers to any domain capable of providing, directly or indirectly, a stimulatory signal that enhances or increases the effectiveness of signal transduction mediated by the TCR complex to enhance at least some aspects of T cell signaling. A domain capable of providing a stimulatory signal can provide this signal directly, for example, a domain capable of providing a stimulatory signal is a primary stimulatory domain or a costimulatory domain. Alternatively, or in addition, a domain capable of providing a stimulatory signal can act indirectly. For example, the domain can be a scaffold that provides an enzymatic activity, such as a kinase activity, that recruits stimulatory proteins to the TCR or acts through a downstream target to provide a stimulatory signal.

[0037] As used herein, a "domain capable of providing an inhibitory signal" refers to any domain capable of providing an inhibitory signal that directly or indirectly inhibits or reduces efficacy signaling mediated by the TCR complex. A domain capable of providing an inhibitory signal can completely or partially reduce or block at least some aspects of T cell signaling or function. A domain capable of providing an inhibitory signal can provide this signal directly, e.g., a domain capable of providing an inhibitory signal provides a primary inhibitory signal. Alternatively, or in addition, a domain capable of providing a stimulatory signal can act indirectly. For example, the domain can recruit additional inhibitory proteins to the TCR or provide an enzymatic activity that acts through a downstream target to provide an inhibitory signal.

[0038] 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. Thus, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the like, as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98%, or 99% identity, including 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.

[0039] Generally, "sequence identity" or "sequence homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity 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, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, an advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990), and is discussed in Altschul, et al., J. Mol. Biol. 215: 403-410 (1990), Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine the percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with, for example, the blastp program, on short query sequences. The desired range of sequence identity is approximately 80% to 100%, and integer values ​​therebetween.Typically, the percent identity between the disclosed and claimed sequences is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0040] As used herein, a "subsequence" refers to a length of contiguous amino acids or nucleotides that form a portion of a sequence described herein. A subsequence, when aligned to a full-length sequence, may be identical to a portion of the full-length sequence, or may be less than 100% identical to the portion of the full-length sequence to which it aligns (e.g., it may be 50% to 90% identical to the full sequence).

[0041] The term "exogenous" is used herein to refer to any molecule, including nucleic acids, proteins or peptides, small molecules, etc., that originate outside of an organism. In contrast, the term "endogenous" refers to any molecule that originates within an organism (i.e., is naturally produced by the organism).

[0042] 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 that encode them are operably linked, preferably in the same open reading frame.

[0043] A "promoter" is a DNA sequence required to turn a gene on or off. A promoter is located immediately upstream of and / or overlapping with a transcription initiation site, and is usually about 100 to several hundred base pairs in length.

[0044] All publications and patents mentioned in this specification are incorporated herein 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, this application, including any definitions herein, will control. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not and should not be considered as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0045] In this description, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value in the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated. The term "about," when immediately preceding a number or numerical value, means that the number or numerical value is within a range of plus or minus 10%.

[0046] antigen The disclosed activator receptors can specifically bind to the erb-b2 receptor tyrosine kinase 2 (HER2) antigen. The HER2 antigen, also known as Neu, ErbB2, and other designations, is a receptor tyrosine kinase that upon activation forms homo- or heterodimers with other ErbB family members to promote intracellular signaling leading to survival and proliferation. HER2 is overexpressed or has differential expression of splice variants and isoforms, which contribute to the mechanisms driving various cancers, such as breast cancer, lung cancer, and other cancers.

[0047] In some embodiments, the target antigen is a peptide antigen of HER2 in a complex with the major histocompatibility class I complex (MHC I) or the major histocompatibility class II complex (MHC II). In some embodiments, the HER2 peptide bound to MHC I comprises KIFGSLAFL (SEQ ID NO: 309). In some embodiments, the HER2 peptide bound to MHC II comprises GSPYVSRLLGICL (SEQ ID NO: 310). In some embodiments, the HER2 antigen comprises a sequence or subsequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or identical to any one of SEQ ID NOs: 2-29. However, all HER2 isoforms and sequences and subsequences thereof that act as HER2 antigens are contemplated within the scope of the present disclosure. In some embodiments, the HER2 antigen comprises a protein comprising any one of SEQ ID NOs: 2-29, or a peptide fragment of any one of SEQ ID NOs: 2-29. In some embodiments, the HER2 antigen comprises a peptide fragment of any one of SEQ ID NOs: 2-29 in complex with the major histocompatibility class I complex (MHC-I).

[0048] Any cancer that contains cancer cells that express the HER2 antigen is a HER2 positive (HER2+) cancer. In some embodiments, the cancer cells are breast cancer cells, bladder cancer cells, ovarian cancer cells, gastric cancer cells, salivary duct cancer cells, non-small cell lung cancer cells, pancreatic cancer cells, or colon cancer cells. In some embodiments, the cancer cells are breast cancer cells or gastric cancer cells.

[0049] Engineered receptors The present disclosure provides activator receptors and inhibitory receptors comprising an extracellular region, the extracellular region comprising a first ligand binding domain capable of specifically binding to a first ligand that activates or promotes the activation of an effector cell expressing the receptor. The present disclosure further provides inhibitory receptors comprising a second ligand binding domain capable of binding to a second ligand, the binding of the second ligand by the second ligand binding domain inhibits, reduces, or prevents activation of the effector cell even in the presence of the first receptor bound to the first ligand. The first ligand may also be referred to as an activator ligand. The second ligand may also be referred to as an inhibitory ligand. Activator receptors and inhibitory receptors that bind to these ligands may also be generally referred to herein as engineered receptors. Engineered receptors may refer to any of the chimeric antigen receptors (CARs) and T cell receptors (TCRs) described in the present disclosure. Engineered receptors may also refer to any receptors designed using the binding domains, hinge regions, transmembrane domains, and / or cytoplasmic domains described herein. The engineered receptors are sometimes referred to herein as fusion proteins.

[0050] Activator Ligand The present disclosure provides a first engineered receptor comprising a first ligand, an activator, and a first ligand binding domain that binds to the first activator ligand. In some embodiments, the first engineered receptor is an activator receptor. In some embodiments, the activator receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the first activator ligand is a HER2 antigen. In some embodiments, the HER2 antigen comprises a sequence or subsequence that shares at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of or is identical to any one of SEQ ID NOs: 2-29. Exemplary HER2 sequences are shown in Table 1. [Table 1]

[0051] In some embodiments, the activator antigen is a peptide antigen of HER2 in complex with the major histocompatibility class I complex (MHC I). In some embodiments, the activator antigen is a peptide antigen of HER2 that is not complexed with the major histocompatibility class I complex (MHC I).

[0052] As used herein, "activator" or "activator ligand" refers to a first ligand that binds to a first activator ligand binding domain (LBD) of an engineered receptor of the present disclosure, such as a CAR or TCR, thereby mediating activation of a T cell expressing the activator receptor. An activator ligand is expressed by a target cell, e.g., a cancer cell, and may also be expressed more broadly than just on target cells. For example, an activator may be expressed on some or all types of normal non-target cells, such as epithelial cells.

[0053] In some embodiments, the activator ligand is expressed by a target cell and not expressed by a non-target cell (i.e., a normal cell that is not targeted by adoptive cell therapy). In some embodiments, the target cell is a cancer cell and the non-target cell is a non-cancer cell.

[0054] In some embodiments, the activator ligand has high cell surface expression on target cells. This high cell surface expression confers the ability to deliver a large activation signal. Methods for measuring cell surface expression are known to those of skill in the art and include, but are not limited to, immunohistochemistry using an appropriate antibody against the activator ligand followed by microscopy or fluorescence activated cell sorting (FACS).

[0055] The activator ligand is present on all target cells, hi some embodiments, the target cells are cancer cells, such as HER2+ cancer cells in HER2+ breast cancer.

[0056] In some embodiments, the activator ligand is present on a plurality of target cells. In some embodiments, the target cells are cancer cells. In some embodiments, the activator ligand is present on at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% of the target cells. In some embodiments, the activator ligand is present on at least 95% of the target cells. In some embodiments, the activator ligand is present on at least 99% of the target cells.

[0057] In some embodiments, the first activator ligand is expressed by a plurality of target cells and a plurality of non-target cells, hi some embodiments, the plurality of non-target cells express both the first activator ligand and the second repressor ligand.

[0058] In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:100 to about 100:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:50 to about 50:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:40 to about 40:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:30 to about 30:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:20 to about 2:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:10 to about 10:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:5 to about 5:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:3 to about 3:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of the first ligand to the second ligand of about 1:2 to about 2:1. In some embodiments, the first activator ligand and the second repressor ligand are present on the plurality of non-target cells in a ratio of about 1:1.

[0059] Extracellular Ligand Binding Domain The present disclosure provides a chimeric antigen receptor (CAR) comprising a polypeptide. In some embodiments, the polypeptide comprises a ligand binding domain, such as an antigen binding domain. Suitable antigen binding domains include, but are not limited to, antigen binding domains derived 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 contemplated within the scope of the present disclosure.

[0060] "Extracellular domain" as used herein refers to the extracellular portion of a protein. For example, the TCR alpha and beta chains each contain an extracellular domain that includes constant and variable regions involved in peptide-MHC recognition. An "extracellular domain" can also include a fusion domain, e.g., a fusion between an endogenous extracellular domain of a TCR subunit and an additional domain that can bind to and target a specific antigen.

[0061] The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins, or fragments thereof, of polyclonal or monoclonal origin, and can be derived from natural or recombinant sources.

[0062] The term "antibody fragment" or "antibody binding domain" refers to at least a portion of an antibody or a recombinant variant thereof that contains the antigen-binding domain, i.e., the antigen-determining variable region of an intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and a defined epitope thereof. 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 as "sdAb") (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.

[0063] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, wherein the light and heavy chain variable regions are linked contiguously 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.

[0064] "Heavy chain variable region" or "VH" (or "VHH" in the case of single domain antibodies, e.g., nanobodies) with respect to antibodies refers to the fragment of a heavy chain containing the three CDRs interposed between adjacent stretches known as framework regions, which are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0065] As used herein, unless otherwise specified, an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and an scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0066] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa ("K") and lambda ("λ") light chains refer to the two major antibody light chain isotypes.

[0067] The term "recombinant antibody" refers to an antibody produced using recombinant DNA techniques, e.g., an antibody expressed by a bacteriophage or yeast expression system. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence techniques that are available and well known in the art.

[0068] In some embodiments, for example, those embodiments in which the receptor comprises a first and a second polypeptide and the antigen binding domain is isolated or derived from a T cell receptor (TCR) extracellular domain or an antibody.

[0069] In some embodiments, the extracellular ligand binding domain of the activator receptor comprises an antibody fragment, a single chain Fv antibody fragment (scFv), a beta chain variable domain (Vβ), or a TCR alpha chain variable domain and a TCR beta chain variable domain. In some embodiments, the extracellular ligand binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the VH and VL regions comprise complementarity determining regions (CDRs) selected from the group of CDRs disclosed in Table 2. [Table 2]

[0070] In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of GFNIKDTYIH (SEQ ID NO: 36), ARIYPTNGYTRYADSVKG (SEQ ID NO: 38), and SRWGGDGFYAMD[Y / V] (SEQ ID NO: 40) or (SEQ ID NO: 41), and the VL region comprises one or more CDR sequences selected from the group consisting of RASQDVNTAVA (SEQ ID NO: 30), SASFLY (SEQ ID NO: 32), and QQHYTTPP (SEQ ID NO: 34).

[0071] In some embodiments, the VH region comprises one or more CDR sequences selected from the group consisting of NYGMN (SEQ ID NO: 37), WINTSTGESTFADDFKG (SEQ ID NO: 39) and WEVYHGYVPY (SEQ ID NO: 42), and the VL region comprises one or more CDR sequences selected from the group consisting of KASQDVYNAVA (SEQ ID NO: 31), SASSRYT (SEQ ID NO: 33), and QQHFRTPFT (SEQ ID NO: 35).

[0072] In some embodiments, the VH region comprises the CDR sequences of GFNIKDTYIH (SEQ ID NO: 36), ARIYPTNGYTRYADSVKG (SEQ ID NO: 38), and SRWGGDGFYAMDY (SEQ ID NO: 40) or SRWGGDGFYAMDV (SEQ ID NO: 41), and the VL region comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO: 30), SASFLY (SEQ ID NO: 32), and QQHYTTPP (SEQ ID NO: 34).

[0073] In some embodiments, the VH region comprises the CDR sequences of NYGMN (SEQ ID NO: 37), WINTSTGESTFADDFKG (SEQ ID NO: 39), and WEVYHGYVPY (SEQ ID NO: 42), and the VL region comprises the CDR sequences of KASQDVYNAVA (SEQ ID NO: 31), SASSRYT (SEQ ID NO: 33), and QQHFRTPFT (SEQ ID NO: 35).

[0074] In some embodiments, the VH region comprises the CDR sequences DTYIH (SEQ ID NO: 313), RIYPTNGYTRYADSVKG (SEQ ID NO: 314), and WGGDGFYAMDV (SEQ ID NO: 315), and the VL region comprises the CDR sequences RASQDVNTAVA (SEQ ID NO: 30), SASFLYS (SEQ ID NO: 311), and QQHYTTPPT (SEQ ID NO: 312).

[0075] In some embodiments, the full length VH and VL regions comprise sequences disclosed in Table 3. In some embodiments, the binding domain comprises full length VH and VL regions on a single polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a full length VH region and a full length VL region. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a full length VL region and a full length VH region. In some embodiments, the full length VH and VL comprise sequences selected from Table 3. In some embodiments, the binding domain comprises SEQ ID NO:43 and SEQ ID NO:44. In some embodiments, the binding domain comprises SEQ ID NO:45 and SEQ ID NO:46. In some embodiments, the binding domain comprises SEQ ID NO:47 and SEQ ID NO:48. In some embodiments, the binding domain comprises SEQ ID NO:45 and SEQ ID NO:49. In some embodiments, the binding domain comprises SEQ ID NO:50 and SEQ ID NO:49. [Table 3]

[0076] In some embodiments, the binding domain is a HER2 scFv domain. In some embodiments, the HER2 scFv domain is selected from the sequences listed in Table 4. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO:51. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO:52. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO:53. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO:54. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO:55. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO:56. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO:57. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO:58. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO:59. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO:60. In some embodiments, the HER2 scFv domain comprises a polypeptide comprising SEQ ID NO: 61. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 62. In some embodiments, the HER2 scFv domain is encoded by a polynucleotide sequence comprising SEQ ID NO: 21803. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0077] In some embodiments, the HER2 scFv domain comprises the sequence of any one of SEQ ID NOs: 51, 53, 55, 57, 59 or 61, or a sequence having or being at least 90%, at least 95%, at least 97%, at least 99% identical thereto.

[0078] Inhibitory receptors The present disclosure provides a second inhibitory receptor comprising a second ligand, an inhibitor, and a second ligand-binding domain that binds to the inhibitor ligand. In some embodiments, the second inhibitory ligand is an antigen, and the second inhibitory receptor comprises an antigen-binding domain that specifically recognizes the second inhibitory antigen.

[0079] The present disclosure provides a second inhibitory receptor comprising an extracellular region comprising a second ligand binding domain capable of specifically binding to a second ligand that inhibits activation of an effector cell expressing the first and second receptors, the effector cell being activated by binding of the first ligand to the first activator receptor.

[0080] As used herein, "inhibitor" or "inhibitor ligand" refers to a second ligand that binds to the second ligand binding domain (inhibitor LBD) of an engineered receptor of the present disclosure, but inhibits activation of immune cells expressing the engineered receptor. Inhibitors are not expressed by target cells. Inhibitor ligands are also expressed in a plurality of normal non-target cells, including normal non-target cells that express activator ligands, thereby protecting these cells from the cytotoxic effects of adoptive cell therapy. Without wishing to be bound by theory, inhibitor ligands can block activation of effector cells through various mechanisms. For example, binding of an inhibitor ligand to an inhibitor LBD can block transmission of a signal that occurs upon binding of an activator ligand to an activator LBD, which results in activation of immune cells expressing an engineered receptor described herein in the absence of the inhibitor.

[0081] Alternatively, or in addition, binding of the inhibitory ligand to the second engineered receptor may cause loss of cell surface expression of the first activating receptor from the surface of immune cells comprising the two receptor systems described herein. Without wishing to be bound by theory, it is believed that upon immune cell engagement of the activator ligand and the inhibitory ligand on a normal cell, the inhibitory receptor causes removal of nearby activator receptor molecules from the immune cell surface. This process locally desensitizes the immune cell and reversibly raises its activation threshold. Immune cells that only engage with the activator ligand on a target cell trigger a local activation signal that is not impeded by the signal from the second inhibitory receptor. This local activation increases until the release of cytotoxic granules leads to selective cell death of the target cell. However, modulation of surface receptor expression levels may not be the only mechanism that inhibits immune cell activation by the first activator receptor. Without wishing to be bound by theory, other mechanisms may come into play, including but not limited to crosstalk between the activator receptor signaling pathway and the inhibitory receptor signaling pathway.

[0082] In some embodiments, the second ligand is not expressed by a target cell and is expressed by a non-target cell, hi some embodiments, the target cell is a cancer cell and the non-target cell is a non-cancer cell.

[0083] In some embodiments, the second inhibitor ligand is an antigen and the second ligand binding domain comprises an scFv domain.

[0084] In some embodiments, the second inhibitor ligand binding domain comprises a Vβ-only ligand binding domain.

[0085] In some embodiments, the second inhibitory ligand binding domain comprises an antigen binding domain isolated or derived from a T cell receptor (TCR), e.g., the second inhibitory ligand binding domain comprises TCR α and β chain variable domains.

[0086] In some embodiments, the second inhibitory ligand binding domain is an antigen-binding domain. Suitable antigen-binding domains include, but are not limited to, antigen-binding domains derived from antibodies, antibody fragments, scFvs, antigen-binding domains derived from T cell receptors, and the like.

[0087] repressor target The present disclosure provides an inhibitory receptor. In some embodiments, the inhibitory receptor comprises an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by cancer cells due to loss of heterozygosity in the cancer cells. In some embodiments, the non-target antigen is an HLA class I allele or a minor histocompatibility antigen (MiHA). In some embodiments, the HLA class I allele comprises HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the HLA class I allele is HLA-A*02. In some embodiments, the HLA-A*02 non-target antigen is expressed by healthy cells of the subject.

[0088] In some embodiments, the inhibitory receptor comprises an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by cancer cells due to loss of heterozygosity in the cancer cells.

[0089] Alternatively, or in addition, expression of an activator target and a repressor target may be correlated, ie, the two are expressed at similar levels on non-target cells.

[0090] In some embodiments, the second inhibitory ligand is a peptide ligand. In some embodiments, the second inhibitory ligand is a peptide antigen complexed with a major histocompatibility (MHC) class I complex (peptide MHC, or pMHC). Inhibitory ligands comprising a peptide antigen complexed with pMHC comprising any of HLA-A, HLA-B, HLA-C, or HLA-E are contemplated within the scope of the present disclosure.

[0091] In some embodiments, the repressor ligand is encoded by a gene that is absent or polymorphic in many tumors.

[0092] Methods for distinguishing between differential expression of the repressor ligand between target and non-target cells will be readily apparent to one of skill in the art. For example, the presence or absence of the repressor ligand in non-target and target cells can be assayed by immunohistochemistry with an antibody that binds the repressor ligand, followed by microscopy or FACS, RNA expression profiling of target and non-target cells, or DNA sequencing of non-target and target cells to determine whether the repressor ligand locus contains a mutation in either the target or non-target cells.

[0093] Loss of alleles due to loss of heterozygosity (LOH) Homozygous deletions in primary tumors are rare and small, making them unlikely to yield target B candidates. For example, in an analysis of 2218 primary tumors across 21 human cancer types, the top four candidates were cyclin-dependent kinase inhibitor 2A (CDKN2A), RB transcriptional corepressor 1 (RB1), phosphatase and tensin homolog (PTEN), and N3PB2. However, CDKN2A (P16) was deleted in only 5% of homozygous deletions 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 a gene in cancer cells by loss of hemizygosity occurs much more frequently.

[0094] In some embodiments, the second repressor ligand comprises an allele of a gene that is lost in the target cell by loss of heterozygosity. In some embodiments, the target cell comprises a cancer cell. Cancer cells undergo frequent genomic rearrangements, including duplications and deletions. These deletions can lead to the loss of one copy of one or more genes in the cancer cell.

[0095] As used herein, "loss of heterozygosity (LOH)" refers to a genetic alteration that occurs frequently in cancer, whereby one of the two alleles is deleted, leaving a single monoallelic (hemizygous) locus.

[0096] HLA class I alleles In some embodiments, the second inhibitory ligand comprises an HLA class I allele. Major histocompatibility complex (MHC) class I is a protein complex that presents antigens to cells of the immune system and induces an immune response. Human leukocyte antigens (HLA) corresponding to MHC class I are HLA-A, HLA-B, HLA-C, and HLA-E.

[0097] In some embodiments, the second inhibitory ligand comprises an HLA class I allele. In some embodiments, the second inhibitory ligand comprises an allele of HLA class I that is lost in the target cell via LOH. HLA-A is a group of human leukocyte antigens (HLA) of the major histocompatibility complex (MHC) encoded by the HLA-A locus. HLA-A is one of the three major types of human MHC class I cell surface receptors. The receptor is a heterodimer comprising a heavy α chain and a smaller β chain. The α chain is encoded by a variant of HLA-A, while the β chain (β2-microglobulin) is invariant. There are thousands of variant HLA-A genes, all of which fall within the scope of this disclosure.

[0098] In some embodiments, the second inhibitory 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 given a specific number (such as HLA-B27).

[0099] In some embodiments, the second inhibitory ligand comprises an HLA-C allele. HLA-C belongs to the HLA class I heavy chain paralogs. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). Over 100 HLA-C alleles have been described.

