Cell surface receptors that respond to loss of heterozygosity

Abstract

The present disclosure relates to a system of two engineered receptors, each with a ligand binding domain, co-designed to target cells identified by loss of heterozygosity and used to treat a disease or disorder, such as cancer. The present disclosure provides immune cells expressing two engineered receptors, methods for preparing them, and polynucleotides and vectors encoding them.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 885,093 filed on August 9, 2019 and U.S. Provisional Patent Application Serial No. 62 / 005,670 filed on April 6, 2020, the contents of each of which are hereby incorporated by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application is submitted in electronic format along with a sequence listing. The sequence listing is provided as a file named A2BI-00903WO_SeqList.txt, created on August 5, 2020, and 404 kilobytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. Background Art

[0005] Cell therapy is a powerful tool for treating various diseases (especially cancer). In conventional adoptive cell therapy, immune cells are engineered to express specific receptors, such as chimeric antigen receptors (CAR) or T cell receptors (TCR), which direct the activity of immune cells to cell targets through the interaction of receptors with ligands expressed by target cells. The identification of suitable target molecules remains challenging. The art needs compositions and methods that can be used for treating diseases (especially cancer) by cell therapy. Summary of the invention

[0006] The present disclosure generally relates to a dual receptor system expressed in engineered immune cells (e.g., immune cells for adoptive cell therapy) that can be used to target these immune cells to tumor cells that exhibit loss of heterozygosity. In this dual receptor system, a first receptor is used to activate or promote activation of immune cells, while a second receptor is used to inhibit activation by the first receptor. The ligands of the first receptor and the second receptor mediate activation of immune cells by target cells that express the first activator ligand but do not express the second inhibitory ligand, for example, through differential expression of loss of heterozygosity of a locus encoding an inhibitory ligand.

[0007] The present disclosure provides an immune cell, which comprises: (a) a first engineered receptor, which comprises a transmembrane region and an extracellular region, wherein the extracellular region comprises a first ligand binding domain capable of specifically binding to a first ligand; and (b) a second engineered receptor, which comprises a transmembrane region and an extracellular region, wherein the extracellular region comprises a second ligand binding domain capable of specifically binding to a second ligand, wherein binding of the first ligand binding domain to the first ligand activates or promotes activation of the immune cell by the receptor, and wherein binding of the second ligand binding domain to the second ligand inhibits activation of the immune cell by the first receptor.

[0008] In some embodiments of the immune cells of the present disclosure, the second ligand is not expressed in the target cell due to loss of heterozygosity of the gene encoding the second ligand. In some embodiments, the second ligand is an HLA class I allele or a minor histocompatibility antigen (MiHA).

[0009] In some embodiments of the immune cells of the present disclosure, the second ligand is MiHA. In some embodiments, the MiHA is selected from a group of MiHAs in Table 8 and Table 9. In some embodiments, the MiHA is HA-1.

[0010] In some embodiments of the immune cells of the present disclosure, the second ligand is an HLA class I allele. In some embodiments, the HLA class I allele includes HLA-A, HLA-B, or HLA-C. In some embodiments, the HLA class I allele is an HLA-A*02 allele.

[0011] In some embodiments of the immune cells of the present disclosure, the second ligand is not expressed in the target cell due to loss of chromosome Y. In some embodiments, the second ligand is encoded by a Y chromosome gene.

[0012] In some embodiments of the immune cells of the present disclosure, the first ligand and the second ligand are not the same. In some embodiments, the first ligand is expressed by a target cell. In some embodiments, the first ligand is expressed by a target cell and a non-target cell. In some embodiments, the second ligand is not expressed by a target cell and is expressed by a plurality of non-target cells. In some embodiments, a plurality of non-target cells express both the first ligand and the second ligand.

[0013] In some embodiments, the target cell is a cancer cell and the non-target cell is a non-cancerous cell.