[0100] In some embodiments, HLA class I alleles have widespread or ubiquitous RNA expression.

[0101] In some embodiments, the HLA class I alleles have a known or generally high minor allele frequency.

[0102] In some embodiments, HLA class I alleles do not require peptide-MHC antigens, for example, when the HLA class I allele is recognized by a pan-HLA ligand binding domain.

[0103] In some embodiments, the second inhibitory ligand comprises an HLA-A allele. In some embodiments, the HLA-A allele comprises HLA-A*02. A variety of single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*02 are suitable for use in the embodiments. Such scFvs include, but are not limited to, the following murine and humanized scFv antibodies that bind to HLA-A*02 in a peptide-independent manner, as shown, for example, in Tables 5 and 6 below (complementarity determining regions are underlined): [Table 5-1] [Table 5-2]

[0104] In some embodiments, the second antigen binding domain comprises an scFv domain that binds to the HLA-A*02 antigen. In some embodiments, the scFv domain comprises the sequence of SEQ ID NO: 207, 209 or 211, or a sequence having or being at least 90%, at least 95%, at least 97%, at least 99% identical thereto. In some embodiments, the scFv domain comprises the sequence of SEQ ID NO: 207, 209 or 211. [Table 6-1] [Table 6-2] [Table 6-3]

[0105] In some embodiments, the non-target antigen comprises HLA-B*07. A variety of single variable domains known in the art or disclosed herein that bind to and recognize HLA-B*07 are suitable for use in the embodiments.

[0106] In some embodiments, the non-target antigen comprises HLA-A*11. A variety of single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*11 are suitable for use in the embodiments.

[0107] In some embodiments, the non-target antigen comprises HLA-A*01. Various single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*01 are suitable for use in the embodiments.

[0108] In some embodiments, the non-target antigen comprises HLA-A*03. A variety of single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*03 are suitable for use in the embodiments.

[0109] In some embodiments, the non-target antigen comprises HLA-C*07. A variety of single variable domains known in the art or disclosed herein that bind to and recognize HLA-C*07 are suitable for use in the embodiments.

[0110] Such scFvs include, for example, but are not limited to, the following murine and humanized scFv antibodies that bind non-target antigens (e.g., HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-B*07, or HLA-C*07) in a peptide-independent manner as shown in Table 7 below (where indicated, complementarity determining regions are underlined): [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11] [Table 7-12]

[0111] Exemplary heavy and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR-L1, CDR-L2 and CDR-L3, respectively) for the HLA-A*02 antigen binding domain are shown in Table 8 below. [Table 8]

[0112] In some embodiments, the scFv comprises a complementarity determining region (CDR) of any one of SEQ ID NOs: 87-102. In some embodiments, the scFv comprises a sequence at least 95% identical to ... heavy chain of the antibody comprises a heavy chain CDR of any one of SEQ ID NOs: 95-102, and the light chain of the antibody comprises a light chain CDR of any one of SEQ ID NOs: 87-94. In some embodiments, the heavy chain comprises a sequence at least 95% identical to a heavy chain portion of any one of SEQ ID NOs: 95-102, and the light chain comprises a sequence at least 95% identical to a light chain portion of any one of SEQ ID NOs: 87-94.

[0113] Additional exemplary heavy and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, or CDR-L1, CDR-L2 and CDR-L3, respectively) for the HLA-A*03, HLA-B*07, HLA-A*11, HLA-C*07 and HLA-A*01 ligand binding domains are shown in Table 9 below. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8]

[0114] In some embodiments, the second inhibitory ligand is HLA-A*02 and the inhibitory ligand binding domain comprises an HLA-A*02 antigen binding domain. In some embodiments, the second antigen binding domain binds to HLA-A*02 independently of a peptide in a pMHC complex that comprises HLA-A*02. In some embodiments, the HLA-A*02 antigen binding domain comprises an scFv domain. In some embodiments, the HLA-A*02 ligand binding domain comprises any one of SEQ ID NOs: 63-74, 207, 209, or 211. In some embodiments, the HLA-A*02 ligand binding domain comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 63-74, 207, 209, or 211.

[0115] In some embodiments, the non-target antigen comprises HLA-B*07 and the ligand binding domain of the second receptor comprises an HLA-B*07 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-B*07 independently of the peptide in a pMHC complex that comprises HLA-B*07. In some embodiments, the HLA-B*07 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-B*07 ligand binding domain comprises the sequence of any one of the B7 binding domains shown in Table 7. In some embodiments, the HLA-B*07 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-B*07 binding domains shown in Table 7Table 9. In some embodiments, the HLA-B*07 scFv comprises any one of the HLA-B*07 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-B*07 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-B*07 scFv sequences shown in Table 7.

[0116] In some embodiments, the non-target antigen comprises HLA-A*11 and the ligand binding domain of the second receptor comprises an HLA-A*11 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-A*11 independently of the peptide in a pMHC complex comprising HLA-A*11. In some embodiments, the HLA-A*11 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*11 ligand binding domain comprises the sequence of any one of the HLA-A*11 binding domains shown in Table 7. In some embodiments, the HLA-A*11 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-A*11 binding domains shown in Table 7. In some embodiments, the HLA-A*11 scFv comprises any one of the HLA-A*11 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*11 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*11 scFv sequences shown in Table 7.

[0117] In some embodiments, the non-target antigen comprises HLA-A*01 and the ligand binding domain of the second receptor comprises an HLA-A*01 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-A*01 independently of the peptide in a pMHC complex comprising HLA-A*01. In some embodiments, the HLA-A*01 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*01 ligand binding domain comprises the sequence of any one of the HLA-A*01 binding domains shown in Table 7. In some embodiments, the HLA-A*01 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-A*01 binding domains shown in Table 7. In some embodiments, the HLA-A*01 scFv comprises any one of the HLA-A*01 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*01 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*01 scFv sequences shown in Table 7.

[0118] In some embodiments, the non-target antigen comprises HLA-A*03 and the ligand binding domain of the second receptor comprises an HLA-A*03 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-A*03 independently of the peptide in a pMHC complex comprising HLA-A*03. In some embodiments, the HLA-A*03 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*03 ligand binding domain comprises the sequence of any one of the HLA-A*03 binding domains shown in Table 7. In some embodiments, the HLA-A*03 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-A*03 binding domains shown in Table 7. In some embodiments, the HLA-A*03 scFv comprises any one of the HLA-A*03 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*03 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*03 scFv sequences shown in Table 7.

[0119] In some embodiments, the non-target antigen comprises HLA-C*07 and the ligand binding domain of the second receptor comprises an HLA-C*07 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-C*07 independently of the peptide in a pMHC complex comprising HLA-C*07. In some embodiments, the HLA-C*07 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-C*07 ligand binding domain comprises the sequence of any one of the HLA-C*07 binding domains shown in Table 7. In some embodiments, the HLA-C*07 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-C*07 binding domains shown in Table 7. In some embodiments, the HLA-C*07 scFv comprises any one of the HLA-C*07 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-C*07 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-C*07 scFv sequences shown in Table 7.

[0120] Minor histocompatibility antigen In some embodiments, the second inhibitory ligand comprises a minor histocompatibility antigen (MiHA). In some embodiments, the second inhibitory ligand comprises an allele of MiHA that is lost in the target cell via LOH.

[0121] MiHA is a peptide derived from a protein that contains nonsynonymous differences between alleles and is presented by common HLA alleles. The nonsynonymous differences may result from SNPs, deletions, frameshift mutations or insertions in the coding sequence of the gene encoding MiHA. An exemplary MiHA may be about 9-12 amino acids long and can bind to MHC class I and MHC class II proteins. Binding of a TCR to an MHC complex that presents MiHA can activate T cells. The genetic and immunological properties of MiHA will be known to those of skill in the art. Candidate MiHAs are known peptides presented by known HLA class I alleles and known to elicit T cell responses in the clinic (e.g., in graft-versus-host disease, or transplant rejection), allowing patient selection by simple SNP genotyping.

[0122] In some embodiments, MiHA has broad or ubiquitous RNA expression.

[0123] In some embodiments, MiHA has a high minor allele frequency.

[0124] In some embodiments, MiHA comprises a peptide derived from a Y chromosome gene.

[0125] In some embodiments, the second repressor ligand comprises a MiHA selected from the group of MiHAs disclosed in Tables 10 and 11.

[0126] Illustrative, but non-limiting examples of MiHAs contemplated within the scope of the present invention are disclosed below in Table 10. The columns of Table 10, from left to right, show the name of the MiHA, the gene from which it is derived, the MHC class I variant in which the MiHA can be displayed, and the sequence of the peptide variant [A / B variants shown in brackets]. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6]

[0127] Illustrative, but non-limiting examples of MiHAs contemplated within the scope of the present invention are disclosed below in Table 11. The columns of Table 11, from left to right, show the name of the MiHA, the gene from which it is derived, the MHC class I variant in which the MiHA can be displayed, and the sequence of the peptide variant [A / B variants shown in brackets]. [Table 11-1] [Table 11-2]

[0128] In some embodiments, MiHA comprises HA-1, a peptide antigen having the sequence VL[H / R]DDLLEA (SEQ ID NO: 105) and derived from the Rho GTPase activating protein 45 (HA-1) gene.

[0129] An exemplary ligand binding domain that selectively binds to the HA-1 variant H peptide (VLHDDLLEA (SEQ ID NO: 195)) is shown below in Table 12. The TCR alpha and TCR beta sequences are separated by a P2A self-cleaving polypeptide of sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 196) with an N-terminal GSG linker. [Table 12-1] [Table 12-2]

[0130] In some embodiments, the second inhibitory ligand comprises HA-1(H). In some embodiments, the second inhibitory ligand binding domain 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: 197. In some embodiments, the TCR alpha and TCR beta variable domains separated by a self-cleaving polypeptide sequence comprise SEQ ID NO: 197, 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: 202, or a sequence having at least 80% identity thereto, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR alpha variable domain comprises SEQ ID NO: 198, 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: 199, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto.

[0131] Loss of Y chromosome antigens In some embodiments, the second repressor ligand comprises a peptide encoded by a Y chromosome gene, i.e., a gene on the Y chromosome. In some embodiments, the second repressor ligand comprises a peptide encoded by a Y chromosome gene that is lost in target cells due to loss of the Y chromosome (LoY). For example, about one-third of the MiHAs characterized are from the Y chromosome. The Y chromosome contains more than 200 protein-encoding genes, all of which are contemplated within the scope of the present disclosure.

[0132] As used herein, "loss of Y" or "LoY" refers to a genetic alteration that occurs frequently in tumors whereby one copy of part or all of the Y chromosome is deleted, resulting in the loss of Y chromosome-encoded genes.

[0133] Loss of the Y chromosome is known to occur in certain cancers. For example, a 40% somatic loss of the Y chromosome has been reported in renal clear cell carcinoma (Arseneault et al., Sci. Rep. 7:44876 (2017)). Similarly, clonal loss of the Y chromosome was reported in 5 of 31 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)). Furthermore, Y chromosome loss was associated with X chromosome disomy in 4 of 7 male patients with gastric cancer (Saal et al., Virchows Arch B Cell Pathol (1993)). Thus, the Y chromosome gene can be lost in various cancers and used as an inhibitory ligand with the engineered receptors of the present disclosure to target cancer cells.

[0134] Chimeric antigen receptors (CARs) In some embodiments, either the first or second engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the first and second engineered receptors are chimeric antigen receptors. All CAR architectures are contemplated within the scope of the present disclosure.

[0135] Extracellular domain In some embodiments, the extracellular domain of the CAR comprises an antigen-binding domain as described above.

[0136] Hinge Area In some embodiments, the CAR of the present disclosure comprises an extracellular hinge region. The incorporation of the hinge region can affect cytokine production from CAR-T cells and improve the expansion of CAR-T cells in vivo. Exemplary hinges can be isolated or derived from IgG, CD28 or CD8, for example, IgG1, among others. Exemplary hinge domains are provided in Table 13.

[0137] In some embodiments, the hinge is isolated or derived from CD8α or CD28. In some embodiments, the CD8α hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 219). In some embodiments, the CD8α hinge comprises SEQ ID NO: 219. In some embodiments, the CD8α hinge consists essentially of SEQ ID NO: 219. In some embodiments, the CD8α hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the following sequence: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 220).

[0138] In some embodiments, the CD8α hinge is encoded by SEQ ID NO:220.

[0139] In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 221). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 221. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the following sequence: TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 222).

[0140] In some embodiments, the CD28 hinge is encoded by SEQ ID NO:222.

[0141] In some embodiments, the activator receptor comprises a hinge sequence isolated or derived from CD8, CD28, IgG1, or IgG4, or a synthetic hinge. [Table 13-1] [Table 13-2]

[0142] In some embodiments, e.g., embodiments in which the CAR is an activator receptor, the hinge comprises a sequence of SEQ ID NO: 1, 223, 225, 228, or 230, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% thereto, or a sequence identical thereto. In some embodiments, e.g., embodiments in which the CAR is an activator receptor, the hinge comprises a sequence of SEQ ID NO: 1, 223, 225, 228, or 230.

[0143] Transmembrane domain The CAR of the present disclosure can be designed to include a transmembrane domain that is fused to the extracellular domain of the CAR. In some embodiments, a transmembrane domain that naturally associates with one of the domains in the CAR is used. For example, a CAR that includes a CD28 costimulatory domain may also use a CD28 transmembrane domain. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or different surface membrane protein and to minimize interaction with other members of the receptor complex.

[0144] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region may be isolated from or derived from (i.e., including at least the transmembrane region thereof) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or an immunoglobulin such as IgG4. Alternatively, the transmembrane domain may be synthetic, in which case it comprises mainly hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0145] In some embodiments of the CAR of the present disclosure, the CAR comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 232). In some embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 232. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of TTCTGGGTGCTGGTCGTTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 233).

[0146] In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO:233.

[0147] In some embodiments of the CAR of the present disclosure, the CAR comprises an IL-2R beta transmembrane domain. In some embodiments, the IL-2R beta transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 234). In some embodiments, the IL-2R beta transmembrane domain comprises or consists essentially of SEQ ID NO: 234. In some embodiments, the IL-2R beta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of ATTCCGTGGCTCGGCCACCTCCTCGTGGGCCTCAGCGGGGCTTTTGGCTTCATCATCTTAGTGTACTTGCTGATC (SEQ ID NO: 235).

[0148] In some embodiments, the IL-2R beta transmembrane domain is encoded by SEQ ID NO:235.

[0149] In some embodiments, the CAR comprises a transmembrane domain isolated or derived from CD8 or CD28. In some embodiments, the CAR comprises a transmembrane domain sequence in Table 14. In some embodiments, the CAR comprises a transmembrane domain sequence of SEQ ID NO:232 or SEQ ID NO:236, or a sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical thereto. In some embodiments, the CAR comprises a transmembrane domain sequence of SEQ ID NO:232 or SEQ ID NO:236. In some embodiments, the CAR comprises a transmembrane domain sequence of SEQ ID NO:561. [Table 14]

[0150] Cytoplasmic domain The present disclosure provides an activator receptor comprising an extracellular ligand-binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen. In some embodiments, the activator receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an intracellular domain isolated or derived from CD28, 4-1BB, or CD3z, or a combination thereof.

[0151] The cytoplasmic domain, or otherwise, intracellular signaling domain, of the CAR of the present invention is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to the specialized function of a cell. Effector functions of regulatory T cells include, for example, the suppression or downregulation of the induction or proliferation of effector T cells. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and causes the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be utilized, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion may be used in place of the intact chain, so long as it transduces an effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired function of the CAR-T cells of the present disclosure. Thus, the term intracellular signaling domain is meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce an effector function signal.

[0152] Examples of intracellular signaling domains for use in the CARs of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capability.

[0153] Thus, the intracellular domain of the CAR of the present disclosure comprises at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures that there is T cell receptor (TCR) signaling necessary to activate the effector function of the CAR T cell. In some embodiments, the at least one cytoplasmic activation domain is the CD247 molecule (CD3zeta) activation domain, the stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or the DNAX activation protein of 12 kDa (DAP12) activation domain. In some embodiments, the CD3 zeta 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 identical to the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 238).

[0154] In some embodiments, the CD3 zeta activation domain comprises or consists essentially of SEQ ID NO: 238. In some embodiments, the CD3 zeta activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the following sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGCGTAGAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGACTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO:239). In some embodiments, the CD3 zeta activation domain is encoded by SEQ ID NO:239.

[0155] It is known that signals generated solely through the TCR are often insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0156] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. In some embodiments, an ITAM contains a tyrosine separated from a leucine or isoleucine by any two other amino acids (YxxL) (SEQ ID NO: 240).

[0157] 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.

[0158] In some embodiments, the transmembrane domain is a CD3 ζ activation domain. In some embodiments, the CD3 ζ activation domain comprises a single ITAM. In some embodiments, the CD3 ζ activation domain comprises two ITAMs. In some embodiments, the CD3 ζ activation domain comprises three ITAMs.

[0159] In some embodiments, the CD3 zeta 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 identical to the sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 241). In some embodiments, the CD3 zeta activation domain comprises SEQ ID NO: 241. In some embodiments, the CD3 zeta activation domain comprising a single ITAM consists essentially of the amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 241). In some embodiments, the CD3 zeta 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 identical to the sequence of AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 242). In some embodiments, the CD3 zeta activation domain is encoded by SEQ ID NO: 242.

[0160] Further examples of ITAMs containing primary cytoplasmic signaling sequences that can be used in the CARs of the present disclosure include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the present invention comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0161] Costimulatory domain In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise a CD3ζ signaling domain by itself or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3ζ chain portion and a costimulatory domain. The costimulatory domain refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. Examples of such molecules include the costimulatory domains of IL-2Rβ, Fc receptor gamma (FcRγ), Fc receptor beta (FcRβ), CD3g molecule gamma (CD3γ), CD3δ, CD3ε, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen-related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF The antibody is selected from the group consisting of receptor superfamily member 4 (OX40), 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 domain, or a functional fragment thereof.

[0162] The cytoplasmic domains within the cytoplasmic signaling portion of the CAR of the present disclosure can be linked together in a random or specified order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, may form the linkage. A glycine-serine doublet provides one example of a suitable linker.

[0163] In some embodiments, the intracellular domain of the CAR of the present disclosure comprises at least one costimulatory domain. In some embodiments, the costimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the following sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (sequence number 243).

[0164] In some embodiments, the CD28 costimulatory domain comprises or consists essentially of SEQ ID NO: 243. In some embodiments, the CD28 costimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the following sequence: AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC (SEQ ID NO: 244). In some embodiments, the CD28 costimulatory domain is encoded by SEQ ID NO:44.

[0165] In some embodiments, the intracellular domain of a CAR of the present disclosure comprises an interleukin-2 receptor beta chain (IL-2R beta or IL-2R-beta) cytoplasmic domain. In some embodiments, the IL-2R beta domain is truncated. In some embodiments, the IL-2R beta cytoplasmic domain comprises one or more STAT5 recruitment motifs. In some embodiments, the CAR comprises one or more STAT5 recruitment motifs outside of the IL-2R beta cytoplasmic domain.

[0166] In some embodiments, the IL-2-R beta 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 identical to the following sequence: NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 245). In some embodiments, the IL2R-beta intracellular domain comprises or consists essentially of SEQ ID NO:245. In some embodiments, the IL-2R transmembrane domain is encoded by a nucleotide sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of AACTGCAGGAACACCGGGCCATGGCTGAAGAAGGTCCTGAAGTGTAACACCCCAGACCCCTCGAAGTTCTTTTCCCAGCTGAGCTCAGAGCATGGAGGCGACGTCCAGAAGTGGCTCTCTTCGCCCTTCCCCTCATCGTCCTTCAGCCCTGGCGGCCTGGCACCTGAGATCTCGCCACTAGAAGTGCTGGAGAGGGACAAGGTGACGCAGCTGCTCCCCCTGAACACTGATGCCTACTTGTCTCTCCAAGAACTCCAGGGTCAGGACCCAACTCACTTGGTG (SEQ ID NO: 246). In some embodiments, the IL-2R-beta intracellular domain is encoded by SEQ ID NO: 246.

[0167] In one 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.

[0168] In some embodiments, the STAT5 recruitment motif consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 247).

[0169] In some embodiments, the CAR comprises an intracellular domain isolated or derived from CD28, 4-1BB and / or CD3z, or a combination thereof. Exemplary domains derived from CD28, 4-1BB and / or CD3z are shown in Table 15 below. In some embodiments, the CAR comprises an intracellular domain comprising a sequence of SEQ ID NO: 248, 250 or 252, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% thereto, or identical thereto. In some embodiments, the CAR comprises an intracellular domain comprising the nucleotide sequence of SEQ ID NO: 248, 250 or 252. In some embodiments, the CAR intracellular domain is encoded by a sequence set forth in any one of SEQ ID NOs: 249, 251, 253, or 21804. [Table 15-1] [Table 15-2]

[0170] T cell receptor (TCR) The present disclosure provides a first activator receptor and an immune cell comprising the same. In some embodiments, the first receptor is a T cell receptor (TCR). Exemplary TCRs comprising an intracellular domain for use in the present disclosure are described in PCT / US2020 / 045250, filed September 6, 2020, the contents of which are incorporated herein by reference.

[0171] As used herein, "TCR," sometimes referred to as "TCR complex" or "TCR / CD3 complex," refers to a protein complex that includes one or more of the TCR alpha chain, TCR beta chain, and invariant CD3 chain (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 that binds to peptide-MHC complexes. When the TCR alpha / beta heterodimer engages peptide-MHC, a conformational change in the TCR complex within the associated invariant CD3 subunit is 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, and CD3 epsilon and CD3 gamma for the heterodimer, as well as two CD3 zetas form homodimers.

[0172]

[0204] Any suitable ligand binding domain may be fused to the extracellular domain, hinge domain or transmembrane of a TCR as described herein. For example, the ligand binding domain may be an antibody or an antigen binding domain of a TCR, or may comprise an antibody fragment, a Vβ-only domain, a linear antibody, a single chain variable fragment (scFv), or a single domain antibody (sdAb).

[0173] In some embodiments, the ligand binding domain is fused to one or more extracellular or transmembrane domains of one or more TCR subunits. The TCR subunits may be TCR alpha, TCR beta, CD3 delta, CD3 epsilon, CD3 gamma, or CD3 zeta. For example, the ligand binding domain may be fused to TCR alpha, or TCR beta, or the ligand binding portion may be fused to two subunits, e.g., the ligand binding domain may be fused to both TCR alpha and TCR beta.

[0174] TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma, and CD3 epsilon. Any one or more of the TCR alpha, TCR beta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta, or fragments or derivatives thereof, may be fused to one or more domains capable of providing a stimulatory signal of the present disclosure, thereby enhancing TCR function and activity.

[0175] TCR transmembrane domains isolated or derived from any source are contemplated within the scope of this disclosure. The transmembrane domains may be derived from either natural or recombinant sources. If the source is natural, the domains may be derived from any membrane-bound or transmembrane protein.

[0176] In some embodiments, the transmembrane domain can transmit a signal to the intracellular domain whenever the TCR complex is bound to a target. A transmembrane domain of particular use in the present disclosure can include, for example, at least the transmembrane region of the alpha, beta, or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0177] In some embodiments, the transmembrane domain can be attached to the extracellular region of a polypeptide of a TCR, e.g., the antigen binding domain of a TCR alpha or beta chain, via a hinge, e.g., a hinge derived from a human protein. For example, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0178] In some embodiments, the extracellular ligand binding domain is linked to one or more transmembrane domains of the TCR. In some embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain, a TCR beta transmembrane domain, or both. In some embodiments, the transmembrane comprises a CD3 zeta transmembrane domain.

[0179] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 15 amino acids of the extracellular region), and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 15 amino acids of the intracellular region).

[0180] In some embodiments, transmembrane domains may be selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0181] If present, the transmembrane domain may be a native TCR transmembrane domain, a native transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain may be a membrane anchor domain. Without being limited thereto, a native or artificial transmembrane domain may contain a hydrophobic a-helix of about 20 amino acids, often with a positive charge adjacent to the transmembrane segment. The transmembrane domain may have one transmembrane segment or more than one transmembrane segment. Predictions of transmembrane domains / segments may be made using published prediction tools (e.g., TMHMM, Krogh et al. Journal of Molecular Biology 2001;305(3):567-580, or TMpred, Hofmann & Stoffel Biol. Chem. Hoppe-Seyler 1993;347:166). Non-limiting examples of membrane anchor systems include the platelet-derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (post-translationally added to a signal sequence), and the like.

[0182] 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 identical to the following sequence: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 316). In some embodiments, the TCR alpha transmembrane domain comprises or consists essentially of SEQ ID NO: 316. In some embodiments, the TCR alpha transmembrane domain is encoded by the following sequence: GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG (sequence number 317).

[0183] 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 identical to the following sequence: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 318). In some embodiments, the TCR beta transmembrane domain comprises or consists essentially of SEQ ID NO: 318. In some embodiments, the TCR beta transmembrane domain is encoded by the following sequence: ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (sequence number 319).

[0184] The TCR of the present disclosure can include one or more intracellular domains. In some embodiments, the intracellular domain includes one or more domains capable of providing a stimulatory signal to the transmembrane domain. In some embodiments, the intracellular domain includes 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 includes a first, second, and third intracellular domain capable of providing a stimulatory signal. The intracellular domain capable of providing a stimulatory signal is selected from the group consisting of a CD28 molecule (CD28) domain, an LCK proto-oncogene, a Src family tyrosine kinase (Lck) domain, a TNF receptor superfamily member 9 (4-1BB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, a Src family tyrosine kinase (Fyn) domain, a zeta chain of T cell receptor-associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, a lymphocyte cytoplasmic protein 2 (SLP76) domain, (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, and CD3 epsilon intracellular domain.

[0185] In some embodiments, the intracellular domain comprises at least one intracellular signaling domain. The intracellular signaling domain generates a signal that promotes immune effector function of a functional cell, e.g., a TCR-containing cell, e.g., a TCR-expressing T cell. In some embodiments, the intracellular domain of the first receptor of the present disclosure comprises at least one intracellular signaling domain. For example, the intracellular domain of CD3 gamma, delta, or epsilon comprises a signaling domain.

[0186] In some embodiments, the extracellular domain, transmembrane domain and intracellular domain are isolated from or derived from the same protein, e.g., T cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma, CD3 epsilon or CD3 zeta.

[0187] Examples of intracellular domains for use in the activator receptors of the present disclosure include the cytoplasmic sequences of TCR alpha, TCR beta, CD3 zeta, and 4-1BB, as well as intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences having the same functional capabilities.

[0188] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from proteins responsible for primary, or antigen-dependent, stimulation.

[0189] In some embodiments, the intracellular domain comprises a CD3 delta intracellular domain, a CD3 epsilon intracellular domain, a CD3 gamma intracellular domain, a CD3 zeta intracellular domain, a TCR alpha intracellular domain, or a TCR beta intracellular domain.

[0190] In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain. In some embodiments, the TCR alpha intracellular domain comprises Ser-Ser. In some embodiments, the TCR alpha intracellular domain is encoded by the sequence TCCAGC.

[0191] In some embodiments, the intracellular domain comprises a TCR beta intracellular domain. In some embodiments, the TCR beta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, or identical to the following sequence: MAMVKRKDSR (SEQ ID NO: 320). In some embodiments, the TCR beta intracellular domain comprises or consists essentially of SEQ ID NO: 320. In some embodiments, the TCR beta intracellular domain is encoded by the sequence ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 321).

[0192] 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 CD3 delta, CD3 gamma, and CD3 epsilon intracellular domains, and one additional stimulatory intracellular domain, e.g., a costimulatory domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, such as CD3 delta, CD3 gamma, and CD3 epsilon intracellular domains, and two additional stimulatory intracellular domains.

[0193] Exemplary costimulatory intracellular signaling domains include those derived from proteins responsible for costimulatory signals or antigen-independent stimulation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll ligand receptors, and DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18) 4-1BB (CD137, TNF receptor superfamily member 9), and CD28 molecules (CD28). Costimulatory proteins can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and NK cell activating receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83, a ligand that specifically binds to CD4, etc. A costimulatory domain can comprise the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional variant thereof.

[0194] In some embodiments, the stimulatory domain comprises a costimulatory domain. In some embodiments, the costimulatory domain comprises a CD28 or 4-1BB costimulatory domain. CD28 and 4-1BB are well-characterized costimulatory molecules required for full T cell activation and are known to enhance T cell effector function. For example, CD28 and 4-1BB have been utilized in chimeric antigen receptors (CARs) to enhance cytokine release, cytolytic function, and persistence over first generation CARs that contain only CD3 zeta signaling domains. Similarly, the inclusion of costimulatory domains, such as CD28 and 4-1BB domains, in TCRs can increase T cell effector function and specifically allow costimulation in the absence of costimulatory ligands that are typically downregulated on the surface of tumor cells. In some embodiments, the stimulatory domain comprises a CD28 intracellular domain or a 4-1BB intracellular domain.

[0195] Inhibitory receptors The present disclosure provides a second receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is lost in a cancer cell, for example, an allelic variant of a gene. The non-target allelic variant may be lost in the cancer cell through any mechanism, including, but not limited to, epigenetic changes that affect the expression of the non-target allelic variant, mutations to the gene encoding the non-target allelic variant, disruption of cell signaling that regulates the expression of the non-target allelic variant, chromosomal loss, partial or complete deletion of a genomic locus, gene silencing due to modification of nucleic acid or heterochromatin, or loss of expression due to other mechanisms. In a variation of the compositions and methods disclosed herein, the treated cells or subjects may show loss of expression of the non-target allelic variant due to non-genetic changes. Thus, the present disclosure provides compositions and methods for killing cells and / or treating subjects that lack expression of the non-target antigen from any cause, including, but not limited to, loss of heterozygosity.

[0196] Exemplary inhibitory receptors are described in PCT / US2020 / 045228, filed September 6, 2020, PCT / US2020 / 064607, filed December 11, 2020, PCT / US2021 / 029907, filed April 29, 2021, and PCT / US2020 / 059856, filed November 10, 2020, the contents of each of which are incorporated herein by reference.

[0197] As used herein, the term "inhibitory chimeric antigen receptor" or "inhibitory receptor" refers to an antigen binding domain fused to an intracellular signaling domain that can transduce an inhibitory signal that inhibits or suppresses the immune activity of an immune cell. Inhibitory receptors have immune cell inhibitory potential and are distinct from and distinguishable from CARs, which are receptors that have immune cell activation potential. For example, CARs contain an intracellular stimulatory domain and / or a co-stimulatory domain, making them activating receptors. Inhibitory receptors are inhibitory receptors that contain an intracellular inhibitory domain.

[0198] As used herein, "inhibitory signal" refers to a change in signal transduction or protein expression in an immune cell that results in suppression of an immune response (e.g., reduced cytokine production or reduced immune cell activation). The suppression or suppression of an immune cell may be selective and / or reversible, or may not be selective and / or reversible. An inhibitory receptor responds to a non-target antigen (e.g., HLA-A*02). For example, when a non-target antigen (e.g., HLA-A*02) binds to or contacts the inhibitory receptor, the inhibitory receptor is responsive and activates an inhibitory signal in an immune cell expressing the inhibitory receptor upon binding of the non-target antigen by the extracellular ligand-binding domain of the inhibitory receptor.

[0199] A non-target antigen may be a non-target allelic variant. As used herein, "non-target allelic variant" refers to an allele (e.g., an allelic variant of an MHC class I gene) that is reduced or eliminated in a target cell (e.g., a cancer cell) by loss of heterozygosity or another mechanism of loss of expression, but is expressed or detectably expressed in a normal or healthy cell. A non-target antigen may be a protein in complex with major histocompatibility complex class I (MHC-I), or an antigenic peptide thereof, and the non-target antigen includes polymorphisms. Because a non-target antigen is polymorphic, loss of a single copy of a gene encoding the non-target antigen, which may occur by loss of heterozygosity in a cancer cell, results in a cancer cell that retains other polymorphic variants of the gene but has lost the non-target antigen. For example, a subject with HLA-A*02 and HLA-A*01 alleles at the HLA locus may have a cancer in which only the HLA-A*02 allele is lost. In such subjects, the HLA-A*01 protein is still present, but is not recognized by the inhibitory receptors of immune cells that encounter cancer cells because the inhibitory receptors are designed to be specific for HLA-A*02 (or other non-target antigens). In normal non-malignant cells, HLA-A*02 (or other non-target antigens) are present and suppress the activation of engineered immune cells. In cancer cells with loss of heterozygosity, the HLA-A*02 allele variant (or other non-target antigen) is lost. Because the inhibitory receptor only responds to HLA-A*02 (or other non-target antigens) that are not present in cancer cells, immune cells engineered to express the inhibitory receptor do not receive an inhibitory signal from the inhibitory receptor. By this mechanism, immune cells are selectively activated and selectively kill cancer cells that express HER2 but have lost HLA-A*02 (or another non-target antigen) due to loss of heterozygosity. HLA-A is used as an example herein. Similar polymorphic variations occur in other populations of MHC genes, as well as in other non-MHC genes. Thus, in some embodiments, the non-target antigen comprises a polymorphic variant of COLEC12, APCDD1 or CXCL16, or HLA-A*02.In some embodiments, the non-target antigen is an HLA class I allele or a minor histocompatibility antigen (MiHA). In some embodiments, the HLA class I allele comprises HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the HLA class I allele is HLA-A*02. In some embodiments, the HLA-A*02 non-target antigen is expressed by healthy cells of the subject. In some embodiments, the non-target antigen is a non-target allele variant. In some embodiments, the non-target antigen is not expressed in cancer cells of the subject. In some embodiments, the non-target antigen is not expressed in a portion of cells in a tumor in the subject. In some embodiments, the cancer cells in the subject have lost expression of the non-target antigen. Loss of expression or lack of expression of the non-target antigen in the cells may be due to any mechanism, including, but not limited to, epigenetic changes affecting non-target gene expression, mutations to genes encoding the non-target antigen, or disruption of cell signaling regulating expression of the non-target gene.

[0200] In some embodiments, the second receptor is an inhibitory chimeric antigen receptor (i.e., an inhibitory receptor). In some embodiments, the second receptor is an inhibitory receptor. In some embodiments, the second receptor is humanized.

[0201] In some embodiments, the second receptor comprises SEQ ID NO: 323, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the second receptor comprises SEQ ID NO: 324, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0202] The present disclosure provides a second receptor that is an inhibitory receptor, comprising extracellular ligand binding that can distinguish single amino acid variant alleles of non-target antigens.This ability to distinguish allelic variants of non-target antigens allows the second receptor to suppress the activation of immune cells that comprise the second receptor in the presence of non-target cells that express the allele recognized by the ligand binding domain.However, the activation of immune cells is not suppressed in the presence of target cells that have lost the allele, for example, cancer cells that have lost one allele of a gene due to loss of heterozygosity.

[0203] The present disclosure provides a second receptor that is an inhibitory receptor that comprises an extracellular ligand binding that can distinguish between different levels of expression of a non-target antigen, thereby allowing the second receptor to inhibit activation of an immune cell that comprises the second receptor in the presence of a non-target cell that expresses a ligand for the second receptor, but allows activation of the immune cell in the presence of cancer cells that express low levels or no ligand for the second receptor.

[0204] Inhibitor Ligand In some embodiments, the non-target antigen is not expressed by the target cells and is expressed by the non-target cells. In some embodiments, the non-target antigen is expressed by healthy cells, i.e., cells that are not cancer cells. In some embodiments, the target cells are a plurality of cancer cells that have lost expression of the non-target antigen through loss of heterozygosity (LOH). In some embodiments, the non-target cells are a plurality of healthy cells (i.e., non-cancer cells, normal cells, or healthy cells) that express both the target and non-target antigens.

[0205] Any cell surface molecule expressed by non-target cells that is not expressed by target cells can be a suitable non-target antigen for the second receptor extracellular ligand binding domain. For example, cell adhesion molecules, intercellular signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane, receptors for neurotransmitters, or voltage-gated ion channels can be used as non-target antigens.

[0206] In some embodiments, the target antigen is a peptide antigen of a cancer cell-specific antigen in complex with major histocompatibility complex class I (MHC-I).

[0207] In some embodiments, the non-target antigen is lost in the cancer cells due to loss of heterozygosity. Exemplary non-target antigens lost in the cancer cells due to loss of heterozygosity include COLEC12, APCDD1, CXCL16, and HLA-A*02. In some embodiments, the non-target antigen is selected from the group consisting of polymorphic variants of COLEC12, APCDD1, and CXCL166, or peptide antigens thereof in complex with major histocompatibility complex class I (MHC-I), or HLA-A*02. In some embodiments, the non-target antigen is an antigenic peptide comprising polymorphic residues of COLEC12, APCDD1, and CXCL16 in complex with major histocompatibility complex class I (MHC-I).

[0208] Non-target MHC-1 (MHC) antigens including any of HLA-A, HLA-B, or HLA-C are contemplated within the scope of the present disclosure. In some embodiments, the non-target antigen comprises HLA-A. In some embodiments, the non-target antigen comprises human leukocyte antigen A*02 allele product (HLA-A*02). In some embodiments, the non-target antigen comprises human leukocyte antigen A*69. In some embodiments, the non-target antigen comprises HLA-B. In some embodiments, the non-target antigen comprises HLA-C.

[0209] In some embodiments, the non-target antigen comprises HLA-A*02.

[0210] In some embodiments, the non-target antigen comprises CXC motif chemokine ligand 16 (CXCL16) or an antigenic peptide thereof in complex with MHC-I. Human CXCL16 precursor is described in NCBI Accession No. NP_001094282.1, the contents of which are incorporated herein by reference in their entirety. In some embodiments, CXCL16 comprises the following amino acid sequence: [ka]

[0211] In some embodiments, the non-target antigen comprises a polymorphism of CXCL16. For example, the non-target antigen comprises a peptide derived from CXCL16 that comprises a polymorphic residue of CXCL16. The polymorphic residue of CXCL16 comprises positions 142 and 200 of SEQ ID NO: 325. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises amino acid 142 or 200 of SEQ ID NO: 325. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises an A at amino acid 200 of SEQ ID NO: 325. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises a V at amino acid 200 of SEQ ID NO: 325. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises an I at amino acid 142 of SEQ ID NO: 325. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises a T at amino acid 142 of SEQ ID NO: 325.

[0212] In some embodiments, the non-target antigen comprises a polymorphism of CXCL16. In some embodiments, the non-target antigen comprises a peptide of CXCL16 comprising an A at amino acid 200 of SEQ ID NO: 325, and the second receptor comprises a ligand binding domain that has a higher affinity for a CXCL16 ligand having an A at position 200 of SEQ ID NO: 325 than for a CXCL16 ligand having a V at position 200 of SEQ ID NO: 325. In some embodiments, the non-target antigen comprises a peptide of CXCL16 comprising a V at amino acid 200 of SEQ ID NO: 325, and the second receptor comprises a ligand binding domain that has a higher affinity for a CXCL16 ligand having a V at position 200 of SEQ ID NO: 325 than for a CXCL16 ligand having an A at position 200 of SEQ ID NO: 325. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises an I at amino acid 142 of SEQ ID NO: 325, and the second receptor comprises a ligand binding domain that has a higher affinity for a CXCL16 ligand that has an I at position 142 of SEQ ID NO: 325 than for a CXCL16 ligand that has a T at position 142 of SEQ ID NO: 325. In some embodiments, the non-target antigen comprises a peptide of CXCL16 that comprises a T at amino acid 142 of SEQ ID NO: 325, and the second receptor comprises a ligand binding domain that has a higher affinity for a CXCL16 ligand that has a T at position 142 of SEQ ID NO: 325 than for a CXCL16 ligand that has an I at position 142 of SEQ ID NO: 325.

[0213] In some embodiments, the non-target antigen comprises collectin subfamily member 12 (COLEC12) or an antigenic peptide thereof in complex with MHC-I. Human COLEC12 is described in NCBI Accession No. NP_569057.2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, COLEC12 comprises the following amino acid sequence: [ka]

[0214] In some embodiments, the non-target antigen comprises a polymorphism of COLEC12. For example, the non-target antigen comprises a peptide derived from COLEC12 that comprises a polymorphic residue of COLEC12. The polymorphic residue of COLEC12 comprises position 522 of SEQ ID NO:326. In some embodiments, the non-target antigen comprises a peptide of COLEC12 that comprises amino acid 522 of SEQ ID NO:326. In some embodiments, the non-target antigen comprises a peptide of COLEC12 that comprises an S at amino acid 522 of SEQ ID NO:326. In some embodiments, the non-target antigen comprises a peptide of COLEC12 that comprises a P at amino acid 522 of SEQ ID NO:326.

[0215] In some embodiments, the non-target antigen comprises APC downregulation 1 (APCDD1) or an antigenic peptide thereof in a complex with MHC-I. An exemplary human APCDD1 is described in UniProtKB Accession No. Q8J025, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the APCDD1 comprises the following amino acid sequence: [ka]

[0216] In some embodiments, the non-target antigen comprises a polymorphism of APCDD1. Exemplary polymorphisms of APCDD1 include rs3748415, which can be a V, I, or L at position 150 of SEQ ID NO:327. In some embodiments, the non-target antigen comprises a peptide of APCDD1 comprising amino acid 150 of SEQ ID NO:327. In some embodiments, the non-target antigen comprises a peptide of APCDD1 comprising a V at amino acid 150 of SEQ ID NO:327. In some embodiments, the non-target antigen comprises a peptide of APCDD1 comprising an I at amino acid 150 of SEQ ID NO:327. In some embodiments, the non-target antigen comprises a peptide of APCDD1 comprising an L at amino acid 150 of SEQ ID NO:327.

[0217] Further exemplary human APCDD1 is described in UniProtKB Accession No. V9GY82, the contents of which are incorporated herein by reference in their entirety. In some embodiments, APCDD1 comprises the following amino acid sequence: [ka]

[0218] Exemplary polymorphisms of APCDD1 include rs1786683, which can be a Y or an S at position 165 of SEQ ID NO: 328. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes amino acid 165 of SEQ ID NO: 328. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes a Y at amino acid 165 of SEQ ID NO: 328. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes an S at amino acid 165 of SEQ ID NO: 328.

[0219] A further exemplary human APCDD1 is set forth in UniProt Accession No. J3QSE3, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the APCDD1 comprises the following amino acid sequence: [ka]

[0220] Exemplary polymorphisms of APCDD1 include rs9952598, which can be Q or R at position 28 of SEQ ID NO: 329. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes amino acid 28 of SEQ ID NO: 329. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes a Q at amino acid 28 of SEQ ID NO: 329. In some embodiments, the non-target antigen comprises a peptide of APCDD1 that includes an R at amino acid 28 of SEQ ID NO: 329.

[0221] In some embodiments, APCDD1 comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 94%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 327-329. Polymorphic residues of APCDD1 are marked as bold and underlined in SEQ ID NOs: 327-329.

[0222] In some embodiments, the non-target antigen comprises HLA-A*02, and the ligand binding domain of the second receptor comprises an HLA-A*02 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-A*02 independently of the peptide in a pMHC complex comprising HLA-A*02. In some embodiments, the HLA-A*02 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*02 ligand binding domain comprises any one of SEQ ID NOs: 63-74. In some embodiments, the HLA-A*02 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 63-74.