[0014] In some embodiments of the immune cells of the present disclosure, the first ligand is selected from the group consisting of: cell adhesion molecules, cell-cell signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane proteins, neurotransmitter receptors, and voltage-gated ion channels. In some embodiments, the first ligand is selected from a group of antigens in Table 5. In some embodiments, the first ligand is selected from the group consisting of: transferrin receptor (TFRC), epidermal growth factor receptor (EGFR), CEA cell adhesion molecule 5 (CEA), CD19 molecule (CD19), erb-b2 receptor tyrosine kinase 2 (HER2) and mesothelin (MSLN) or their peptide antigens. In some embodiments, the first ligand comprises HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G. In some embodiments, the first ligand is a pan-HLA ligand.

[0015] In some embodiments of the immune cells of the present disclosure, the second ligand is selected from the group consisting of: HLA class I alleles, minor histocompatibility antigens (MiHA), and Y chromosome genes. In some embodiments, the expression of the second ligand is lost in the target cell due to loss of heterozygosity. In some embodiments, MiHA is HA-1. In some embodiments, the HLA class I allele is an HLA-A*02 allele.

[0016] In some embodiments of the immune cells of the present disclosure, the first engineered receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the second engineered receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0017] In some embodiments of the immune cells of the present disclosure, the first ligand binding domain comprises a single-chain Fv antibody fragment (ScFv), a β chain variable domain (Vβ), a TCRα chain variable domain and a TCRβ chain variable domain, or a variable heavy chain (VH) domain and a variable light chain (VL) domain. In some embodiments, the second ligand binding domain comprises a ScFv, a Vβ domain, a TCRα chain variable domain and a TCRβ chain variable domain, or a variable heavy chain (VH) domain and a variable light chain (VL) domain.

[0018] In some embodiments of the immune cells of the present disclosure, the first ligand is EGFR or a peptide antigen thereof. In some embodiments, the first ligand binding domain comprises a sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118 or SEQ ID NO: 391, or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the first ligand binding domain comprises a CDR selected from SEQ ID NO: 131-166.

[0019] In some embodiments of the immune cells of the present disclosure, the first ligand is MSLN or a peptide antigen thereof. In some embodiments, the first ligand binding domain comprises a sequence of SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90 or SEQ ID NO: 92, or a sequence having at least 90%, at least 95% or at least 99% identity thereto.

[0020] In some embodiments of the immune cells of the present disclosure, the first ligand is CEA or a peptide antigen thereof. In some embodiments, the first ligand binding domain comprises SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 282, SEQ ID NO: 284 or SEQ ID NO: 286, or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the first ligand binding domain comprises a CDR selected from SEQ ID NO: 294-302.

[0021] In some embodiments of the immune cells of the present disclosure, the first ligand is CD19 or a peptide antigen thereof, and the first ligand binding domain comprises SEQ ID NO: 275 or SEQ ID NO: 277, or a sequence that is at least 90%, at least 95%, or at least 99% identical thereto.

[0022] In some embodiments of the immune cells of the present disclosure, the first ligand is a pan-HLA ligand. In some embodiments, the first ligand binding domain comprises a sequence of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto.

[0023] In some embodiments of the immune cells of the present disclosure, the second ligand comprises HA-1. In some embodiments, and wherein the second ligand binding domain comprises a TCR alpha variable domain and a TCR beta variable domain, the TCR alpha variable domain comprises SEQ ID NO: 199 or a sequence having at least 90%, at least 95% or at least 99% identity thereto, and the TCR beta variable domain comprises SEQ ID NO: 200 or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the second ligand binding domain comprises a TCR alpha variable domain comprising SEQ ID NO: 199 and a TCR beta variable domain comprising SEQ ID NO: 200.

[0024] In some embodiments of the immune cells of the present disclosure, the second ligand comprises an HLA-A*02 allele. In some embodiments, the second ligand binding domain comprises any one of SEQ ID NOs: 53-64 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the second ligand binding domain comprises a CDR selected from SEQ ID NOs: 41-52.

[0025] In some embodiments of the immune cells of the present disclosure, the second engineered receptor comprises an immunoreceptor tyrosine-based inhibition motif (ITIM).

[0026] In some embodiments of the immune cells of the present disclosure, the second engineered receptor comprises a LILRB1 intracellular domain or a functional variant thereof. In some embodiments, the LILRB1 intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO: 76. In some embodiments, the second engineered receptor comprises a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO: 85. In some embodiments, the second engineered receptor comprises a LILRB1 hinge domain or a functional fragment or variant thereof. In some embodiments, the LILRB1 hinge domain comprises a sequence that is at least 95% identical to SEQ ID NO: 84, SEQ ID NO: 77 or SEQ ID NO: 78. In some embodiments, the second engineered receptor comprises a LILRB1 intracellular domain and a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 intracellular domain and the LILRB1 transmembrane domain comprise SEQ ID NO: 80 or a sequence that is at least 95% identical to SEQ ID NO: 80. In some embodiments, the second engineered receptor comprises a first polypeptide comprising SEQ ID NO:80, or a sequence at least 95% identical thereto, fused to a TCRα variable domain, and a second polypeptide comprising SEQ ID NO:80, or a sequence at least 95% identical thereto, fused to a TCRβ variable domain.

[0027] In some embodiments of the immune cells of the present disclosure, the first receptor and the second receptor are expressed on the surface of the immune cell at a first receptor to second receptor ratio of between about 1:10 to 10: 1. In some embodiments, the first receptor and the second receptor are expressed on the surface of the immune cell at a first receptor to second receptor ratio of between about 1:3 to 3: 1. In some embodiments, the first receptor and the second receptor are expressed on the surface of the immune cell at a ratio of about 1:1.

[0028] In some embodiments of the immune cells of the present disclosure, the immune cells are selected from the group consisting of: T cells, B cells, and natural killer (NK) cells. In some embodiments, the immune cells are non-natural. In some embodiments, the immune cells are isolated.

[0029] The present disclosure provides immune cells expressing the dual receptor system of the present disclosure for use as a medicament. In some embodiments, the medicament is used to treat cancer.

[0030] The present disclosure provides a pharmaceutical composition comprising the immune cells of the present disclosure. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of immune cells.

[0031] The present disclosure provides a method of increasing the specificity of adoptive cell therapy in a subject, the method comprising administering to the subject a plurality of immune cells or a pharmaceutical composition of the present disclosure.

[0032] The present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immune cell or a pharmaceutical composition of the present disclosure.

[0033] In some embodiments of the methods of the present disclosure, the subject has cancer. In some embodiments, the cancer cells express the first ligand. In some embodiments, the cancer cells do not express the second ligand due to loss of heterozygosity or loss of the Y chromosome.

[0034] The present disclosure provides a method for preparing the immune cells of the present disclosure, the method comprising (a) providing a plurality of immune cells; and (b) transforming the immune cells with a vector encoding a first engineered receptor and a vector encoding a second engineered receptor, wherein the first engineered receptor comprises a transmembrane region and an extracellular region, the extracellular region comprises a first ligand binding domain capable of specifically binding to a first ligand, and the second engineered receptor comprises a transmembrane region and an extracellular region, the extracellular region comprises a second ligand binding domain capable of specifically binding to a second ligand; wherein binding of the first ligand binding domain to the first ligand activates or promotes activation of the immune cell, and wherein binding of the second ligand binding domain to the second ligand inhibits activation of the immune cell by the first ligand.

[0035] The present disclosure provides a kit comprising the immune cell or pharmaceutical composition of the present disclosure.

[0036] The present disclosure provides inhibitory receptors comprising an extracellular ligand binding domain capable of specifically binding to HA-1 minor histocompatibility antigen (MiHA) and an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibition motif (ITIM).

[0037] In some embodiments of the inhibitory receptors disclosed herein, the extracellular ligand binding domain has a higher affinity for the HA-1 (H) peptide of VLHDDLLEA (SEQ ID NO: 191) than for the HA-1 (R) peptide of VLRDDLLEA (SEQ ID NO: 266). In some embodiments, the inhibitory receptor is activated by the HA-1 (H) peptide of VLHDDLLEA (SEQ ID NO: 191), but not by the HA-1 (R) peptide of VLRDDLLEA (SEQ ID NO: 266) or by it to a lesser extent. In some embodiments, the extracellular ligand binding domain comprises a TCR alpha variable domain and a TCR beta variable domain, the TCR alpha variable domain comprising SEQ ID NO: 199 or a sequence having at least 90%, at least 95% or at least 99% identity thereto, and the TCR beta variable domain comprising SEQ ID NO: 200 or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain comprises a TCR alpha variable domain comprising SEQ ID NO:199 and a TCR beta variable domain comprising SEQ ID NO:200.