[0223] In some embodiments, the HLA-A*02 scFv comprises a complementarity determining region (CDR) of any one of SEQ ID NOs: 87-102. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ ID NOs: 87-102. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOs: 87-102. In some embodiments, the heavy chain of the antigen-binding domain comprises a heavy chain CDR of any one of SEQ ID NOs: 87-102, and the light chain of the antigen-binding domain comprises a light chain CDR of any one of SEQ ID NOs: 87-102. In some embodiments, the HLA-A*02 antigen-binding domain comprises a heavy chain and a light chain, and the heavy chain comprises a CDR selected from SEQ ID NOs: 95-102, and the light chain comprises a CDR selected from SEQ ID NOs: 87-94. In further embodiments of any of the ligand-binding domains, each CDR sequence may have one, two, three or more substitutions, insertions, or deletions. The CDR sequences may tolerate substitutions, deletions, or insertions. Using sequence alignment tools, routine experimentation, and known assays, one of skill in the art can generate and test variant sequences having one, two, three or more substitutions, insertions, or deletions in the CDR sequences without undue experimentation.

[0224] In some embodiments, the HLA-A*02 antigen binding domain comprises a heavy chain and a light chain, wherein the heavy chain comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 63-74, and the light chain comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOs: 63-74.

[0225] In some embodiments, the heavy chain comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 63-74, and the light chain comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 63-74.

[0226] In some embodiments, the non-target antigen comprises HLA-B*07 and the ligand binding domain of the second receptor comprises an HLA-B*07 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-B*07 independently of the peptide in a pMHC complex that comprises HLA-B*07. In some embodiments, the HLA-B*07 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-B*07 ligand binding domain comprises the sequence of any one of the B7 binding domains shown in Table 7. In some embodiments, the HLA-B*07 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-B*07 binding domains shown in Table 7. In some embodiments, the HLA-B*07 scFv comprises any one of the HLA-B*07 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-B*07 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-B*07 scFv sequences shown in Table 7.

[0227] In some embodiments, the non-target antigen comprises HLA-A*11 and the ligand binding domain of the second receptor comprises an HLA-A*11 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-A*11 independently of the peptide in a pMHC complex comprising HLA-A*11. In some embodiments, the HLA-A*11 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*11 ligand binding domain comprises the sequence of any one of the HLA-A*11 binding domains shown in Table 7. In some embodiments, the HLA-A*11 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-A*11 binding domains shown in Table 7. In some embodiments, the HLA-A*11 scFv comprises any one of the HLA-A*11 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*11 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*11 scFv sequences shown in Table 7.

[0228] In some embodiments, the non-target antigen comprises HLA-A*01 and the ligand binding domain of the second receptor comprises an HLA-A*01 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-A*01 independently of the peptide in a pMHC complex comprising HLA-A*01. In some embodiments, the HLA-A*01 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*01 ligand binding domain comprises the sequence of any one of the HLA-A*01 binding domains shown in Table 7. In some embodiments, the HLA-A*01 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-A*01 binding domains shown in Table 7. In some embodiments, the HLA-A*01 scFv comprises any one of the HLA-A*01 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*01 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*01 scFv sequences shown in Table 7.

[0229] In some embodiments, the non-target antigen comprises HLA-A*03 and the ligand binding domain of the second receptor comprises an HLA-A*03 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-A*03 independently of the peptide in a pMHC complex comprising HLA-A*03. In some embodiments, the HLA-A*03 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-A*03 ligand binding domain comprises the sequence of any one of the HLA-A*03 binding domains shown in Table 7. In some embodiments, the HLA-A*03 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-A*03 binding domains shown in Table 7. In some embodiments, the HLA-A*03 scFv comprises any one of the HLA-A*03 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*03 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-A*03 scFv sequences shown in Table 7.

[0230] In some embodiments, the non-target antigen comprises HLA-C*07 and the ligand binding domain of the second receptor comprises an HLA-C*07 ligand binding domain. In some embodiments, the ligand binding domain binds to HLA-C*07 independently of the peptide in a pMHC complex comprising HLA-C*07. In some embodiments, the HLA-C*07 ligand binding domain comprises an scFv domain. In some embodiments, the HLA-C*07 ligand binding domain comprises the sequence of any one of the HLA-C*07 binding domains shown in Table 7. In some embodiments, the HLA-C*07 ligand binding domain comprises a sequence at least 90%, at least 95%, or at least 99% identical to the sequence of any one of the HLA-C*07 binding domains shown in Table 7. In some embodiments, the HLA-C*07 scFv comprises any one of the HLA-C*07 complementarity determining regions (CDRs) shown in Table 9. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-C*07 scFv sequences shown in Table 7. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of the HLA-C*07 scFv sequences shown in Table 7.

[0231] Inhibitory receptors of the present disclosure can include an extracellular ligand-binding domain. Any type of ligand-binding domain capable of regulating the activity of the receptor in a ligand-dependent manner is contemplated within the scope of the present disclosure.

[0232] In some embodiments, the ligand binding domain is an antigen binding domain. Exemplary antigen binding domains include, inter alia, scFv, SdAb, Vβ-only domains, and TCR antigen binding domains derived from TCR α and β chain variable domains.

[0233] Any type of antigen binding domain is contemplated within the scope of this disclosure.

[0234] In some embodiments, the extracellular ligand-binding domain of the second receptor binds to and recognizes COLEC12, APCDD1, CXCL16, or a polymorphic variant of an antigenic peptide thereof in complex with major histocompatibility complex class I (MHC-I), or HLA-A*02. In some embodiments, the extracellular ligand-binding domain of the second receptor is an scFv.

[0235] In some embodiments, the extracellular ligand-binding domain of the second receptor is fused to the extracellular domain of the inhibitory receptor.

[0236] In some embodiments, the inhibitory receptors of the present disclosure include an extracellular hinge region. Exemplary hinges can be isolated or derived from IgD and CD8 domains, e.g., IgG1. In some embodiments, the hinge is isolated or derived from CD8α or CD28.

[0237] Inhibitory receptors of the present disclosure can be designed to include a transmembrane domain fused to the extracellular domain of the inhibitory receptor. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins and to minimize interactions with other members of the receptor complex.

[0238] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region may be isolated from or derived from (i.e., including at least the transmembrane region thereof) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or an immunoglobulin such as IgG4. Alternatively, the transmembrane domain may be synthetic, in which case it comprises mainly hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular domain of the inhibitory receptor. A glycine-serine doublet provides a particularly suitable linker.

[0239] The present disclosure provides an inhibitory receptor comprising an intracellular domain. The intracellular domain of the inhibitory receptor of the present disclosure is responsible for suppressing the activation of immune cells comprising the inhibitory receptor, which would otherwise be activated in response to an activation signal by the first receptor. In some embodiments, for example, in the second inhibitory receptor of the present disclosure that provides an inhibitory signal, the inhibitory signal is transmitted through the intracellular domain of the receptor. In some embodiments, the inhibitory receptor comprises an inhibitory intracellular domain. In some embodiments, the inhibitory engineered receptor is a CAR (inhibitory CAR) that comprises an inhibitory intracellular domain.

[0240] In some embodiments, the inhibitory intracellular domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory intracellular domain comprising an ITIM can be isolated or derived from immune checkpoint inhibitors such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immunoinhibitory receptors expressed on the surface of T cells and play important roles in attenuating or terminating T cell responses.

[0241] The inhibitory domain can be isolated from the human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and CD200 receptor 1. In some embodiments, the TRAIL receptor comprises TR10A, TR10B, or TR10D.

[0242] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, or a combination thereof. In some embodiments, the inhibitory intracellular domain is isolated from a phosphoprotein membrane anchor with glycosphingolipid microdomain 1 (PAG1). In some embodiments, the inhibitory intracellular domain is isolated from leukocyte immunoglobulin-like receptor B1 (LILRB1). In some embodiments, the inhibitory domain is isolated or derived from a human protein, such as a human TRAIL receptor, CTLA-4, or PD-1, PAG1, or LILRB1 protein.

[0243] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region, or a combination thereof. In some embodiments, the inhibitory domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, an inhibitory domain comprising an ITIM can be isolated or derived from immune checkpoint inhibitors such as CTLA-4 and PD-1. Exemplary hinge, transmembrane, and intracellular domains that can be used in the inhibitory receptors described herein are shown in Table 16.

[0244] The inhibitory domain may be isolated from human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and CD200 receptor 1. In some embodiments, the inhibitory domain is isolated or derived from a human protein, such as a human TRAIL receptor, CTLA-4, or PD-1 protein. In some embodiments, the TRAIL receptor comprises TR10A, TR10B, or TR10D.

[0245] Endogenous TRAIL is expressed as a 281-amino acid type II transmembrane protein that is anchored to the plasma membrane and presented on the cell surface. TRAIL is expressed by natural killer cells and can induce TRAIL-dependent apoptosis in target cells after the establishment of cell-cell contact. Physiologically, the TRAIL signaling system has been shown to be essential for immune surveillance, shaping the immune system by regulating T helper cell 1 versus T helper cell 2, as well as the number of "helpless" CD8+ T cells, and for the suppression of spontaneous tumor formation.

[0246] In some embodiments, the inhibitory domain comprises an intracellular domain isolated or derived from the CD200 receptor. The cell surface glycoprotein CD200 receptor 1 (Uniprot reference: Q8TD46) represents another example of an inhibitory intracellular domain of the present invention. This inhibitory receptor of the CD200 / OX2 cell surface glycoprotein limits inflammation by suppressing the expression of proinflammatory molecules, including TNF-alpha, interferons, and inducible nitric oxide synthase (iNOS), in response to selected stimuli.

[0247] In some embodiments, the engineered receptor comprises a killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 2 (KIR3DL2), a killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 3 (KIR3DL3), a leukocyte immunoglobulin-like receptor B1 (LIR1, also called LIR-1 and LILRB1), a programmed cell death 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), a killer cell lectin-like receptor K1 (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N- and C-terminal SH2 domains), or an inhibitory domain isolated or derived from ZAP70 KI_K369A (kinase-inactive ZAP70).

[0248] In some embodiments, the inhibitory domain is isolated or derived from a human protein. In some embodiments, the second inhibitory receptor comprises an inhibitory domain. In some embodiments, the second inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory domain and a transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of the inhibitory receptor. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of the inhibitory receptor.

[0249] In some embodiments, the second inhibitory receptor comprises a cytoplasmic domain and a transmembrane domain isolated or derived from the same protein, e.g., an ITIM-containing protein. In some embodiments, the second inhibitory receptor comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, or a portion thereof, isolated or derived from the same protein, e.g., an ITIM-containing protein. In some embodiments, the second inhibitory receptor comprises an intracellular domain and / or a transmembrane domain, isolated or derived from the same protein, e.g., an ITIM-containing protein, and a hinge region isolated or derived from the same protein, e.g., an ITIM-containing protein. In some embodiments, the second inhibitory receptor comprises an inhibitory domain. In some embodiments, the second inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the second engineered receptor is a CAR (inhibitory CAR) comprising an inhibitory domain. In some embodiments, the inhibitory intracellular domain is fused to the intracellular domain of the CAR. In some embodiments, the inhibitory intracellular domain is fused to the transmembrane domain of the CAR.

[0250] LILRB1 inhibitory receptor The present disclosure provides a second inhibitory receptor comprising a LILRB1 inhibitory domain and, optionally, a LILRB1 transmembrane domain and / or hinge domain, or a functional variant thereof. By including a LILRB1 transmembrane domain and / or a LILRB1 hinge domain in an inhibitory receptor, the inhibitory signal generated by the inhibitory receptor may be increased compared to a reference inhibitory receptor having a different transmembrane domain or a different hinge domain. The second inhibitory receptor comprising a LILRB1 inhibitory domain may be a CAR or a TCR as described herein. Any suitable ligand binding domain described herein may be fused to a LILRB1-based second inhibitory receptor having one or more domains from leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1 or LIR1). In some embodiments, the inhibitory receptor comprises a LILRB1 intracellular domain or a functional variant thereof.

[0251] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), also known as leukocyte immunoglobulin-like receptor B1, as well as ILT2, LIR1, MIR7, PIRB, CD85J, ILT-2 LIR-1, MIR-7, and PIR-B are members of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1 protein belongs to the subfamily B class of LIR receptors. These receptors contain 2-4 extracellular immunoglobulin domains, a transmembrane domain, and 2-4 cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces negative signals that inhibit stimulation of the immune response. LILRB1 is believed to play a role in regulating inflammatory responses and cytotoxicity, and limiting autoreactivity. There are multiple transcriptional variants that encode different isoforms of LILRB1, all of which are contemplated to be within the scope of the present disclosure.

[0252] In some embodiments of the inhibitory receptors described herein, the inhibitory receptor comprises one or more domains isolated or derived from LILRB1. In some embodiments of the receptor having one or more domains isolated or derived from LILRB1, one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to the sequence or subsequence of SEQ ID NO:280. In some embodiments ...

[0253] In some embodiments of a receptor having one or more domains isolated or derived from LILRB1, one or more domains of LILRB1 are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to the sequence or subsequence of SEQ ID NO:556.

[0254] In some embodiments of a receptor having one or more domains of LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence identical to a sequence or subsequence of SEQ ID NO: 556. In various embodiments, a chimeric antigen receptor is provided comprising a polypeptide, the polypeptide comprising one or more of a LILRB1 hinge domain or a functional fragment or variant thereof, a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain or an intracellular domain comprising at least one, or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0255] Intracellular domain The present disclosure provides an inhibitory receptor, the inhibitory receptor comprising a polypeptide. In some embodiments, the polypeptide comprises an intracellular domain. In some embodiments, the inhibitory receptor comprises a LILRB1 intracellular domain or a functional variant thereof.

[0256] 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 a cell and performs a cytoplasmic function. As used herein, "cytoplasmic function" refers to a function of a protein or protein complex that is performed in the cytoplasm of a cell. For example, an intracellular signaling cascade is a cytoplasmic function.

[0257] As used herein, "immunoreceptor tyrosine-based inhibitory motif" or "ITIM" refers to a conserved sequence of amino acids with a consensus sequence, such as S / I / V / LxYxxI / V / L (SEQ ID NO: 260), found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-bearing inhibitory receptors interact with their ligands, the ITIM motif becomes phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or an inositol-phosphatase called SHIP.

[0258] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibition motif (ITIM), at least two ITIMs, at least three ITIMs, at least four ITIMs, at least five ITIMs, or at least six ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0259] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one ITIM selected from the group of ITIMs consisting of NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0260] In further specific embodiments, the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibition motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO:256), VTYAEV (SEQ ID NO:257), VTYAQL (SEQ ID NO:258), and SIYATL (SEQ ID NO:259).

[0261] In some embodiments, the intracellular domain comprises both the ITIMs NLYAAV (SEQ ID NO:256) and VTYAEV (SEQ ID NO:257). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO:260. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO:281.

[0262] In some embodiments, the intracellular domain comprises both the ITIMs VTYAEV (SEQ ID NO:257) and VTYAQL (SEQ ID NO:258). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO:261. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO:261.

[0263] In some embodiments, the intracellular domain comprises both the ITIMs VTYAQL (SEQ ID NO:258) and SIYATL (SEQ ID NO:259). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO:262. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO:262.

[0264] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO:256), VTYAEV (SEQ ID NO:257), and VTYAQL (SEQ ID NO:258). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO:263. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO:263.

[0265] In some embodiments, the intracellular domain comprises the ITIM VTYAEV (SEQ ID NO:257), VTYAQL (SEQ ID NO:258), and SIYATL (SEQ ID NO:259). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO:264. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO:264.

[0266] In some embodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ ID NO:256), VTYAEV (SEQ ID NO:257), VTYAQL (SEQ ID NO:258), and SIYATL (SEQ ID NO:259). In embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO:265. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO:265.

[0267] In some embodiments, the intracellular domain comprises a sequence at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 266). In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to the LILRB1 intracellular domain (SEQ ID NO: 266).

[0268] The LILRB1 intracellular domain or functional variant thereof of the present disclosure can have at least 1, at least 2, at least 4, at least 4, at least 5, at least 6, at least 7, or at least 8 ITIMs. In some embodiments, the LILRB1 intracellular domain or functional variant thereof has 2, 3, 4, 5, or 6 ITIMs.

[0269] In certain embodiments, the polypeptide comprises an intracellular domain comprising two, three, four, five, or six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO:256), VTYAEV (SEQ ID NO:257), VTYAQL (SEQ ID NO:258), and SIYATL (SEQ ID NO:259).

[0270] In certain embodiments, the polypeptide comprises an intracellular domain comprising at least three immunoreceptor tyrosine-based inhibition motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0271] In certain embodiments, the polypeptide comprises an intracellular domain comprising three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0272] In certain embodiments, the polypeptide comprises an intracellular domain comprising four immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0273] In certain embodiments, the polypeptide comprises an intracellular domain comprising five immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0274] In certain embodiments, the polypeptide comprises an intracellular domain comprising six immunoreceptor tyrosine-based inhibitory motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0275] In certain embodiments, the polypeptide comprises an intracellular domain comprising at least seven immunoreceptor tyrosine-based inhibition motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0276] In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain. In some embodiments, the intracellular domain comprises a TCR alpha intracellular domain and a LILRB1 intracellular domain described herein. In some embodiments, the TCR alpha intracellular domain comprises Ser-Ser. In some embodiments, the TCR alpha intracellular domain is encoded by a sequence of TCCAGC.

[0277] In some embodiments, the intracellular domain comprises a TCR beta intracellular domain. In some embodiments, the intracellular domain comprises a TCR beta intracellular domain and a LILRB1 intracellular domain, as described herein. In some embodiments, the TCR beta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, or identical to the following sequence: MAMVKRKDSR (SEQ ID NO: 267). In some embodiments, the TCR beta intracellular domain comprises or consists essentially of MAMVKRKDSR (SEQ ID NO: 267). In some embodiments, the TCR beta intracellular domain is encoded by the sequence ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 268).

[0278] The LILRB1 protein has four immunoglobulin (Ig)-like domains, designated D1, D2, D3, and D4. In some embodiments, the LILRB1 hinge domain comprises a LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1 D3D4 domain comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or identical to, SEQ ID NO:274. In some embodiments, the LILRB1 D3D4 domain comprises or essentially consists of SEQ ID NO:274.

[0279] Transmembrane domain The present disclosure provides an inhibitory receptor, the receptor comprising a polypeptide. In some embodiments, the polypeptide comprises a transmembrane domain. In some embodiments, the inhibitory receptor comprises a LILRB1 hinge domain, or a functional variant thereof. In some embodiments, the inhibitory receptor comprises a LILRBI transmembrane domain, or a functional variant thereof. In some embodiments, the inhibitory receptor comprises a LILRB1 hinge domain and a transmembrane domain, or a functional variant thereof.

[0280] "Transmembrane domain" as used herein refers to a domain of a protein that spans the membrane of a cell. Transmembrane domains typically consist of primarily non-polar amino acids and may cross the lipid bilayer once or several times. Transmembrane domains usually contain an alpha helix, a configuration that maximizes internal hydrogen bonding.

[0281] Transmembrane domains isolated or derived from any source are contemplated within the scope of the fusion proteins of the present disclosure.

[0282] In certain embodiments, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof.

[0283] In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to SEQ ID NO: 269. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence at least 95% identical to SEQ ID NO: 269. In some embodiments, the LILRB1 transmembrane domain comprises a sequence identical to SEQ ID NO: 269. In embodiments, the LILRB1 transmembrane domain consists essentially of a sequence identical to SEQ ID NO: 269.

[0284] In some embodiments of the chimeric antigen receptor of the present disclosure, the transmembrane domain is not a LILRB1 transmembrane domain, hi some embodiments, the transmembrane domain is associated with one of the other domains of the fusion protein or is isolated or derived from the same protein as one of the other domains of the fusion protein.

[0285] The transmembrane domain may be derived from either natural or recombinant sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains may include, for example, at least the transmembrane region of the alpha, beta, or zeta chain of 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.

[0286] In some embodiments, the transmembrane 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 identical to the following sequence: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 270). In some embodiments, the TCR alpha transmembrane domain comprises or consists essentially of VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 270). In some embodiments, the TCR alpha transmembrane domain is encoded by the following sequence: GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG (SEQ ID NO: 271).

[0287] In some embodiments, the transmembrane 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 identical to the following sequence: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 272). In some embodiments, the TCR beta transmembrane domain comprises or consists essentially of TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 272). In some embodiments, the TCR beta transmembrane domain is encoded by the sequence ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO: 273).

[0288] In some embodiments, the TCR alpha and / or TCR beta transmembrane domains comprise one or more mutations that attenuate or abolish the interaction of the TCR with the TCR CD3 subunit. In some embodiments, the TCR alpha transmembrane domain comprises a R253L mutation. In some embodiments, the TCR beta transmembrane domain comprises a K288L mutation.

[0289] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 232). In some embodiments, the CD28 transmembrane domain comprises or consists essentially of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 232). In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of TTCTGGGTGCTGGTCGTTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 233).

[0290] In some embodiments, the transmembrane domain can be attached to the extracellular region chimeric antigen receptor, e.g., the antigen binding domain or the ligand binding domain, via a hinge, e.g., a hinge derived from a human protein. For example, in some embodiments, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, a CD8a hinge, or a LILRB1 hinge.

[0291] Hinge Domain The present disclosure provides an inhibitory receptor, the receptor comprising a polypeptide. In some embodiments, the polypeptide comprises a hinge domain. In some embodiments, the hinge domain is a LILRB1 hinge domain or a functional variant thereof.