[0038] In some embodiments of the inhibitory receptors disclosed herein, the intracellular domain comprises a LILRB1 intracellular domain or a functional variant thereof. In some embodiments, the LILRB1 intracellular domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO:76. In some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO:85. In some embodiments, the inhibitory receptor comprises a LILRB1 intracellular domain and a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 intracellular domain and the LILRB1 transmembrane domain comprise SEQ ID NO:80 or a sequence that is at least 95% identical to SEQ ID NO:80. In some embodiments, the inhibitory receptor comprises a first polypeptide comprising SEQ ID NO: 80 or a sequence at least 95% identical thereto fused to a TCR alpha variable domain, and a second polypeptide comprising SEQ ID NO: 80 or a sequence at least 95% identical thereto fused to a TCR beta variable domain. In some embodiments, the inhibitory receptor comprises a polypeptide of SEQ ID NO: 195 or a polypeptide at least 95% identical thereto and a polypeptide of SEQ ID NO: 197 or a polypeptide at least 95% identical thereto.

[0039] The present disclosure provides an immune cell, which comprises: (a) a first engineered receptor, which comprises a transmembrane region and an extracellular region, wherein the extracellular region comprises a first ligand binding domain capable of specifically binding to a CD19 ligand; and (b) a second engineered receptor, which comprises a transmembrane region and an extracellular region, wherein the extracellular region comprises a second ligand binding domain capable of specifically binding to an HLA-A*02 allele, wherein binding of the first ligand binding domain to the CD19 ligand activates or promotes activation of the immune cell by the first receptor, and wherein binding of the second ligand binding domain to the HLA-A*02 allele inhibits activation of the immune cell by the first receptor.

[0040] The present disclosure provides an immune cell, which comprises: (a) a first engineered receptor, which comprises a transmembrane region and an extracellular region, wherein the extracellular region comprises a first ligand binding domain capable of specifically binding to an EGFR ligand; and (b) a second engineered receptor, which comprises a transmembrane region and an extracellular region, wherein the extracellular region comprises a second ligand binding domain capable of specifically binding to an HLA-A*02 allele, wherein binding of the first ligand binding domain to the EGFR ligand activates or promotes activation of the immune cell by the receptor, and wherein binding of the second ligand binding domain to the HLA-A*02 allele inhibits activation of the immune cell by the first receptor.

[0041] The present disclosure provides an immune cell, which comprises: (a) a first engineered receptor, which comprises a transmembrane region and an extracellular region, wherein the extracellular region comprises a first ligand binding domain capable of specifically binding to a mesothelin (MSLN) ligand; and (b) a second engineered receptor, which comprises a transmembrane region and an extracellular region, wherein the extracellular region comprises a second ligand binding domain capable of specifically binding to an HLA-A*02 allele, wherein binding of the first ligand binding domain to the MSLN ligand activates or promotes activation of the immune cell by the first receptor, and wherein binding of the second ligand binding domain to the HLA-A*02 allele inhibits activation of the immune cell by the first receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] A better understanding of the features and advantages of the present invention may be obtained by reference to the following detailed description which sets forth illustrative embodiments, in which the principles of the invention are utilized, and in which the accompanying drawings are:

[0043] Figure 1is a diagram showing hemizygous tumor cells forming tumors against a background of heterozygous cells that make up normal tissue. Hemizygous tumor cells express only target A and have lost target B due to loss of heterozygosity (LOH), while normal cells express both targets A and B. This genetic difference can be used to create tumor-selective cytotoxic therapeutics that are blocked by target B and activated by target A, thereby selectively killing tumors.

[0044] Figure 2A is a diagram showing an exemplary structural system of a dual-targeted therapeutic based on LOH in tumors. In this example, there is a cell-based integration of activator and blocker signals.

[0045] Figure 2B is a series of figures showing various activator and receptor forms and combinations.

[0046] Figure 3A is a pair of diagrams showing exemplary dual receptor constructs of the present disclosure in the form of a TCR. In this example, the activator and inhibitor (blocker) LBDs are each fused to the CD3γ subunit of the TCR.

[0047] Figure 3B are figures and tables of exemplary dual receptor constructs of the present disclosure in the form of CARs. Exemplary ITIM and inhibitor domains of inhibitor CARs are shown in the table on the right.

[0048] Figure 4A is a plot showing RNA-Seq expression of transferrin receptor (TFRC) in human tissues from the GTEx database. Transferrin receptor (TFRC) is a candidate for target A (activator). Expression of TFRC at the RNA level is ubiquitous and relatively uniform. TRFC is an essential gene: loss-of-function homozygous mutations are embryonic lethal in mice.

[0049] Figure 4B is a plot showing RNA-Seq expression profiles of HLA-A and HLA-B.

[0050] Figures 5A-5H It was shown that LIR-1 blockers are modular and mediate large EC50 shifts.

[0051] Figure 5A A schematic diagram of the T2-Jurkat experiment evaluating blocker constructs is shown.

[0052] Figure 5BShown is the effect of various NY-ESO-1 scFv LBD blocking modules (PD-1, CTLA-4, LIR-1) on the EC50 of MAGE-A3 CAR activator (MP1-CAR) when loaded with NY-ESO-1 blocking peptides. Error bars represent ± SD (n = 2).

[0053] Figure 5C Shown is the effect of LIR-1 blocker modules with various scFv LBDs (ESO, MP1 LBD 1, MP1 LBD 2, HPV E6 LBD 1, HPV E6 LBD 2, HPV E7) on the EC50 of MAGE-A3 CAR activator (MP1-CAR) when loaded with the corresponding peptides. Error bars represent ± SD (n = 2).

[0054] Figure 5D Shown is the effect of the LIR-1 blocker module with NY-ESO-1 scFv LBD on the EC50 of different MAGE-A3 CAR activators (MP1-CAR or MP2-CAR) when loaded with NY-ESO-1 blocker peptide. Error bars represent ± SD (n = 2).

[0055] Figure 5E The effect of the LIR-1 blocker module with NY-ESO-1 scFv LBD on the EC50 of different TCR activators (MP1-TCR, MP2-TCR, HPV E6-TCR) when loaded with NY-ESO-1 blocker peptide is shown. Error bars represent ±SD (n=2). Three different TCR activators were blocked by NY-ESO-LIR-1, which has ESO scFv, LIR-1 hinge, LIR-1 TM and LIR-1 ICD.

[0056] Fig. 5F The effect of the LIR-1 blocker module with NY-ESO-1 Ftcr LBD on the EC50 of MAGE-A3 CAR and TCR activators (MP1-CAR, MP1-TCR). Error bars represent ± SD (n = 2). Both third-generation CAR activators and conventional TCR activators can be blocked by NY-ESO-1 Ftcr-LIR-1, which has TCRa ECD, LIR-1 TM, LIR-1 ICD and TCRb ECD, LIR-1 TM and LIR-1 ICD.

[0057] Figure 5GJurkat cells transfected with HPV E7-CAR or HPV E7-CAR and A2-LIR-1 are shown co-cultured with beads displaying various ratios of activator (HPV E7) to blocker (NY-ESO-1) antigens, demonstrating cis but not trans blocking.

[0058] Figure 5H The A2-LIR-1 blocker module blocks CD19-CAR activators at various activator to blocker ratios. E:T ratio: effector:target ratio.

[0059] Fig. 6A , Figure 6C-6E We show that primary T cells expressing LIR-1 blockade selectively kill tumor cells with both pMHC and non-pMHC proof-of-concept targets.

[0060] Fig. 6A Shown is an approximately 100-fold shift in the EC50 of primary T cells transduced with HPV E7-TCR activator and ESO-LIR-1 blocker in the primary T cell killing assay. Error bars represent + / - SD (n=2).

[0061] Figure 6B HLA-A*02-LIR-1 is shown to block NY-ESO-1 CAR activator in Jurkat cells at various activator:blocker DNA ratios.