[0292] The LILRB1 protein has four immunoglobulin (Ig)-like domains, designated D1, D2, D3, and D4. In some embodiments, the LILRB1 hinge domain comprises a LILRB1 D3D4 domain or a functional variant thereof. In some embodiments, the LILRB1 D3D4 domain comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or identical to, SEQ ID NO:274. In some embodiments, the LILRB1 D3D4 domain comprises or essentially consists of SEQ ID NO:274.

[0293] In some embodiments, the polypeptide comprises a LILRB1 hinge domain, or a functional fragment or variant thereof. In embodiments, the LILRB1 hinge domain, or a functional fragment or variant thereof comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to, or identical to, SEQ ID NO:275, SEQ ID NO:274, or SEQ ID NO:276. In embodiments, the LILRB1 hinge domain, or a functional fragment or variant thereof comprises a sequence that is at least 95% identical to, SEQ ID NO:275, SEQ ID NO:274, or SEQ ID NO:276.

[0294] In some embodiments, the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO:275, SEQ ID NO:274, or SEQ ID NO:276.

[0295] In some embodiments, the LILRB1 hinge domain consists essentially of a sequence identical to SEQ ID NO:275, SEQ ID NO:274, or SEQ ID NO:276.

[0296] In some embodiments, the chimeric antigen receptor, polypeptide of the disclosure comprises a hinge that is not isolated or derived from LILRB1.

[0297] In some embodiments, the hinge is isolated or derived from CD8α or CD28. In some embodiments, the CD8α hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 219). In some embodiments, the CD8α hinge comprises TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 219). In some embodiments, the CD8α hinge consists essentially of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 219). In some embodiments, the CD8 alpha hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or identical to the sequence accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 220).

[0298] In some embodiments, the CD28 hinge comprises an amino acid sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 221). In some embodiments, the CD28 hinge comprises or consists essentially of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 221). In some embodiments, the CD28 hinge is encoded by a nucleotide sequence that has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity, or is identical to the following sequence: tgtaccattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 222).

[0299] LILRB1 domain combinations In some embodiments, an inhibitory receptor of the present disclosure comprises a polypeptide comprising more than one LILRB1 domain or a functional equivalent thereof, for example, in some embodiments, the polypeptide comprises a LILRB1 transmembrane domain and an intracellular domain, or a LILRB1 hinge domain, a transmembrane domain and an intracellular domain.

[0300] In certain embodiments, the polypeptide comprises a LILRB1 hinge domain, or a functional fragment or variant thereof, and a LILRB1 transmembrane domain, or a functional variant thereof. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO:277. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:277. In some embodiments, the polypeptide comprises a sequence that is identical to SEQ ID NO:277.

[0301] In further embodiments, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibitory motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO:256), VTYAEV (SEQ ID NO:257), VTYAQL (SEQ ID NO:258), and SIYATL (SEQ ID NO:259). In some embodiments, the polypeptide comprises a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least two ITIMs, wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:256), VTYAEV (SEQ ID NO:257), VTYAQL (SEQ ID NO:258), and SIYATL (SEQ ID NO:259).

[0302] In some embodiments, the polypeptide comprises a LILRB1 transmembrane domain and an intracellular domain. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO:278. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:278. In some embodiments, the polypeptide comprises a sequence identical to SEQ ID NO:278.

[0303] In a preferred embodiment, the polypeptide comprises a LILRB1 hinge domain, or a functional fragment or variant thereof, a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibition motifs (ITIMs), each ITIM independently selected from NLYAAV (SEQ ID NO: 256), VTYAEV (SEQ ID NO: 257), VTYAQL (SEQ ID NO: 258), and SIYATL (SEQ ID NO: 259).

[0304] In some embodiments, the polypeptide comprises a sequence at least 95% identical to SEQ ID NO:254 or SEQ ID NO:279, or at least 99% identical to SEQ ID NO:254 or SEQ ID NO:279, or identical to SEQ ID NO:254 or SEQ ID NO:279.

[0305] In some embodiments, the polypeptide comprises a sequence at least 99% identical to SEQ ID NO:277, or at least 99% identical to SEQ ID NO:277, or identical to SEQ ID NO:277.

[0306] In some embodiments, the polypeptide comprises a sequence at least 99% identical to SEQ ID NO:278, or at least 99% identical to SEQ ID NO:278, or identical to SEQ ID NO:278.

[0307] In some embodiments, the inhibitory receptor comprises a LIRLRB1 hinge, transmembrane domain and intracellular domain. In some embodiments, the LIRLRB1 hinge, transmembrane domain and intracellular domain comprise a sequence of SEQ ID NO: 254, or a sequence that is 90%, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical thereto. [Table 16]

[0308] Further sequences of LILRB1-based inhibitory receptors of the present disclosure are shown in Table 17 below. [Table 17-1] [Table 17-2] [Table 17-3]

[0309] Assay Provided herein are assays that can be used to measure the activity of the engineered receptors of the present disclosure.

[0310] The activity of engineered receptor can be assayed using engineered cell line to express receptor activity reporter, such as luciferase reporter.Exemplary cell line includes Jurkat T cell, but any suitable cell line known in the art can be used.For example, Jurkat cell expressing luciferase reporter under the control of NFAT promoter can be used as effector cell.The expression of luciferase by this cell line reflects TCR-mediated signal transduction.

[0311] Reporter cells can be transfected with each of the various fusion protein constructs described herein, combinations of fusion protein constructs, or controls.

[0312] Expression of the fusion protein in the reporter cells can be confirmed by detecting expression of the fusion protein using a fluorescently labeled MHC tetramer, for example, an Alexa Fluor 647 labeled HER2-MHC tetramer.

[0313] To assay the activity of engineered receptors, target cells are loaded with antigen before exposure to effector cells that contain reporter and engineered receptor. For example, target cells can be loaded with antigen for at least 12, 14, 16, 18, 20, 22, or 24 hours before exposure to effector cells. Exemplary target cells include A375 cells, but any suitable cells known in the art can be used. In some cases, target cells can be loaded with serial dilution concentrations of antigen, such as HER2 antigen. Effector cells can then be co-cultured with target cells for a suitable period of time, for example, 6 hours. Luciferase is then measured by luminescence reading after co-culture. Luciferase luminescence can be normalized to maximum and minimum intensity to allow comparison of activation peptide concentrations of each engineered receptor construct.

[0314] Provided herein is a method for determining the relative EC50 of an engineered receptor of the present disclosure. As used herein, "EC50" refers to the concentration of an inhibitor or agent at which the response (or binding) is halved. The EC50 of an engineered receptor of the present disclosure refers to the concentration of an antigen at which the binding of the engineered receptor to the antigen is halved. Binding of an antigen or probe to an engineered receptor can be measured by staining with a labeled peptide or a labeled peptide-MHC complex, such as an MHC:HER2 pMHC complex conjugated with a fluorophore. The EC50 can be obtained by nonlinear regression curve fitting of the reporter signal with peptide titration. Probe binding and EC50 can be normalized to the level of a benchmark TCR without the fusion protein (e.g., HER2).

[0315] Polynucleotides The present disclosure provides a polynucleotide system comprising one or more polynucleotides comprising a polynucleotide sequence encoding an activator receptor comprising an extracellular ligand binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen, and an inhibitory receptor comprising an extracellular ligand binding domain specific for a non-target antigen that is not expressed by cancer cells due to loss of heterozygosity in the cancer cells.

[0316] The present disclosure provides vectors comprising the polynucleotide systems described herein.The present disclosure provides vectors comprising the polynucleotides described herein.

[0317] The present disclosure provides polynucleotides encoding the activator receptor and inhibitory receptor sequences described herein. In some embodiments, the activator and / or inhibitory receptor sequences, or fusion proteins of the activator and / or inhibitory receptor, are operably linked to a promoter. In some embodiments, the sequence encoding the activator receptor, or a polypeptide thereof, is operably linked to a first promoter, and the sequence encoding the inhibitory receptor, or a fusion protein thereof, is operably linked to a second promoter.

[0318] In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 52, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 54, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 56, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 58, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 60, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 62, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 21803, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0319] In some embodiments, the polynucleotide or polynucleotide system comprises any of SEQ ID NOs: 75-86, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 76, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 77, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 78, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 79, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 80, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 81, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 82, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO:83, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 84, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 85, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 86, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 208, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 210, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the polynucleotide or polynucleotide system comprises SEQ ID NO: 212, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0320] In some embodiments, the polynucleotide or polynucleotide system comprises any one of SEQ ID NOs: 224, 226, 227, 229, or 231, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0321] In some embodiments, the polynucleotide or polynucleotide system comprises any one of SEQ ID NOs: 292, 294, 296, 298, 300, 302, 304, 306, or 308, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0322] The present disclosure provides a coding sequence or vector encoding any of the engineered receptors described herein. In some embodiments, the activating and / or inhibitory receptor sequences are operably linked to a promoter. In some embodiments, the activating receptor coding sequence is operably linked to a first promoter and the inhibitory receptor coding sequence is operably linked to a second promoter.

[0323] In some embodiments, the activating receptor is encoded by a first vector and the inhibitory receptor is encoded by a second vector, hi some embodiments, both engineered receptors are encoded by a single vector.

[0324] In some embodiments, the activator receptor and the inhibitory receptor are encoded by a single vector. Methods for encoding multiple polypeptides using a single vector are known to those of skill in the art and may include, among others, encoding multiple polypeptides under the control of different promoters or, when using a single promoter to control the transcription of multiple polypeptides, the use of sequences encoding internal ribosome entry sites (IRES) and / or self-cleaving peptides. Exemplary self-cleaving peptides include T2A, P2A, E2A and F2A self-cleaving peptides. In some embodiments, the T2A self-cleaving peptide comprises the sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 288). In some embodiments, the P2A self-cleaving peptide comprises the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 196). In some embodiments, the E2A self-cleaving peptide comprises the sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 289). In some embodiments, the F2A self-cleaving peptide comprises the sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 290). Any of the foregoing may also include an N-terminal GSG linker. For example, the T2A self-cleaving peptide may also include the sequence GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 557), which may be encoded by the sequence GGATCCGGAGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCC (SEQ ID NO: 558).

[0325] In some embodiments, the vector comprises SEQ ID NO:52, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO:54, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO:56, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO:58, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO:60, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 62, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 21803, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0326] In some embodiments, the vector comprises SEQ ID NO: 75, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 76, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 77, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 78, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 79, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 80, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 81, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 82, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 83, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 84, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.In some embodiments, the vector comprises SEQ ID NO:85, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO:86, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO:208, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO:210, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO:212, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0327] In some embodiments, the vector comprises SEQ ID NO: 224, 226, 227, 229, or 231, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector comprises SEQ ID NO: 292, 294, 296, 298, 300, 302, 304, 306, or 308, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0328] In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:51, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:53, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:55, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:57, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:59, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:61, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:63, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:64, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO:65, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 66, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 67, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 68, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 69, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 70, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 72, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 73, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 74, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0329] In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 207, 209, or 211, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the vector encodes a polypeptide sequence comprising SEQ ID NO: 291, 293, 295, 297, 299, 301, 303, 305, 307, 21805, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0330] In some embodiments, the polynucleotide sequences provided herein are codon optimized, which is the process of introducing silent nucleotide changes into a nucleic acid sequence that increase production of the protein encoded by the nucleic acid sequence without changing the amino acid sequence of the protein.

[0331] In some embodiments, the vector is an expression vector, ie, one for expression of an engineered receptor of the present disclosure in a suitable cell.

[0332] Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, because they allow long-term stable integration of transgenes and their proliferation in daughter cells.Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of low immunogenicity.

[0333] Expression of natural or synthetic nucleic acids encoding engineered receptors is typically achieved by operably linking the nucleic acid encoding the fusion protein or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for eukaryotic replication and integration. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0334] The polynucleotide encoding the fusion protein can be cloned into several types of vectors. For example, the polynucleotide can be cloned into a vector, including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0335] Furthermore, the expression vector can be provided to cells, such as immune cells, in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, 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., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0336] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of interest either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0337] Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 base pairs (bp) upstream of the start site, although many promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0338] An 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 operably linked thereto. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α). Another example of a suitable promoter is the U6 promoter. Another example of a suitable promoter is the H1 promoter. However, other constitutive promoter sequences may also be used, including, but not limited to, Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter. An exemplary sequence of a U6 promoter is set forth in SEQ ID NO:21800. An exemplary sequence of a H1 promoter is set forth in SEQ ID NO:21801.

[0339] In certain embodiments, the vector includes a termination sequence, such as a T6 or T7 termination sequence.

[0340] To assess the expression of the engineered receptor, the expression vector introduced into the cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected via the viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA or used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0341] Reporter genes are used to identify potentially transfected or transduced cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. At a suitable time after the DNA is introduced into the recipient cells, the expression of the reporter gene is assayed. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0342] Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0343] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0344] 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 lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0345] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0346] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to an inhibitor of the invention, various assays can be performed to confirm the presence of the recombinant DNA sequence within the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents falling within the scope of the invention.

[0347] immune cells Provided herein are immune cells comprising the polynucleotides, vectors, fusion proteins and engineered receptors described herein.

[0348] The present disclosure provides an immune cell responsive to loss of heterozygosity in a cancer cell, the immune cell comprising an activating receptor comprising an extracellular ligand-binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen and an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen not expressed by the cancer cell due to loss of heterozygosity in the cancer cell. In some embodiments, the non-target antigen is an HLA class I allele or a minor histocompatibility antigen (MiHA). In some embodiments, the HLA class I allele comprises HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the non-target antigen is selected from CXCL16, COLEC12, and APCDD1, HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-B*07, HLA-C*07, or an antigenic peptide thereof. In some embodiments, the HLA class I allele is HLA-A*02. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is a peptide antigen of HER2 in a complex with the major histocompatibility class I complex (MHC I) or MHC II. In some embodiments, the cancer cell expresses HER2. In some embodiments, the cancer cell is a breast cancer cell, a bladder cancer cell, an ovarian cancer cell, a gastric cancer cell, a salivary duct cancer cell, a non-small cell lung cancer cell, a pancreatic cancer cell, or a colon cancer cell. In some embodiments, the cancer cell is a breast cancer cell or a gastric cancer cell.

[0349] In some embodiments, the HLA-A*02 non-target antigen is expressed by healthy cells of the subject. In some embodiments, the healthy cells of the subject express both the target antigen and the HLA-A*02 non-target antigen. In some embodiments, both the activator receptor and the inhibitory receptor specifically activate immune cells in the presence of cancer cells.

[0350] In some embodiments, the immune cells are T cells. In some embodiments, the T cells are CD8+CD4- T cells. In some embodiments, the immune cells are autologous. In some embodiments, the immune cells are allogeneic.

[0351] As used herein, the term "immune cell" refers to a cell involved in the innate or adaptive (acquired) immune system. Exemplary innate immune cells include phagocytes such as neutrophils, monocytes, and macrophages, natural killer (NK) cells, polymorphonuclear leukocytes such as neutrophils, eosinophils, and basophils, and mononuclear cells such as monocytes, macrophages, and mast cells. Immune cells that play a role in acquired immunity include lymphocytes such as T cells and B cells.

[0352] As used herein, "T cells" refers to a type of lymphocyte derived from myeloid precursors that develop in the thymus. There are several different types of T cells that develop upon migration to the thymus, including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells, and stem memory T cells. Different types of T cells can be distinguished by those skilled in the art based on their expression of markers. Methods for distinguishing types of T cells will be readily apparent to those skilled in the art.

[0353] In some embodiments, the engineered immune cells express the first and second receptors in a ratio of about 100:1 to 1:100 of the first receptor to the second receptor. In some embodiments, the engineered immune cells express the first and second receptors in a ratio of about 50:1 to 1:50 of the first receptor to the second receptor. In some embodiments, the engineered immune cells express the first and second receptors in a ratio of about 10:1 to 1:10 of the first receptor to the second receptor. In some embodiments, the engineered immune cells express the first and second receptors in a ratio of about 5:1 to 1:5 of the first receptor to the second receptor. In some embodiments, the engineered immune cells express the first and second receptors in a ratio of about 3:1 to 1:3 of the first receptor to the second receptor. In some embodiments, the engineered immune cells express the first and second receptors in a ratio of the first receptor to the second receptor of about 2:1 to 1:2. In some embodiments, the engineered immune cells express the first and second receptors in a ratio of about 1:1.

[0354] In some embodiments, the first receptor comprises SEQ ID NO: 291, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 303, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 291, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 305, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 291 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 307 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 291 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 21805 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0355] In some embodiments, the first receptor comprises SEQ ID NO: 295, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 303, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 295, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 305, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 295 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 307 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 295 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 21805 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0356] In some embodiments, the first receptor comprises SEQ ID NO: 299, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 303, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 299, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 305, or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 299 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 307 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith. In some embodiments, the first receptor comprises SEQ ID NO: 299 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith, and the second receptor comprises SEQ ID NO: 21805 or a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity therewith.

[0357] In some embodiments, the immune cells further comprise a T2A autocleaving peptide, wherein the T2A autocleaving peptide comprises SEQ ID NO: 288 or 557, or a sequence sharing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0358] In some embodiments, the engineered immune cells comprising the engineered receptors of the present disclosure are T cells. In some embodiments, the T cells are effector T cells or regulatory T cells. As used herein, "T cells" refers to a type of lymphocyte derived from myeloid precursors that develop in the thymus. There are several different types of T cells that develop upon migration to the thymus, including helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells, NK T cells, γδ T cells, mucosal-associated invariant T (MAIT) cells, and stem memory T cells. The different types of T cells can be distinguished by one of skill in the art based on their expression of markers. Methods for distinguishing types of T cells will be readily apparent to one of skill in the art.

[0359] In some embodiments, the immune cell is selected from the group consisting of a T cell, a B cell, and a natural killer (NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is CD8-. In some embodiments, the immune cell is CD8+. In some embodiments, the immune cell is CD4+. In some embodiments, the immune cell is CD4-. In some embodiments, the immune cell is CD8- / CD4+. In some embodiments, the immune cell is a CD8+CD4- T cell.

[0360] In some embodiments, the immune cell is a gamma delta (γδ) T cell. In some embodiments, the immune cell is an invariant T cell. In some embodiments, the immune cell is an invariant natural killer T cell (iNKT cell).

[0361] In some embodiments, the immune cells comprise a modification to an MHC class I gene or a B2M gene. In some embodiments, the MHC class I gene is HLA-A*02, or an allelic variant thereof. In some embodiments, the modification reduces or eliminates expression of the MHC class I gene or the B2M gene.

[0362] In some embodiments, the immune cell comprises a polynucleotide or polynucleotide system described herein. In some embodiments, the polynucleotide or polynucleotide system comprises a sequence encoding a first receptor described herein. In some embodiments, the polynucleotide or polynucleotide system comprises a sequence encoding a second receptor described herein. In some embodiments, the polynucleotide or polynucleotide system encodes an shRNA described herein. In some embodiments, the polynucleotide or polynucleotide system comprises a sequence encoding a first receptor described herein and a second receptor described herein. In some embodiments, the polynucleotide or polynucleotide system comprises a sequence encoding a first receptor described herein, a second receptor described herein, and an shRNA described herein.

[0363] It will be readily apparent to one of skill in the art how to transduce a population of immune cells, such as T cells, using the vectors of the present disclosure. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi) following the manufacturer's instructions. T cells can be cultured at a density of 1x10^6 cells / mL in X-Vivo 15 medium supplemented with 5% human A / B serum and 1% Pen / strep in the presence of CD3 / 28 Dynabeads (1:1 cell to bead ratio) and 300 units / mL IL-2 (Miltenyi). After 2 days, T cells can be transduced with a viral vector, such as a lentiviral vector, using methods known in the art. In some embodiments, the viral vector is transduced at a multiplicity of infection (MOI) of 5. The cells can then be cultured in IL-2 or other cytokines, such as a combination of IL-7 / 15 / 21, for an additional 5 days before enrichment. Methods for isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells, will be readily apparent to those skilled in the art. This method outlines potential approaches, but it should be noted that these methodologies are rapidly evolving. For example, successful viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of expansion to generate a cell population that is more than 99% CD3+ highly transduced.

[0364] Methods for activating and culturing populations of T cells containing an engineered TCR, CAR, fusion protein, or vector encoding a fusion protein of the present disclosure will be readily apparent to one of skill in the art.

[0365] The T cells, whether before or after genetic modification of the T cells to express an engineered TCR, can be generated using any of the methods described in, e.g., U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318 ... Nos. 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, 10040846, and U.S. Patent Application Publication No. 2006 / 0121005.

[0366] In some embodiments, the T cells of the present disclosure are expanded and activated in vitro. In general, the T cells of the present disclosure are expanded in vitro by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, a population of T cells can be stimulated as described herein, for example, by contact with an anti-CD3 antibody. For costimulation of an accessory molecule on the surface of the T cells, a ligand that binds to 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 to stimulate proliferation of the T cells. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate proliferation of either CD4+ T cells or CD8+ T cells. Exemplary anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and can be used as well as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0367] In some embodiments, the primary stimulatory signal and the costimulatory signal for T cells may be provided by different protocols. For example, the agents providing each signal may be in solution or may be bound to a surface. When bound to a surface, the agents may be bound to the same surface (i.e., in a "cis" formation) or to separate surfaces (i.e., in a "trans" formation). Alternatively, one agent may be bound to a surface and the other agent may be in solution. In some embodiments, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents may be in solution. In another embodiment, the agents may be in soluble form and then crosslinked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that binds to the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810 for artificial antigen presenting cells (aAPCs) contemplated for use in activating and expanding T cells in the present invention.

[0368] In some embodiments, the two agents are immobilized on the beads, either on the same bead, i.e., "cis", or on separate beads, i.e., "trans". As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents are co-immobilized on the same bead with comparable molecular weights. In one embodiment, a 1:1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell proliferation is used. In some embodiments, the ratio of CD3:CD28 antibodies bound to the beads ranges from 100:1 to 1:100 and all integer values ​​therebetween. In one aspect of the invention, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the ratio of CD3:CD28 is less than 1. In certain embodiments of the invention, the ratio of anti-CD28 antibodies to anti-CD3 antibodies bound to the beads is greater than 2:1.