[0062] Figure 6C Primary T cells transduced with CD19 CAR activator and HLA-A*02 blocker were shown to discriminate "tumor" cells from "normal" cells in an in vitro cytotoxicity assay and demonstrated selective killing of "tumor" cells in a mixed target cell assay at 3:1 E:T. A2-LIR-1: LIR-1 based receptor with HLA-A2*02 LBD.

[0063] Figure 6D-6E Primary T cells transduced with a CD19 CAR activator and an HLA-A*02 blocker demonstrated reversible blockade in an in vitro cytotoxicity assay at 3:1 E:T after 3 rounds of antigen exposure (AB–A–AB and A–AB–A). Fig.6D ) and activation ( Fig. 6E ). Primary T cell cytotoxicity assay was reproduced with three HLA-A*02 negative donors.

[0064] Figures 7A-7E Modified CAR-T cells (i.e., CAR-T cells expressing both activator and blocker receptors) were shown to selectively kill tumors in a xenograft model.

[0065] Fig. 7A It was shown that primary T cells transduced with CD19 CAR activator and HLA-A*02 blocker exhibited approximately 20-fold expansion within 10 days with CD3 / 28 stimulation.

[0066] Figure 7B Schematic diagram showing the in vivo study design: HLA-A*02 NSG mice were subcutaneously administered with either “tumor cells” (A2-negative Raji cells) or “normal cells” (A2-positive Raji cells), and when the Raji xenografts averaged approximately 70 mm 3 Primary T cells (human, HLA-A*02-negative donor) were injected into the tail vein at 4 ℃ and 1 ℃.

[0067] Figure 7C-7E Tumor size readings measured by caliper are shown ( Figure 7C ), human blood T cell counts by flow cytometry ( Fig.7D ) and survival ( Fig. 7E ). Error bars are standard error of the mean (sem). UTD: untransduced.

[0068] Figure 8 Peptide loading shifts in activation EC50 are shown to be generally less than about 10x. The effect of blocker peptide loading (50 uM each for NY-ESO-1, MAGE-A3, HPV E6, and HPV E7) on activation of MAGE-A3 CAR (MP2 CAR) is shown.

[0069] Fig. 9 It is shown that the LIR-1 blocker module is ligand-dependent. The effect of NY-ESO-1-LIR-1 blocker on the EC50 of activating MAGE-A3 CAR (MP1-CAR) when loaded with various concentrations of NY-ESO-1 blocker peptide is shown.

[0070] Fig.10 Blockers without ICD or with mutated non-functional ICD are shown not to block activation. The effect of modified LIR-1 blocker modules without ICD (blue) or with mutated ICD with NY-ESO-1 scFv LBD (purple) on the EC50 of MAGE-A3 CAR activator (MP2-CAR) when loaded with 10 uM NY-ESO-1 blocker peptide is shown.

[0071] Fig.11CD19 activation and A2-LIR-1 blockade of Jurkat activation in HLA-A*02+(A2+)Raji cells are shown. Jurkat cells transfected with CD19 or CD19 and A2-LIR-1 were co-cultured with WT(A2-)Raji cells or A2+Raji cells at various cell ratios.

[0072] Fig.12 are four panels showing the correlation of hCD3+ T cells in mouse blood with tumor growth. A graph showing hCD3+ T cells relative to tumor volume 10 and 17 days after T cell injection with A2- and A2+ Raji cells is shown.

[0073] Fig.13 It is shown that Jurkat cells expressing EGFR CAR activator and HLA-A*02 LIR-1 blocker are activated by EGFR+ / HLA-A*02-HeLa target cells, but not by EGFR+ / HLA-A*02+HeLa target cells.

[0074] Fig.14A The expression of HLA-A*02 on HeLa cells and HCT116 cells transduced with HLA-A*02 is shown. HeLa and HCT1116 cells were labeled with the anti-HLA-A2 antibody BB7.2 and FAC sorted. Green: unlabeled HeLa; orange: unlabeled HCT116; blue: wild-type HCT116 labeled with BB7.2; red: HeLa cells transduced with HLA-A*02 and labeled with BB7.2.