[0369] To stimulate T cells or other target cells, particle to cell ratios of 1:500 to 500:1 and any integer value therebetween may be used. As one of ordinary skill in the art can readily appreciate, the particle to cell ratio may depend on the particle size relative to the target cells. For example, small sized beads may only bind to a few cells, while larger beads may bind to many cells. In certain embodiments, the cell to particle ratio ranges from 1:100 to 100:1 and any integer value therebetween, and in further embodiments, the ratio includes 1:9 to 9:1 and any integer value therebetween, and may also be used to stimulate T cells. In some embodiments, a 1:1 cell to bead ratio is used. One of ordinary skill in the art will appreciate that various other ratios may be suitable for use in the present invention. In particular, the ratio will vary depending on the particle size, as well as the size and type of cells.

[0370] In a further embodiment of the invention, cells such as T cells are combined with agent-coated beads, followed by separation of the beads and cells, and then culturing the cells. In an alternative embodiment, the agent-coated beads and cells are not separated but are cultured together prior to culturing. 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.

[0371] As an example, cell surface proteins can be ligated by allowing anti-CD3 and anti-CD28 to bind and contact the T cells with paramagnetic beads. In one embodiment, cells (e.g., CD4+ T cells) and beads (e.g., DYNABEADS CD3 / CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer. Again, one of skill in the art can readily appreciate that any cell concentration can be used. In certain embodiments, it is desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, more than 100 million cells / ml is used. In further embodiments, 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, a concentration of 125 or 150 million cells / ml may be used. In some embodiments, cells cultured at a density of 1×106 cells / mL are used.

[0372] In some embodiments, the mixture may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value of time units therebetween. In another embodiment, the beads and T cells are cultured together for 2 to 3 days. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for the growth of cells known to those of skill in the art. Other additives for the growth of cells include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in amounts sufficient for T cell growth and expansion. In some embodiments, the media comprises X-VIVO-15 medium supplemented with 5% human A / B serum, 1% penicillin / streptomycin (pen / strep), and 300 units / ml of IL-2 (Miltenyi).

[0373] The T cells are maintained under the necessary conditions to support proliferation, for example, at an appropriate temperature (eg, 37° C.) and atmosphere (eg, air+5% CO 2 ).

[0374] In some embodiments, the T cells comprising the engineered TCR, CAR, and / or inhibitory receptors of the present disclosure are autologous. Prior to expansion and genetic modification, a source of T cells is obtained from the subject. Immune cells such as T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art can be used. In certain embodiments of the present disclosure, the T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation.

[0375] In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, may lack magnesium, or may lack many, but not all, divalent cations. As one of skill in the art would readily appreciate, the washing steps may be accomplished by methods known to those of skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca2+-free PBS, Mg2+-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0376] In some embodiments, immune cells such as T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugation. Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD4 conjugated beads for a period of time sufficient for positive selection of the desired T cells.

[0377] Enrichment of immune cell populations, such as T cell populations, by negative selection can be achieved by a combination of antibodies directed to surface markers specific to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0378] For isolation of a desired population of immune cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and beads.

[0379] In some embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at either 2-10° C. or at room temperature.

[0380] PBMCs from which immune cells such as stimulatory T cells or T cells are isolated can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogenous product by removing granulocytes, and to some extent monocytes, in the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium 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 medium containing, for example, Hespan and PlasmaLyte A, and the cells are then 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, as well as uncontrolled instantaneous freezing at -20°C or liquid nitrogen, may also be used.

[0381] The present disclosure provides immune cells expressing the activating and / or inhibitory receptors described herein, wherein the immune cells have reduced expression and / or function of major histocompatibility (MHC) class I complexes.

[0382] In some embodiments, the immune cells are autologous. For example, the immune cells are isolated or derived from the same subject who receives the cells as part of a therapeutic regimen. The modification of autoimmune cells to reduce MHC class I expression and / or function by inhibitory receptors may be advantageously specific to MHC class I antigens. Without wishing to be bound by theory, the modification of autoimmune cells to reduce MHC class I expression and / or function reduces the binding of inhibitory receptors by MHC class I expressed by the immune cells, either in cis or trans.

[0383] In some embodiments, the immune cells are all allogeneic. Allogeneic immune cells may be derived from a donor other than the subject to whom the immune cells are administered. Because allogeneic cells may be prepared and stored for use in subjects of various genotypes, allogeneic immune cells are commonly referred to as "off the shelf" or "universal" in cell therapy.

[0384] Any suitable method of reducing expression and / or function of an MHC class I complex is contemplated within the scope of the present disclosure, including, inter alia, expression of an interfering RNA that knocks down one or more RNAs encoding an MHC class I component, or modification of a gene encoding an MHC class I component.

[0385] The major histocompatibility complex (MHC) is a genetic locus on the vertebrate genome that encodes a set of polypeptides required for the adaptive immune system. Among these are MHC class I polypeptides, including HLA-A, HLA-B, and HLA-C, and their alleles. MHC class I alleles are highly polymorphic and expressed in all nucleated cells. MHC class I polypeptides encoded by HLA-A, HLA-B, and HLA-C and their alleles form heterodimers with β2 microglobulin (B2M) and are present in complex with antigens on the surface of cells. As referred to herein, MHC class I genes or polypeptides may refer to any polypeptide found in the MHC or the corresponding gene encoding the polypeptide. In some embodiments, the immune cells of the present disclosure are inactivated by inhibitory ligands, including MHC class I polypeptides, for example, HLA-A, HLA-B, and HLA-C, and their alleles. An HLA-A allele can be, for example, but not limited to, HLA-A*02, HLA-A*02:01, HLA-A*02:01:01, HLA-A*02:01:01:01, and / or any gene encoding a protein identical or similar to an HLA-A*02 protein. Thus, to prevent the autocrine signaling / binding described herein, it is desirable to remove or reduce expression of polypeptides encoded by HLA-A, HLA-B, and HLA-C, and their alleles, in immune cells.

[0386] Immune cells with reduced expression of MHC class I polypeptides In some embodiments, the immune cells described herein are modified to inactivate or reduce or eliminate expression or function of endogenous genes encoding alleles of endogenous MHC class I polypeptides. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A, HLA-B, and / or HLA-C. HLA-A, HLA-B, and HLA-C are encoded by the HLA-A, HLA-B, and HLA-C loci. Each of HLA-A, HLA-B, and HLA-C includes many variant alleles, all of which are contemplated within the scope of the present disclosure. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A*02. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A*02:01. In some embodiments, the genes encoding MHC class I polypeptides are HLA-A*02:01:01. In some embodiments, the gene encoding the MHC class I polypeptide is HLA-A*02:01:01:01.

[0387] In some embodiments, the engineered immune cells described herein are modified to reduce or eliminate expression of the B2M gene product. The beta-2 microglobulin (B2M) gene encodes a protein that associates with the major histocompatibility complex (MHC) class I, i.e., the MHC-I complex. The MHC-I complex is required for the presentation of antigens on the cell surface. The MHC-I complex is disrupted and non-functional when B2M is deleted (Wang D et al. Stem Cells Transl Med. 4:1234-1245 (2015)). Furthermore, the B2M gene can be disrupted with high efficiency using gene editing techniques known in the art (Ren et al. Clin. Cancer Res. 23:2255-2266 (2017)). Reducing or eliminating B2M can reduce or eliminate functional MHC I on the surface of immune cells.

[0388] The present disclosure provides a gene editing system for editing endogenous target genes in immune cells. The present disclosure provides an interfering RNA specific to the sequence of the target gene. Gene editing systems such as CRISPR / Cas system, TALEN and zinc finger can be used to generate double-strand breaks, which can be used to introduce mutations through gene repair mechanisms such as homology-directed repair or non-homologous end joining (NHEJ). NHEJ after resection of broken ends or improper end joining can be used to introduce deletions. In some embodiments, the target gene comprises a gene encoding a subunit of the MHC-I complex.

[0389] Target gene sequences include, but are not limited to, promoters, enhancers, introns, exons, intron / exon junctions, transcripts (pre-mRNA, mRNA, and splice variants), and / or 3' and 5' untranslated regions (UTRs). Any genetic element or combination of genetic elements can be targeted for the purposes of gene editing in immune cells as described herein. Modifications to target genes can be achieved by editing the target gene, resulting in alteration or disruption of expression or function of the target gene or gene product, using any method known in the art.

[0390] In some embodiments, modifying a gene encoding an MHC class I polypeptide comprises deleting all or a portion of the gene. In some embodiments, modifying a gene encoding an MHC class I polypeptide comprises introducing a mutation into the gene. In some embodiments, the mutation comprises a deletion, an insertion, a substitution, or a frameshift mutation. In some embodiments, modifying the gene comprises using a nucleic acid-guided endonuclease.

[0391] Gene sequences for the target genes described herein are known in the art. The sequences can be found in public databases such as NCBI GenBank or the NCBI nucleotide database. The sequences can be found using gene identifiers, for example, the HLA-A gene has NCBI gene ID: 3105, the HLA-B gene has NCBI gene ID: 3106, the HLA-C gene has NCBI gene ID: 3107, and the B2M gene has NCBI gene ID: 567 and NCBI reference sequence: NC_000015.10. Gene sequences can also be found by searching public databases using keywords. For example, HLA-A alleles can be found in the NCBI nucleotide database by searching the keywords "HLA-A*02", "HLA-A*02:01", "HLA-A*02:01:01", or "HLA-A*02:01:01:01". These sequences can be used for targeting in various gene editing techniques known in the art. Table 18 provides non-limiting exemplary sequences of HLA-A alleles and B2M gene sequences targeted for modification as described herein. [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5] [Table 18-6] [Table 18-7] [Table 18-8]

[0392] One of skill in the art will understand that T can be substituted for U to convert an RNA sequence to a DNA sequence, and vice versa, and both are contemplated as target gene sequences in the present disclosure.

[0393] In some embodiments, the target gene is edited in the immune cells described herein using a nucleic acid-guided endonuclease. Exemplary nucleic acid-guided endonucleases include class II endonucleases such as CRISPR / Cas9.

[0394] As used herein, "CRISPR" or "CRISPR gene editing" refers to a set of clustered regularly interspaced short palindromic repeats or a system that includes such a set of repeats. "Cas" as used herein refers to a CRISPR-associated protein. "CRISPR / Cas" system refers to a system derived from CRISPR and Cas that can be used to silence, knock out, or mutate target genes. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR / Cas systems have been modified for use in gene editing. This is accomplished by introducing into a eukaryotic cell one or more specifically designed guide nucleic acids (gNAs), typically guide RNAs (gRNAs), and an appropriate Cas endonuclease that forms a ribonucleoprotein complex with the gNA. The gNA guides the gNA-endonuclease protein complex to the target genomic location, and the endonuclease introduces a strand break at the target genomic location. This double-stranded break can be repaired by cellular mechanisms such as non-homologous end joining (leading to deletions) or homologous repair (which can generate insertions), thereby introducing a genetic modification into the host cell genome.

[0395] CRISPR / Cas systems are classified into classes and types. Class 2 systems represent a single interfering protein that is currently classified into three different types (type II, type V, and type VI). Any class 2 CRISPR / Cas system suitable for gene editing, such as type II, type V, or type VI systems, is contemplated within the scope of the present disclosure. Exemplary class 2 type II CRISPR systems include Cas9, Csn2, and Cas4. Exemplary class 2 type V CRISPR systems include Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, and Cas12k (C2c5). Exemplary class 2 type VI systems include Cas13, Cas13a (C2c2) Cas13b, Cas13c, and Cas13d.

[0396] CRISPR sequence, sometimes called CRISPR locus, comprises alternating repeats and spacers. In naturally occurring CRISPR, the spacer usually comprises a sequence foreign to bacteria, such as a plasmid or phage sequence. As described herein, the spacer sequence may also be referred to as "targeting sequence". In the CRISPR / Cas system for genetic manipulation, the spacer is derived from the target gene sequence (gNA).

[0397] Exemplary class 2 type II CRISPR system relies on protein Cas9, which is a nuclease with two active cleavage sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi (2013) Science 341:833-836. In some embodiments, the Cas protein used to modify immune cells is Cas9.

[0398] Thus, CRISPR / Cas system can be used to edit target genes, such as the genes targeted for editing in immune cells described herein, by adding or deleting base pairs, or by introducing early stops, thereby reducing the expression of the target. Alternatively, CRISPR / Cas system can be used like RNA interference to reversibly turn off target genes. In mammalian cells, for example, RNA can guide Cas protein to target gene promoters and sterically block RNA polymerase.

[0399] The Cas protein may be derived from any bacterial or archaeal Cas protein. Any suitable CRISPR / Cas system is contemplated within the scope of the present disclosure. In other embodiments, the Cas protein is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a (Cpf1), Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, The Cas protein may comprise one or more of Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, CasY, homologs thereof, or modified forms thereof. In some embodiments, the Cas protein is a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, or a combination or complex thereof. In some embodiments, the Cas protein is a Cas9 protein.

[0400] The artificial CRISPR / Cas system that suppresses target genes can be generated using techniques known in the art, such as those described in US Publication No. 2014 / 0068797 and Cong (2013) Science 339:819-823. For example, those described in Tsai (2014) Nature Biotechnol.,32:6 569-576, U.S. Patent Nos. 8,871,445, 8,865,406, 8,795,965, 8,771,945, and 8,697,359, but other artificial CRISPR / Cas systems that suppress target genes known in the art can also be generated. Methods for designing suitable gNAs for specific Cas proteins will be known to those skilled in the art.

[0401] The present disclosure provides gene-targeting guide nucleic acids (gNAs) that can guide the activity of an associated polypeptide (e.g., a nucleic acid-guided endonuclease) to a specific target gene sequence within a target nucleic acid genome. The genome-targeting nucleic acid can be an RNA. The genome-targeting RNA is referred to herein as a "guide RNA" or "gRNA." The guide RNA can include at least a targeting sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In some type II systems, the gRNA also includes a second RNA, called a tracrRNA sequence, also referred to herein as a "scaffold" sequence. In type II guide RNAs (gRNAs), the CRISPR repeat sequence and the scaffold sequence hybridize to each other to form a duplex. In type V guide RNAs (gRNAs), the crRNA forms a duplex. In both systems, the duplex can bind to a site-directed polypeptide such that the guide RNA and the site-directed polypeptide form a complex. The gene-targeting nucleic acid can provide target specificity to the complex by association with the site-directed polypeptide. Thus, the gene targeting nucleic acid can direct the activity of a site-specific polypeptide.

[0402] In some embodiments, the present disclosure provides a guide RNA comprising a targeting sequence and a guide RNA scaffold sequence, wherein the targeting sequence is complementary to a sequence of a target gene.

[0403] Exemplary guide RNAs include a targeting sequence of about 15-20 bases. As will be appreciated by those skilled in the art, each gRNA can be designed to include a targeting sequence that is complementary to its genomic target sequence. For example, the targeting sequence may be an RNA version of a DNA sequence that is present.

[0404] Table 19 (excluding the three 3' nucleotides representing its PAM site) can be placed into a single RNA chimera or crRNA.

[0405] The gene targeting nucleic acid may be a bimolecular guide RNA.The gene targeting nucleic acid may be a single-molecule guide RNA.The gene targeting nucleic acid may be any known configuration of guide RNA known in the art, including, for example, paired gRNA, or multiple gRNAs used in a single step.The coding sequence and splice junction are clear from the genome sequence, but other features required for gene expression may be specific and unclear.

[0406] The bimolecular guide RNA can comprise two strands of RNA. The first strand comprises a 5' to 3' sequence, an optional spacer extension sequence, a targeting sequence, and a minimal CRISPR repeat sequence. The second strand can comprise a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3'tracrRNA sequence, and an optional tracrRNA extension sequence.

[0407] The single molecule guide RNA (sgRNA) in the type II system can include, from 5' to 3', an optional spacer extension sequence, a targeting sequence, a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension can include elements that contribute additional functionality (e.g., stability) to the guide RNA. The single molecule guide linker can link the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can include one or more hairpins.

[0408] In some embodiments, the guide RNA or single molecule guide RNA (sgRNA) can include a targeting sequence and a scaffold sequence. In some embodiments, the scaffold sequence is a Cas9 gRNA sequence. In some embodiments, the scaffold sequence is encoded by a DNA sequence comprising a sequence that shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 575). In some embodiments, the scaffold sequence is encoded by a DNA sequence comprising GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 575).

[0409] In some embodiments, for example those embodiments where the CRISPR / Cas system is a Cas9 system, the sgRNA can comprise a targeting sequence of 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a targeting sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a targeting sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a variable length targeting sequence having 17-30 nucleotides at the 5' end of the sgRNA sequence.

[0410] Suitable scaffold sequences, and the location of the scaffold target sequence, will depend on the choice of endonuclease and will be known to one of skill in the art.

[0411] A single molecule guide RNA (sgRNA) in a Type II system, such as Cas9, can comprise, from 5' to 3', a minimal CRISPR repeat sequence and a targeting sequence.

[0412] By way of example, guide RNAs or other smaller RNAs used in the CRISPR / Cas9 or CRISPR / Cpf1 system can be easily synthesized by chemical means, as exemplified below and described in the art. While chemical synthesis procedures are continually expanding, as polynucleotide lengths increase significantly beyond about 100 nucleotides, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult. One approach used to generate longer RNAs is to produce two or more molecules that are ligated together. Much longer RNAs, such as those that code for Cas9 or Cpf1 endonucleases, are more easily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic generation of RNA, for example, modifications that improve stability, reduce the likelihood or extent of innate immune responses, and / or improve other attributes, as described in the art.

[0413]

[0423] The targeting sequence of gRNA hybridizes to a sequence in the target nucleic acid of interest. The targeting sequence of genome targeting nucleic acid can interact with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The nucleotide sequence of the targeting sequence can vary depending on the sequence of the target nucleic acid of interest.

[0414] In the Cas9 system described herein, the targeting sequence can be designed to hybridize to the target nucleic acid located 5' of the reverse complement of the PAM of the Cas9 enzyme used in the system. The targeting sequence can be a perfect match to the target sequence or can have a mismatch. Each CRISPR / Cas system protein may have a specific PAM sequence in a specific orientation and position that is recognized in the target DNA. For example, S. pyogenes Cas9 recognizes a PAM in the target nucleic acid that contains the sequence 5'-NRG-3', where R contains either A or G, and N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the targeting sequence. The selection of an appropriate PAM sequence will be apparent to one of skill in the art.

[0415] The target sequence is complementary to and hybridizes with the targeting sequence of the gRNA. The target nucleic acid sequence can include 20 nucleotides. The target nucleic acid can include less than 20 nucleotides. The target nucleic acid can include more than 20 nucleotides. The target nucleic acid can include at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides or more. In some embodiments, for example, in those embodiments where the CRISPR / Cas system is a Cas9 system, the target nucleic acid sequence can include 20 nucleotides immediately 5' of the first nucleotide of the reverse complement of the PAM sequence. This target nucleic acid sequence is often referred to as the PAM strand or target strand, and the complementary nucleic acid sequence is often referred to as the non-PAM strand or non-target strand. Those skilled in the art will recognize that the targeting sequence hybridizes to the non-PAM strand of the target nucleic acid. See, for example, US2019 / 0185849A1.

[0416] In some instances, the percent complementarity between the targeting sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some instances, the percent complementarity between the targeting sequence and the target nucleic acid is up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%. In some examples, the percent complementarity between the targeting sequence and the target nucleic acid is 100% over the six contiguous 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. The percent complementarity between the targeting sequence and the target nucleic acid can be at least 60% over about 20 contiguous nucleotides. The length of the targeting sequence and the target nucleic acid can vary by 1-6 nucleotides, which can be considered a bulge or multiple bulges.

[0417] The targeting sequence can be designed or selected using computer programs known to those skilled in the art. The computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic occurrence frequency (e.g., of sequences that are identical or similar but vary in one or more spots as a result of mismatches, insertions, or deletions), methylation status, presence of SNPs, etc. Available computer programs can take as input NCBI gene IDs, official gene symbols, Ensembl Gene IDs, genomic coordinates, or DNA sequences, and create an output file that includes sgRNAs that target the appropriate genomic regions specified as input. The computer program can also provide summary statistics and scores indicating on-target and off-target binding of the sgRNA for the target gene (Doench et al. Nat Biotechnol. 34:184-191 (2016)). The present disclosure provides a guide RNA that includes a targeting sequence. In some embodiments, the guide RNA further includes a guide RNA scaffold sequence. In some embodiments, the targeting sequence is complementary to a sequence of a target gene selected from the group consisting of HLA-A, HLA-B, HLA-C, B2M, or an allele thereof. In some embodiments, the target gene is an HLA-A gene. In some embodiments, the target gene is an HLA-B gene. In some embodiments, the target gene is an HLA-C gene. In some embodiments, the target gene is HLA-A, HLA-B, HLA-C, or a combination thereof. In some embodiments, the targeting sequence comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity with or is identical to a sequence disclosed in Table 18.

[0418] Table 19.

[0419] In some embodiments, the gNA specifically targets a sequence of endogenous HLA-A. In some embodiments, the gNA that specifically targets a sequence of the HLA-A locus comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with a sequence selected from the sequences disclosed in Table 19.

[0420] In some embodiments, the gNA that specifically targets a sequence in the HLA-A locus comprises a sequence selected from the sequences disclosed in Table 19.

[0421] In some embodiments, the gNA that specifically targets a sequence in the HLA-A locus comprises a sequence set forth in any one of SEQ ID NOs: 576-8650.