[0075] Fig. 14B The expression of EGFR on HeLa cells and HCT116 cells is shown. HeLa and HCT1116 cells were labeled with anti-EGFR antibodies and FAC sorted. Green: unlabeled HeLa; Orange: unlabeled HCT1116; Blue: wild-type HCT116 labeled with anti-EGFR; Red: HeLa cells transduced with HLA-A*02 and labeled with anti-EGFR.

[0076] Fig.15A EGFR CAR activation of Jurkat cells and HCT116 target cells expressing EGFR CAR is shown.

[0077] Fig. 15BIt was shown that EGFR CAR activation of Jurkat cells can be blocked by the HLA-A*02 LIR-1 inhibitory receptor. When Jurkat cells were presented with HCT116 target cells expressing EGFR and HLA-A*02, co-expression of EGFR CAR and HLA-A*02 LIR-1 inhibitory receptor by Jurkat cells resulted in a shift in CAR Emax by approximately 1.8x.

[0078] Fig.16A Titration of activator antigen in a bead-based assay to determine the optimal ratio of activator to blocker antigen is shown.

[0079] Fig. 16B Shown is the titration of blocker (inhibitory) antigen in the presence of a constant amount of activator antigen in a bead-based assay to determine the optimal ratio of activator to blocker antigen.

[0080] Fig.17 are diagrams (left) and drawings (right) showing that NY-ESO-1 ScFv LIR-1 based inhibitory receptor can inhibit the activation of MP1 MAGE-A3 TCR on Jurkat cells using the solid tumor cell line A375 as target cells.

[0081] Fig.18 Schematic (left) and graphic (right) showing that the inhibitory receptor based on pMHC HLA-A*02 ScFvLIR-1 can inhibit the activation of Jurkat cells by CD19 ScFv CAR using the B cell leukemia line NALM6 as target cells.

[0082] Fig.19 Graph (left) and drawing (right) showing that pMHC HLA-A*02 ScFv LIR-1 based inhibitory receptor can inhibit activation of Jurkat cells by NY-ESO-1 ScFv CAR activator in a dose-dependent manner.

[0083] Fig. 20 Shown is that the pan-HLA (pan-class I) ScFv CAR was blocked by the HAL-A*02 LIR-1 blocker expression with adjustable strength when assayed in Jurkat cells using T2 target cells and luciferase assay.

[0084] Fig.21A It was shown that the pMHC HLA-A*02 ScFv LIR-1 based inhibitory receptor can cis-inhibit activation of Jurkat cells in a cell-free bead based assay.

[0085] Fig.21BIt was shown that the inhibitory receptor based on pMHC HLA-A*02 ScFv LIR-1 could inhibit the activation of Jurkat cells by MSLN ScFv CAR using the leukemia cell line K562 as target cells.

[0086] Fig. 22 Graph (left) and drawing (right) showing that the pMHC HLA-A*02 ScFv LIR-1 based inhibitory receptor can inhibit the activation of Jurkat cells by MSLN ScFv CAR as measured by the induction fold of IFNγ using the pMHC HLA-A*02 ScFv LIR-1 based inhibitory receptor and using HLA-A*02+HeLa and SiHa cells as target cells.

[0087] Fig.23 It was shown that the inhibitory receptor based on pMHC HLA-A*02 ScFv LIR-1 inhibited killing by the MSLN CAR activator using HLA-A*02+SiHa cells but not HLA-A*02-SiHa cells.

[0088] Fig.24 It was shown that activation of Jurkat cells expressing EGFR ScFv CAR using a bead-based assay could be blocked by the pMHC HLA-A*02 ScFv LIR-1 based inhibitory receptor when the activator and inhibitor antigens were presented on the beads in cis, but not when the activator and inhibitor antigens were presented on the beads in trans.

[0089] Fig.25A It was shown that activation of Jurkat cells by EGFR ScFv CAR could be blocked by the inhibitory receptor based on pMHC HLA-A*02 ScFv LIR-1 using SiHa target cells expressing HLA-A*02 (SiHa A02), but not by SiHa cells not expressing HLA-A*02 (SiHa WT).