[0422] In some embodiments, the gNA specifically targets a sequence of the HLA-A*02 allele. For example, the gRNA specifically targets and hybridizes to a sequence shared by all HLA-A*02 alleles, but not shared by HLA-A*02 and HLA-A*03 alleles. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets a sequence of the HLA-A*02:01:01:01 allele. In some embodiments, the gNA specifically targets a coding DNA sequence of HLA-A*02.

[0423] In some embodiments, the gNA specifically targets a coding DNA sequence shared by more than 1000 HLA-A*02 alleles. [Table 19-1] [Table 19-2]

[0424] The sequences disclosed in Table 19 include the corresponding genomic sequences, including the PAM sequence. One of skill in the art will understand that the targeting sequence of a gRNA does not include the three 3' terminal nucleotides of the sequences in Table 19, which represent the corresponding PAM site for the gRNA.

[0425] The present disclosure provides a gRNA comprising a targeting sequence specific to the B2M gene. In some embodiments, the gRNA specifically targets the coding sequence (CDS) sequence of the B2M gene. In some embodiments, the gRNA comprises a sequence that targets the B2M gene promoter sequence.

[0426] In some embodiments, the gRNA comprises a targeting sequence and a gRNA scaffold sequence. In some embodiments, the targeting sequence comprises a sequence set forth in Table 20, or a sequence sharing about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity thereto.

[0427] In some embodiments, the targeting sequence is complementary to a sequence of the B2M gene. In some embodiments, the B2M gene comprises a sequence that shares about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity with the B2M sequence shown in Table 18. [Table 20-1] [Table 20-2]

[0428] In some embodiments, the immune cells described herein are edited using TALEN gene editing.

[0429] "TALEN" or "TALEN gene editing" refers to Transcription Activator-Like Effector Nucleases, artificial nucleases used to edit target genes.

[0430] TALENs are artificially produced by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) derived from Xanthomonas bacteria can be engineered to bind to any desired DNA sequence, including a portion of a target gene, such as a TCR subunit, an MHC class I complex component, or CD52. The engineered TALE can be combined with a DNA cleavage domain to produce a restriction enzyme specific to any desired DNA sequence, including a target gene sequence. These can then be introduced into cells and used for genome editing.

[0431] To produce a TALEN, a TALE protein is fused to a nuclease (N), which is a wild-type or mutant Fold endonuclease. Several mutations to FokI have been made for use in the TALEN, which improve, for example, cleavage specificity or activity.

[0432] The FokI domain functions as a dimer, requiring two constructs with their own DNA binding domains to be properly oriented and spaced to a site in the target genome. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain, and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity.

[0433] TALENs specific for sequences within a target gene can be constructed using any method known in the art, including a variety of schemes that use modular components.

[0434] In some embodiments, target genes are edited in immune cells described herein using ZFN gene editing.

[0435] "ZFN" or "zinc finger nuclease" or "ZFN gene editing" refers to zinc finger nucleases, which are artificial nucleases that can be used to edit target genes.

[0436] Similar to TALENs, ZFNs contain a Fold nuclease domain (or a derivative thereof) fused to a DNA-binding domain, which in the case of ZFNs contains one or more zinc fingers.

[0437] Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. Zinc fingers can, for example, contain Cys2His2 and can recognize about 3 bp sequences. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize about 6, 9, 12, 15, or 18 bp sequences. A variety of selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0438] Like TALENs, ZFNs must dimerize to cleave DNA. Thus, a pair of ZFNs is required to target non-palindromic DNA sites. Two individual ZFNs must bind to opposite strands of DNA with their nucleases appropriately spaced apart.

[0439] Also, like TALENs, ZFNs can create double-stranded breaks in DNA, which, if improperly repaired, can create frameshift mutations, resulting in reduced expression and amount of target genes or gene products in cells.ZFNs can also be used in conjunction with homologous recombination to make mutations in target genes.

[0440] ZFNs specific for a sequence within a target gene can be constructed using any method known in the art.

[0441] In some embodiments, the expression and function of one or more MCH-I components are reduced using RNA interference. "RNAi" or "RNA interference" refers to the process of sequence-specific post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA). Double-stranded RNAs, such as siRNA (small interfering RNA), miRNA (microRNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeat-associated siRNA), and modified forms thereof, can all mediate RNA interference. These dsRNA molecules can be commercially available or can be designed and prepared based on known sequence information. The antisense strand of these molecules can include RNA, DNA, PNA, or a combination thereof. DNA / RNA chimeric polynucleotides include, but are not limited to, double-stranded polynucleotides consisting of DNA and RNA that suppress the expression of a target gene. The dsRNA molecule can also include one or more modified nucleotides, as described herein, which can be incorporated into either or both strands.

[0442] In RNAi gene silencing or knockdown, dsRNA is introduced into an organism, comprising a first (antisense) strand that is complementary to a portion of a target gene, and a second (sense) strand that is fully or partially complementary to the first antisense strand. After being introduced into an organism, the target gene-specific dsRNA is processed into relatively small fragments (siRNAs), which are then distributed throughout the organism, reducing the messenger RNA of the target gene and resulting in a phenotype that can be very similar to the phenotype resulting from complete or partial deletion of the target gene.

[0443] Certain dsRNA in cells can be acted upon by the Dicer enzyme, which is a ribonuclease III enzyme. Dicer can process dsRNA into shorter pieces of dsRNA, namely siRNA. RNAi also involves an endonuclease complex known as the RNA-induced silencing complex (RISC). After cleavage by Dicer, siRNA enters the RISC complex and directly cleaves the single-stranded RNA target that has a sequence complementary to the antisense strand of the siRNA duplex. The other strand of the siRNA is the passenger strand. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex. Thus, siRNA can downregulate or knock down gene expression by mediating RNA interference in a sequence-specific manner.

[0444] As used herein with respect to RNA interference, a "target gene" or a "target sequence" refers to a gene or gene sequence whose corresponding RNA is targeted for degradation via the RNAi pathway using dsRNA or siRNA as described herein. Exemplary target gene sequences are shown in Table 18. For example, to target a gene using siRNA, the siRNA comprises an antisense region that is complementary or substantially complementary to at least a portion of the target gene or sequence, and a sense strand that is complementary to the antisense strand. Once introduced into a cell, the siRNA directs the RISC complex to cleave the RNA that contains the target sequence, thereby degrading the RNA. The present disclosure provides an interfering RNA. The double-stranded RNA molecule of the present disclosure may be in the form of any type of RNA interference molecule known in the art. In some embodiments, the double-stranded RNA molecule is a small interfering RNA (siRNA). In other embodiments, the double-stranded RNA molecule is a short hairpin RNA (shRNA) molecule. In other embodiments, the double-stranded RNA molecule is a Dicer substrate that is processed in the cell to produce an siRNA. In other embodiments, the double-stranded RNA molecule is a part of a microRNA precursor molecule.

[0445] In some embodiments, the shRNA is of a length suitable as a Dicer substrate and can be processed to produce a RISC-active siRNA molecule. See, e.g., Rossi et al., US2005 / 0244858.

[0446] A Dicer substrate double-stranded RNA (e.g., shRNA) can be of sufficient length to be processed by Dicer to produce an active siRNA and can further include one or more of the following properties: (i) a Dicer substrate shRNA can be asymmetric, e.g., with a 3' overhang on the antisense strand, (ii) a Dicer substrate shRNA can have a modified 3' end on the sense strand to direct the orientation of Dicer binding and processing of the dsRNA into an active siRNA, e.g., incorporation of one or more DNA nucleotides, and (iii) the first and second strands of a Dicer substrate dsRNA can be 21-30 bp in length.

[0447] In some embodiments, the interfering RNA comprises a sequence complementary to a sequence of B2M mRNA. In some embodiments, the interfering RNA can induce RNAi-mediated degradation of B2M mRNA. In some embodiments, the B2M mRNA sequence comprises a coding sequence. In some embodiments, the B2M mRNA sequence comprises an untranslated region.

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

[0449] In some embodiments, the interfering RNA is a short hairpin RNA (shRNA). In some embodiments, the shRNA comprises a first sequence having a sequence complementary to B2M mRNA at its 5' to 3' end, and a second sequence having a sequence complementary to the first sequence at its 5' to 3' end, and the first sequence and the second sequence form the shRNA.

[0450] In some embodiments, the first sequence is 18, 19, 20, 21, or 22 nucleotides. In some embodiments, the first sequence is complementary to a sequence selected from the sequences shown in Tables 21 and 22. In some embodiments, the first sequence has a GC content of 25% or more and less than 60%. In some embodiments, the first sequence is complementary to a sequence selected from the sequences shown in Tables 21 and 22. In some embodiments, the first sequence does not contain four nucleotides of the same base, or a run of seven C or G nucleotide bases. In some embodiments, the first sequence is 21 nucleotides.

[0451] In some embodiments, the first sequence is complementary to a sequence selected from SEQ ID NOs: 8771-13382. In some embodiments, the first sequence has a GC content of 25% or more and less than 60%. In some embodiments, the first sequence is complementary to a sequence selected from SEQ ID NOs: 8771-13382. In some embodiments, the first sequence does not contain four nucleotides of the same base, or a run of seven C or G nucleotide bases. In some embodiments, the first sequence is complementary to a sequence selected from SEQ ID NOs: 8771-13382. In some embodiments, the first sequence is 21 nucleotides. In some embodiments, the first sequence is complementary to a sequence selected from SEQ ID NOs: 8771-9352. In some embodiments, the first sequence is complementary to a sequence selected from SEQ ID NOs: 8771-9052. In some embodiments, the first sequence is complementary to a sequence selected from SEQ ID NOs: 8771-8908. Exemplary target B2M sequences complementary to the first sequence are shown in SEQ ID NOs:8771-8908.

[0452] Exemplary target B2M sequences complementary to the first sequence are shown in Table 21. In some embodiments, the first sequence targeting B2M comprises a sequence set forth in any one of SEQ ID NOs: 8771-13382.

[0453] In some cases, the first sequence may have 100% identity, i.e., complete identity, homology, complementarity, with the target nucleic acid sequence.In other cases, there may be one or more mismatches between the first sequence and the target nucleic acid sequence.For example, there may be 1, 2, 3, 4, 5, 6, or 7 mismatches between the sense region and the target nucleic acid sequence.

[0454] The sequences shown in Table 21 are presented as DNA sequences. In all sequences shown in Table 21, thymine (T) can be replaced by uracil (U) to arrive at the sequence of the target mRNA sequence. [Table 21-1] [Table 21-2]

[0455] Exemplary sequences encoding B2M shRNAs include the sequence GCACTCAAAGCTTGTTAAGATCGAAATCTTAACAAGCTTTGAGTGC (SEQ ID NO: 13383), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto. Further exemplary sequences encoding B2M shRNAs include the sequence GTTAACTTCCAATTTACATACCGAAGTATGTAAATTGGAAGTTAAC (SEQ ID NO: 13384), or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity thereto.

[0456] In some embodiments, the shRNA comprises a first sequence having a sequence at its 5' to 3' end that is complementary to HLA-A*02 mRNA, and a second sequence having a sequence at its 5' to 3' end that is complementary to the first sequence. In some embodiments, the HLA-A*02 mRNA sequence comprises a coding sequence. In some embodiments, the HLA-A*02 mRNA sequence comprises an untranslated region. In some embodiments, the first and second sequences are present on a polynucleotide, and the first and second sequences are separated by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, and the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides form a loop region in the shRNA. In some embodiments, the shRNA further comprises a 5' flanking sequence and a 3' flanking sequence, wherein the 5' flanking sequence is linked to the 5' end of the first sequence and the 3' flanking sequence is linked to the 3' end of the second sequence.

[0457] In some embodiments, a polynucleotide encoding an shRNA has, from the 5' to the 3' end, a 5' flanking sequence, a first sequence, a loop sequence, a second sequence, and a 3' flanking sequence. In some embodiments, a polynucleotide encoding an shRNA has, from the 5' to the 3' end, a 5' flanking sequence, a second sequence, a loop sequence, a first sequence, and a 3' flanking sequence.

[0458] In some embodiments, the first sequence is 18, 19, 20, 21, or 22 nucleotides. In some embodiments, the first sequence is complementary to a sequence selected from Tables 13385-21779. In some embodiments, the first sequence has a GC content of 25% or more and less than 60%. In some embodiments, the first sequence is complementary to a sequence selected from Tables 13385-16975. In some embodiments, the first sequence does not contain four nucleotides of the same base, or a run of seven C or G nucleotide bases. In some embodiments, the first sequence is complementary to a sequence selected from Tables 13385-16493. In some embodiments, the first sequence is complementary to a sequence selected from Tables 13385-13663. In some embodiments, the first sequence is complementary to a sequence selected from Tables 13385-13470.

[0459] Exemplary target HLA sequences complementary to the first sequence are shown in Table 22. [Table 22-1] [Table 22-2]

[0460] In some embodiments, the first and second sequences are separated by a linker, sometimes referred to as a loop. In some embodiments, both the first and second sequences are encoded by one single-stranded RNA or DNA vector. In some embodiments, the loop is between the first and second sequences. In these embodiments, the first and second sequences hybridize to form a double-stranded region. The first and second sequences are linked by a linker sequence to form a "hairpin" or "stem-loop" structure. The shRNA can have a complementary first and second sequence at opposite ends of a single-stranded molecule, such that the molecule can form a double-stranded region with the complementary sequence portion, and the strands are linked at one end of the double-stranded region by a linker (i.e., loop sequence). The linker, or loop sequence, can be either a nucleotide or a non-nucleotide linker. The linker can interact with the first sequence, and optionally the second sequence, via covalent or non-covalent interactions.

[0461] Any suitable nucleotide loop sequence is contemplated within the scope of the present disclosure. The shRNA of the present disclosure may include a nucleotide, non-nucleotide, or mixed nucleotide / non-nucleotide linker that connects the first sequence of the shRNA to the second sequence of the shRNA. The nucleotide loop sequence may be 2 or more nucleotides in length, for example, about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. Exemplary loop sequences are disclosed in Table 23.

[0462] In some embodiments, the shRNA further comprises a 5' flanking sequence and a 3' flanking sequence, in some embodiments, the 5' flanking sequence is linked to the 5' end of the first sequence and the 3' flanking sequence is linked to the 3' end of the second sequence.

[0463] Without wishing to be bound by theory, it is believed that flanking the shRNA stem-loop sequence with 5' and 3' sequences similar to those found in microRNAs can target the shRNA for processing by endogenous microRNA processing machinery and increase the efficacy of shRNA processing. Alternatively, or in addition, the flanking sequences can increase shRNA compatibility with polymerase II or polymerase III promoters, resulting in more effective regulation of shRNA expression.

[0464] In some embodiments, the 5' flanking sequence is selected from the sequences shown in Table 23. Exemplary flanking sequences are shown in Table 23. [Table 23]

[0465] In some embodiments, the first and second sequences are present on a single stranded polynucleotide, and the first and second sequences are separated by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, and the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides form a loop region in the shRNA. In some embodiments, the loop region comprises a sequence selected from the sequences set forth in Table 24. [Table 24]

[0466] The shRNA of the present disclosure can be produced exogenously by chemical synthesis, by in vitro transcription, or by cleavage of longer double-stranded RNA by Dicer or another suitable nuclease with similar activity. Chemically synthesized siRNA produced from protected ribonucleoside phosphoramidites using conventional DNA / RNA synthesizers can be obtained from commercial sources such as Millipore Sigma (Houston, Tex.), Ambion Inc. (Austin, Tex.), Invitrogen (Carlsbad, Calif.), or Dharmacon (Lafayette, Colo.). siRNA can be purified, for example, by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, siRNA may be used with little or no purification to avoid losses due to sample processing. In some embodiments, the shRNA of the present disclosure can be produced using an expression vector in which a nucleic acid encoding the double-stranded RNA has been cloned, for example, under the control of a suitable promoter.

[0467] Pharmaceutical Compositions The present disclosure provides a pharmaceutical composition comprising an immune cell expressing an engineered receptor of the present disclosure and a pharma- ceutically acceptable diluent, carrier, or excipient. In some embodiments, the pharmaceutical composition is for use as a medicament in the treatment of cancer. In some embodiments, the cancer is breast cancer, bladder cancer, ovarian cancer, gastric cancer, salivary duct cancer, non-small cell lung cancer, pancreatic cancer, or colon cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is gastric cancer.

[0468] Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, and preservatives.

[0469] In some embodiments, the immune cells express both the first receptor and the second receptor. In some embodiments, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the immune cells express both the first receptor and the second receptor. In some embodiments, at least 90% of the immune cells express both the first receptor and the second receptor.

[0470] The present disclosure provides a pharmaceutical composition comprising a plurality of immune cells of the present disclosure and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0471] A formulation of a pharmaceutical composition suitable for parenteral administration typically consists of immune cells combined with a pharma- ceutically acceptable carrier, usually sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, e.g., in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, e.g., aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted- and programmed release.

[0472] In some embodiments, the formulated composition comprising the immune cells is suitable for administration by injection. In some embodiments, the formulated composition comprising the immune cells is suitable for administration by injection.

[0473] The pharmaceutical compositions of the present disclosure may be conveniently presented in unit dosage form and may be prepared according to conventional techniques well known in the pharmaceutical industry, including the step of bringing into association the immune cells with a pharmaceutical carrier or excipient, e.g., a liquid carrier.

[0474] Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0475] The composition of the present disclosure may additionally contain other auxiliary components that are conventionally found in pharmaceutical compositions.Thus, for example, the composition may contain additional compatible pharmacologic active materials, such as, for example, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials that are useful for physically formulating various dosage forms of the composition of the present disclosure, such as dyes, preservatives, antioxidants, opacifiers, thickeners, and stabilizers.However, such materials, when added, should not unduly interfere with the biological activity of immune cells of the composition of the present disclosure.

[0476] The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with immune cells, and each activity does not adversely affect each other.Such active ingredients are preferably present in combination in an amount that is effective for the intended purpose.Thus, in some embodiments, the pharmaceutical composition further comprises other pharmacologic active agents or drugs, such as chemotherapeutic agents.

[0477] In some embodiments, the pharmaceutical composition can use time-release, delayed release, and sustained release delivery systems, so that the delivery of the composition occurs before the sensitization of the treated area and at a sufficient time to cause the sensitization of the treated area.Many types of release delivery systems are available and known.Such systems can avoid repeated administration of the composition, thereby increasing the convenience for subjects and physicians.

[0478] Administration can be in one dose, continuously or intermittently throughout the course of treatment. Single or multiple administrations can be carried out with dose levels and pattern being selected by the treating physician.

[0479] The pharmaceutical composition in some embodiments contains immune cells in an amount effective for treating or preventing cancer, e.g., a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the treated subject. For repeated administration over several days, weeks, or months, depending on the condition, treatment can be repeated until a desired suppression of signs or symptoms of cancer occurs. However, other dosing regimens may be useful and can be determined. The desired dose can be delivered by a single bolus or injection of the composition, or by multiple boluses or injections of the composition.

[0480] How to use Provided herein is a method of selectively killing HER2-positive tumor cells having loss of heterozygosity at alleles encoding non-target antigens in HER2-positive cancer, comprising contacting the HER2-positive tumor cells with immune cells comprising an engineered receptor of the present disclosure. The cells may be, for example, in a tissue (e.g., in vivo) or in a mixed culture. In some embodiments, the non-target antigen is an HLA class I allele or a minor histocompatibility antigen (MiHA). In some embodiments, the HLA class I allele comprises HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the HLA class I allele is HLA-A*02. In some embodiments, the non-target antigen is selected from the group consisting of HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-B*07, and HLA-C*07.

[0481] Provided herein is a method of treatment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising immune cells comprising an engineered receptor of the present disclosure.

[0482] In some embodiments, the method comprises treating a HER2+ cancer in a subject identified as having or suspected of having a loss of heterozygosity in an allele encoding a non-target antigen in the HER2+ cancer, comprising administering to the subject an immune cell as described herein. In some embodiments, the immune cell is autologous. In some embodiments, the immune cell is allogeneic. In some embodiments, the non-target antigen is an HLA class I allele or a minor histocompatibility antigen (MiHA). In some embodiments, the HLA class I allele comprises HLA-A, HLA-B, HLA-C, or HLA-E. In some embodiments, the HLA class I allele is HLA-A*02. In some embodiments, the non-target antigen is selected from the group consisting of HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-B*07, and HLA-C*07.

[0483] The present disclosure provides a method for producing an immune cell therapy. In some embodiments, the method comprises transforming an immune cell with a polynucleotide system described herein. In some embodiments, the polynucleotide system comprises one or more polynucleotides comprising a polynucleotide sequence encoding an activator receptor comprising an extracellular ligand binding domain specific for an erb-b2 receptor tyrosine kinase 2 (HER2) antigen, and an inhibitory receptor comprising an extracellular ligand binding domain specific for a non-target antigen that is not expressed by the cancer cell due to loss of heterozygosity in the cancer cell.

[0484] In some embodiments, a method of treating a subject comprises providing immune cells from a subject suffering from or at risk for HER2 positive (HER2+) cancer, hi some embodiments, the method comprises transducing the immune cells with a polynucleotide system described herein.

[0485] Current methods of adoptive cell therapy using autologous cells include isolating immune cells from the patient's blood, performing a series of modifications on the isolated cells, and administering the cells to the patient (Papathanasiou et al. Cancer Gene Therapy. 27:799-809 (2020)). Providing immune cells from a subject suffering from or at risk for cancer or hematological malignancy requires isolation of immune cells from the patient's blood, which can be accomplished by methods known in the art, for example, leukapheresis. During leukapheresis, blood from the subject is extracted, peripheral blood mononuclear cells (PBMCs) are separated, and the remainder of the blood is returned to the subject's circulation. The PBMCs are stored, either frozen or cryopreserved, as a sample of immune cells and provided for further processing steps, for example, the modifications described herein.

[0486] In some embodiments, the methods of treating a subject described herein include modifications to immune cells from the subject, including a range of modifications including enrichment, activation, genetic modification, expansion, formulation, and cryopreservation.

[0487] The present disclosure provides an enrichment step, which can be, for example, a washing and fractionation method known in the art for preparation of subject PBMCs for downstream procedures, e.g., modifications as described herein. For example, but not limited to, the method can include a device for removing total red blood cell and platelet contaminants, a system for size-based cell fractionation for depletion of monocytes and isolation of lymphocytes, and / or a system that allows enrichment of specific subsets of T cells, such as, for example, CD4+, CD8+, CD25+, or CD62L+ T cells. Following the enrichment step, a target subpopulation of immune cells is isolated from the subject's PBMCs for further processing. One skilled in the art will appreciate that the enrichment step provided herein can also encompass any newly discovered methods, devices, reagents, or combinations thereof.

[0488] The present disclosure provides an activation step that can be any method known in the art for inducing activation of immune cells, e.g., T cells, necessary for their ex vivo expansion. Immune cell activation can be achieved, for example, by culturing the subject's immune cells in the presence of dendritic cells, culturing the subject's immune cells in the presence of artificial antigen presenting cells (AAPCs), or culturing the immune cells in the presence of irradiated K562-derived AAPCs. Other methods for activating the subject's immune cells can be, for example, culturing the immune cells in the presence of isolated activation elements and compositions, e.g., beads, surfaces, or particles functionalized with activation elements. Activation elements can include, for example, antibodies, e.g., anti-CD3 antibodies and / or anti-CD28 antibodies. Activation elements can also be, for example, cytokines, e.g., interleukin (IL)-2 or IL-21. Activation elements can also be, for example, costimulatory molecules, such as CD40, CD40L, CD70, CD80, CD83, CD86, CD137L, ICOSL, GITRL, and CD134L. One of skill in the art will understand that the activating elements provided herein can also encompass any newly discovered activating elements, reagents, compositions, or combinations thereof that are capable of activating immune cells.

[0489] The present disclosure provides a genetic modification step for modifying immune cells of a subject. In some embodiments, the genetic modification comprises transducing immune cells with an engineered receptor. In some embodiments, the method comprises transducing immune cells with a first vector comprising a sequence encoding an activator receptor and a second vector comprising a sequence encoding an inhibitory receptor, thereby producing immune cells expressing the activator receptor and the inhibitory receptor.

[0490] The present disclosure provides an expansion step for genetically modified subject immune cells. The genetically modified subject immune cells can be expanded in any immune cell expansion system known in the art to generate a therapeutic dose of immune cells for administration. For example, a bioreactor bag for use in a system including a controller pump, and a probe that allows for automatic feeding and waste removal can be used for immune cell expansion. A cell culture flask with a gas permeable membrane at the base can be used for immune cell expansion. Any such system known in the art that allows for immune cell expansion for clinical use is encompassed in the expansion step provided herein. The immune cells are expanded in a culture system in a medium specifically formulated for expansion. Expansion can also be facilitated by culturing the immune cells of the present disclosure in the presence of the activating elements described herein. Those skilled in the art will understand that the expansion step provided herein can also encompass any newly discovered culture system, medium, or activating elements that can be used to expand immune cells.

[0491] The present disclosure provides formulation and cryopreservation steps for the expanded genetically modified subject immune cells. The formulation steps provided include, for example, washing away excess components used in the preparation and expansion of the immune cells of the therapeutic methods described herein. Any pharma- ceutically acceptable formulation medium or washing buffer compatible with the immune cells known in the art can be used to wash, dilute / concentrate, and prepare a dose for administration of the immune cells. The formulation medium can be acceptable for administration of the immune cells, such as, for example, a crystalloid fluid for intravenous infusion. Optionally, cryopreservation can be used to store the immune cells for long periods of time. Cryopreservation can be accomplished using methods known in the art, including, for example, storing the cells in a cryopreservation medium containing cryopreservation components. The cryopreservation components can include, for example, dimethyl sulfoxide or glycerol. The immune cells stored in the cryopreservation medium can be cryopreserved by lowering the storage temperature to -80°C to -180°C.

[0492] In some embodiments, the method includes administering an immune cell as described herein. In some embodiments, the method includes administering a conditioning regimen prior to administering the immune cell as described herein. In some embodiments, the conditioning regimen is lymphodepletion. The lymphodepletion regim...

Claims

1. 1. An immune cell responsive to loss of heterozygosity in a cancer cell, comprising: a. an activator receptor comprising an extracellular ligand-binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen; (i) the VH region of the activator receptor comprises the CDR sequences of DTYIH (SEQ ID NO: 313), RIYPTNGYTRYADSVKG (SEQ ID NO: 314), and WGGDGFYAMDV (SEQ ID NO: 315); the VL region of the activator receptor comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO: 30), SASFLYS (SEQ ID NO: 311), and QQHYTTPPT (SEQ ID NO: 312); or (ii) the VH region of the activator receptor comprises the CDR sequences of GFNIKDTYIH (SEQ ID NO: 36), ARIYPTNGYTRYADSVKG (SEQ ID NO: 38), and SRWGGDGFYAMD[Y / V] (SEQ ID NO: 40) or (SEQ ID NO: 41); the VL region of the activator receptor comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO: 30), SASFLY (SEQ ID NO: 32), and QQHYTTPP (SEQ ID NO: 34); or (iii) the VH region of the activator receptor comprises the CDR sequences of NYGMN (SEQ ID NO: 37), WINTSTGESTFADDFKG (SEQ ID NO: 39), and WEVYHGYVPY (SEQ ID NO: 42); and the VL region of the activator receptor comprises one or more CDR sequences selected from the group consisting of KASQDVYNAVA (SEQ ID NO: 31), SASSRYT (SEQ ID NO: 33), and QQHFRTPFT (SEQ ID NO: 35); or (iv) the VH region of the activator receptor comprises the CDR sequences of GFNIKDTYIH (SEQ ID NO: 36), ARIYPTNGYTRYADSVKG (SEQ ID NO: 38), and SRWGGDGFYAMDY (SEQ ID NO: 40); the VL region of the activator receptor comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO: 30), SASFLY (SEQ ID NO: 32), and QQHYTTPP (SEQ ID NO: 34); or (v) an activator receptor, wherein the VH region of the activator receptor comprises the CDR sequences GFNIKDTYIH (SEQ ID NO: 36), ARIYPTNGYTRYADSVKG (SEQ ID NO: 38), and SRWGGDGFYAMDV (SEQ ID NO: 41); and the VL region of the activator receptor comprises the CDR sequences RASQDVNTAVA (SEQ ID NO: 30), SASFLY (SEQ ID NO: 32), and QQHYTTPP (SEQ ID NO: 34); b. an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by the cancer cell due to loss of heterozygosity in the cancer cell.

2. the non-target antigen is an HLA class I allele or a minor histocompatibility antigen (MiHA); Optionally, the HLA class I alleles include HLA-A, HLA-B, HLA-C, or HLA-E.

2. The immune cell of claim 1, wherein the HLA class I allele is HLA-A*02, if desired.

3. the target antigen is HER2 or a peptide antigen of HER2 in complex with the major histocompatibility complex class I complex (MHC I); Optionally, the cancer cells express HER2. and / or optionally, the cancer cells are breast cancer cells, bladder cancer cells, ovarian cancer cells, gastric cancer cells, salivary duct cancer cells, non-small cell lung cancer cells, pancreatic cancer cells, or colon cancer cells; and / or optionally, the cancer cells are breast cancer cells or gastric cancer cells; and / or optionally, the HLA-A*02 non-target antigen is expressed by healthy cells of the subject; and / or optionally, healthy cells of the subject express both the target antigen and the HLA-A*02 non-target antigen. and / or optionally, said activator receptor and said inhibitory receptor together specifically activate said immune cell in the presence of said cancer cells, and optionally, said HER2 antigen comprises a sequence or subsequence at least 95% identical to a sequence or subsequence of any one of SEQ ID NOs: 2-29.

4. The immune cell is a T cell, Optionally, the T cell is a CD8+CD4- T cell.

5. the activator receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR); Optionally, the extracellular ligand binding domain of the activator receptor comprises an antibody fragment, a single chain Fv antibody fragment (scFv), a beta chain variable domain (Vβ), or a TCR alpha chain variable domain and a TCR beta chain variable domain. and / or optionally, the extracellular ligand-binding domain of the activator receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region; Optionally, the VH and VL regions of the activator receptor comprise complementarity determining regions (CDRs) selected from the group consisting of SEQ ID NOs: 30-42. or optionally, the VH region of the activator receptor comprises one or more CDR sequences selected from the group consisting of GFNIKDTYIH (SEQ ID NO: 36), ARIYPTNGYTRYADSVKG (SEQ ID NO: 38), and SRWGGDGFYAMD[Y / V] (SEQ ID NO: 40) or (SEQ ID NO: 41); and the VL region of the activator receptor comprises one or more CDR sequences selected from the group consisting of RASQDVNTAVA (SEQ ID NO: 30), SASFLY (SEQ ID NO: 32), and QQHYTTPP (SEQ ID NO: 34); or optionally, the VH region of the activator receptor comprises one or more CDR sequences selected from the group consisting of NYGMN (SEQ ID NO:37), WINTSTGESTFADDFKG (SEQ ID NO:39), and WEVYHGYVPY (SEQ ID NO:42), and the VL region of the activator receptor comprises one or more CDR sequences selected from the group consisting of KASQDVYNAVA (SEQ ID NO:31), SASSRYT (SEQ ID NO:33), and QQHFRTPFT (SEQ ID NO:35); or optionally, the VH region of the activator receptor comprises the CDR sequences of GFNIKDTYIH (SEQ ID NO:36), ARIYPTNGYTRYADSVKG (SEQ ID NO:38), and SRWGGDGFYAMDY (SEQ ID NO:40), and the VL region of the activator receptor comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO:30), SASFLY (SEQ ID NO:32), and QQHYTTPP (SEQ ID NO:34); or optionally, the VH region of the activator receptor comprises the CDR sequences of GFNIKDTYIH (SEQ ID NO:36), ARIYPTNGYTRYADSVKG (SEQ ID NO:38), and SRWGGDGFYAMDV (SEQ ID NO:41); and the VL region of the activator receptor comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO:30), SASFLY (SEQ ID NO:32), and QQHYTTPP (SEQ ID NO:34); or optionally, the VH region of the activator receptor comprises the CDR sequences of DTYIH (SEQ ID NO:313), RIYPTNGYTRYADSVKG (SEQ ID NO:314), and WGGDGFYAMDV (SEQ ID NO:315), and the VL region of the activator receptor comprises the CDR sequences of RASQDVNTAVA (SEQ ID NO:30), SASFLYS (SEQ ID NO:311), and QQHYTTPPT (SEQ ID NO:312); and / or optionally, the scFv of the activator receptor comprises a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to or identical to a sequence selected from the group consisting of SEQ ID NOs: 51, 53, 55, 57.59 and 61; and / or optionally, the activator receptor is a CAR comprising a hinge sequence isolated or derived from CD8, CD28, IgG1, or IgG4, or a synthetic hinge. and / or optionally, the activator receptor is a CAR comprising a transmembrane domain isolated or derived from CD8 or CD28. And / or optionally, the CAR comprises an intracellular domain isolated or derived from CD28, 4-1BB or CD3z, or a combination thereof.

6. the inhibitory receptor comprises a TCR or a CAR; Optionally, the extracellular ligand-binding domain of the inhibitory receptor comprises an antibody fragment, a single chain Fv antibody fragment (scFv), a beta chain variable domain (Vβ), or a TCR alpha chain variable domain and a TCR beta chain variable domain. and / or optionally, the extracellular ligand-binding domain of the inhibitory receptor comprises a heavy chain variable (VH) region and a light chain variable (VL) region; Optionally, the VH and VL regions of the inhibitory receptor comprise CDRs selected from the group consisting of SEQ ID NOs: 87-102. and / or optionally, the VH region of the inhibitory receptor comprises one or more CDRs selected from the group consisting of ASGYTFTSYHIH (SEQ ID NO: 95), WIYPGNVNTEYNEKFKGK (SEQ ID NO: 98), and EEITYAMDY (SEQ ID NO: 101), and the VL region of the inhibitory receptor comprises one or more CDRs selected from the group consisting of RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFSGVP[D / A]R (SEQ ID NO: 90) or (SEQ ID NO: 91), and FQGSHVPRT (SEQ ID NO: 92); or optionally, the VH region of the inhibitory receptor comprises one or more CDRs selected from the group consisting of SGYTFTSYHMH (SEQ ID NO: 97), WIYPGDGSTQYNEKFKG (SEQ ID NO: 100) and EGTYYAMDY (SEQ ID NO: 102), and the VL region of the inhibitory receptor comprises one or more CDRs selected from the group consisting of RSSQSIVHSNGNTYLD (SEQ ID NO: 89), KVSNRFSGVP[D / A]R (SEQ ID NO: 90) or (SEQ ID NO: 91) and MQGSHVPRT (SEQ ID NO: 94); or optionally, the VH region of the inhibitory receptor comprises the CDR sequences of SYHIH (SEQ ID NO:566), WIYPGNVNTEYNEKFKG (SEQ ID NO:567), and EETYAMDY (SEQ ID NO:101), and the VL region of the inhibitory receptor comprises the CDR sequences of RSSQSIVHSNGNTYLE (SEQ ID NO:87), KVSNRFS (SEQ ID NO:565), and FQGSHVPRT (SEQ ID NO:92); and / or optionally, the scFv of the inhibitory receptor has at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to or comprises a sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 63-74, 207, 209 and 211.

7. An immune cell described in any one of claims 1 to 6, wherein the activator receptor comprises a polypeptide sequence having at least 95% identity to SEQ ID NO:

291.

8. An immune cell described in any one of claims 1 to 7, wherein the inhibitory receptor comprises a polypeptide sequence having at least 95% identity to SEQ ID NO:

305.

9. The inhibitory receptor comprises a LILRB1 intracellular domain or a functional variant thereof; Optionally, the inhibitory receptor comprises a LILRB1 hinge and transmembrane domain, or a functional variant thereof. and / or optionally the inhibitory receptor is The immune cell of any one of claims 1 to 8, comprising a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to or identical to KHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 254).

10. the immune cell is modified to inactivate, or reduce or eliminate, expression or function of an endogenous gene encoding an allele of an endogenous MHC class I polypeptide; 10. The immune cell of any one of claims 1 to 9, wherein optionally the gene encoding the MHC class I polypeptide is HLA-A*02, and / or optionally the immune cell is modified to reduce or eliminate expression of a B2M gene product.

11. A pharmaceutical composition comprising a therapeutically effective amount of an immune cell according to any one of claims 1 to 10, Optionally, further comprising a pharma- ceutically acceptable carrier, diluent, or excipient; and / or optionally a pharmaceutical composition for use as a medicament in the treatment of cancer, or A polynucleotide system comprising: a. an activator receptor comprising an extracellular ligand-binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen; and b. a polynucleotide system comprising one or more polynucleotides comprising a polynucleotide sequence encoding an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by the cancer cells due to loss of heterozygosity in the cancer cells; or A vector comprising the polynucleotide system.

12. A composition comprising an immune cell according to any one of claims 1 to 10, for use in a method of treating HER2+ cancer in a subject identified as having, or suspected of having, loss of heterozygosity in an allele encoding a non-target antigen in a HER2-positive cancer.

13. A method for producing an immune cell therapy, comprising transforming an immune cell with the polynucleotide system or vector according to claim 11, or A kit comprising the immune cell according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, Optionally, the kit further comprises instructions for use.

14. 11. An in vitro method of selectively killing HER2-positive tumor cells having a loss of heterozygosity in an allele encoding a non-target antigen of said HER2-positive cancer comprising contacting said HER2-positive tumor cells with an immune cell according to any one of claims 1 to 10, Optionally, the tumor cells are in a tissue. or optionally, the tumor cells are in a mixed culture. and / or optionally, the non-target antigen is selected from the group consisting of HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-B*07, and HLA-C*07; or An immune cell, a. an activator receptor comprising an extracellular ligand binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen, said extracellular ligand binding domain of said activator receptor comprising: (i) a VH region comprising the CDR sequences of DTYIH (SEQ ID NO: 313), RIYPTNGYTRYADSVKG (SEQ ID NO: 314), and WGGDGFYAMDV (SEQ ID NO: 315); (ii) an activator receptor comprising a VL region comprising the CDR sequences RASQDVNTAVA (SEQ ID NO: 30), SASFLYS (SEQ ID NO: 311), and QQHYTTPPT (SEQ ID NO: 312); b. an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by a cancer cell due to loss of heterozygosity in said cancer cell, said extracellular ligand-binding domain of said inhibitory receptor comprising: (i) a heavy chain variable (VH) region comprising the CDR sequences of SYHIH (SEQ ID NO: 566), WIYPGNVNTEYNEKFKG (SEQ ID NO: 567), and EETYAMDY (SEQ ID NO: 101); and (ii) an inhibitory receptor comprising a light chain variable (VL) region comprising RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFS (SEQ ID NO: 565), and FQGSHVPRT (SEQ ID NO: 92); or an immune cell comprising: a. an activator receptor comprising an extracellular ligand binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen, wherein the extracellular ligand binding domain of the activator receptor is an scFv comprising the sequence of SEQ ID NO:51; b. An immune cell comprising an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by a cancer cell due to loss of heterozygosity in the cancer cell, wherein the extracellular ligand-binding domain of the inhibitory receptor is an scFv comprising the sequence of SEQ ID NO:

209.

15. A composition comprising immune cells according to any one of claims 1 to 10 for use in a method of treating HER2+ cancer in a subject identified as having, or suspected of having, a loss of heterozygosity at an allele encoding HLA-A*02, wherein optionally the immune cells comprise: a. an activator receptor comprising an extracellular ligand binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen, said extracellular ligand binding domain of said activator receptor comprising: (i) a VH region comprising the CDR sequences of DTYIH (SEQ ID NO: 313), RIYPTNGYTRYADSVKG (SEQ ID NO: 314), and WGGDGFYAMDV (SEQ ID NO: 315); (ii) an activator receptor comprising a VL region comprising the CDR sequences RASQDVNTAVA (SEQ ID NO: 30), SASFLYS (SEQ ID NO: 311), and QQHYTTPPT (SEQ ID NO: 312); b. an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by a cancer cell due to loss of heterozygosity in said cancer cell, said extracellular ligand-binding domain of said inhibitory receptor comprising: (i) a heavy chain variable (VH) region comprising the CDR sequences of SYHIH (SEQ ID NO: 566), WIYPGNVNTEYNEKFKG (SEQ ID NO: 567), and EETYAMDY (SEQ ID NO: 101); and (ii) an inhibitory receptor comprising a light chain variable (VL) region comprising the CDR sequences RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFS (SEQ ID NO: 565), and FQGSHVPRT (SEQ ID NO: 92); or 1. A composition for use in a method of treating HER2+ cancer in a subject identified as having, or suspected of having, a loss of heterozygosity at an allele encoding HLA-A*02, comprising immune cells, the immune cells comprising: (a) an activator receptor comprising an extracellular ligand binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen, wherein the extracellular ligand binding domain of the activator receptor is an scFv comprising the sequence of SEQ ID NO:51; (b) an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by a cancer cell due to loss of heterozygosity in the cancer cell, wherein the extracellular ligand-binding domain of the inhibitory receptor is an scFv comprising the sequence of SEQ ID NO:

209.

16. A composition for use in a method of selectively killing HER2 positive tumor cells having loss of heterozygosity at an allele encoding HLA-A*02, comprising an immune cell, said method comprising contacting said HER2 positive tumor cells with an immune cell according to any one of claims 1 to 10, and optionally wherein said immune cell is a. an activator receptor comprising an extracellular ligand binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen, said extracellular ligand binding domain of said activator receptor comprising: (i) a VH region comprising the CDR sequences of DTYIH (SEQ ID NO: 313), RIYPTNGYTRYADSVKG (SEQ ID NO: 314), and WGGDGFYAMDV (SEQ ID NO: 315); (ii) an activator receptor comprising a VL region comprising the CDR sequences RASQDVNTAVA (SEQ ID NO: 30), SASFLYS (SEQ ID NO: 311), and QQHYTTPPT (SEQ ID NO: 312); b. an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by a cancer cell due to loss of heterozygosity in said cancer cell, said extracellular ligand-binding domain of said inhibitory receptor comprising: (i) a heavy chain variable (VH) region comprising the CDR sequences of SYHIH (SEQ ID NO: 566), WIYPGNVNTEYNEKFKG (SEQ ID NO: 567), and EETYAMDY (SEQ ID NO: 101); and (ii) an inhibitory receptor comprising a light chain variable (VL) region comprising the CDR sequences RSSQSIVHSNGNTYLE (SEQ ID NO: 87), KVSNRFS (SEQ ID NO: 565), and FQGSHVPRT (SEQ ID NO: 92); or 1. A composition for use in a method of selectively killing HER2 positive tumor cells having a loss of heterozygosity at an allele encoding HLA-A*02, comprising an immune cell, the method comprising contacting the HER2 positive tumor cells with the immune cell, optionally wherein the immune cell is (a) an activator receptor comprising an extracellular ligand binding domain specific for the erb-b2 receptor tyrosine kinase 2 (HER2) antigen, wherein the extracellular ligand binding domain of the activator receptor is an scFv comprising the sequence of SEQ ID NO:51; (b) an inhibitory receptor comprising an extracellular ligand-binding domain specific for a non-target antigen that is not expressed by a cancer cell due to loss of heterozygosity in the cancer cell, wherein the extracellular ligand-binding domain of the inhibitory receptor is an scFv comprising the sequence of SEQ ID NO: 209.