[0090] Fig.25B It was shown that activation of Jurkat cells by EGFR ScFv CAR could be blocked by the inhibitory receptor based on pMHC HLA-A*02 ScFv LIR-1 using HeLa target cells expressing HLA-A*02 (HeLa A02), but not by HeLa cells not expressing HLA-A*02 (HeLa WT).

[0091] Fig.26It is shown that another ScFv fused to the LIR-1 inhibitory domain inhibits constitutive CAR activators in a dose-dependent manner. Jurkat-NFAT luciferase reporter cells are transfected with an activation CAR construct exhibiting high tonic signaling and an inhibitory construct recognizing various pMHCs. The effect on NFAT-luciferase activation is measured by co-culturing transfected Jurkat cells with T2 cells loaded with various amounts of inhibitory peptides.

[0092] Fig. 27 Schematic (left) and graphic (right) showing that using T2 target cells, an inhibitory receptor containing the MiHA-b surrogate ScFv ligand binding domain (KRAS G12VScFv blocker) inhibits activation of Jurkat effector cells by an activator TCR targeting the MiHA-a surrogate (KRAS G12D TCR, C-891).

[0093] Fig.28 Schematic (left) and graphic (right) showing that an inhibitory receptor containing a MiHA-b surrogate ScFv ligand binding domain (KRASG12D ScFv blocker) fused to the LIR-1 hinge, TM and ICD inhibits activation of Jurkat effector cells by a TCR targeting a MiHA-a surrogate (KRASG12V TCR, C-913) using T2 target cells.

[0094] Fig.29 Schematic (left) and graphic (right) showing that using T2 target cells, inhibitory receptors containing the MiHA-b surrogate Ftcr binding domain (KRAS G12V Ftcr blocker) fused to the LIR1 TM and ICD inhibit activation of Jurkat effector cells by TCR targeting the MiHA-a surrogate (KRAS G12D TCR).

[0095] Fig.30 Schematic (left) and graphic (right) showing that using T2 target cells, an inhibitory receptor containing the MiHA-b surrogate Ftcr binding domain (KRAS G12D Ftcr blocker) fused to the LIR-1 TM and ICD inhibits the activation of Jurkat effector cells by TCR targeting the MiHA-a surrogate (KRAS G12VTCR).

[0096] Fig.31AFigure 2 is a plot showing inhibition of Jurkat cell activation by MiHA-a TCR using an inhibitory receptor comprising the MiHA-b ScFv ligand binding domain that binds one mutant KRAS peptide [KRAS G12D] and the LIR-1 hinge, transmembrane domain, and intracellular domain (ICD) that bind another mutant KRAS peptide (KRAS G12V). Black: C-891 activator; blue: C-891 activator, C-1761 inhibitor; red: C-891 activator, C-2371 and C2369 inhibitors.

[0097] Fig.31B Figure 2 is a plot showing inhibition of Jurkat cell activation by MiHA-a TCR using an inhibitory receptor comprising the MiHA-b Ftcr ligand binding domain and the LIR-1 transmembrane domain and intracellular domain (ICD). Black: C-913 activator; Blue: C-913 agonist, C-1761 inhibitor; Red: C-913 agonist, C2365 and C2367 inhibitors.

[0098] Fig.32 is a drawing showing that mouse MiHA-Y TCR can activate Jurkat effector cells.

[0099] Fig.33A is a graph and table showing that HA-1 Ftcr can specifically block NY-ESO-1 TCR in the presence of HA-1 (H) peptide.

[0100] Fig.33B is a graph and table showing that HA-1 Ftcr does not substantially block NY-ESO-1 TCR in the presence of a non-specific allelic variant HA-1(R) peptide.

[0101] Fig.34A is a drawing and table showing that HA-1 Ftcr can specifically block KRAS TCR in the presence of HA-1 (H) blocker peptide.

[0102] Fig.34B Graph and table showing that HA-1 Ftcr does not substantially block KRAS TCR in the presence of a non-specific allelic variant HA-1(R) peptide.

[0103] Fig.35 is a graph comparing peptide loading of HA-1(R), HA-1(H) and NY-ESO-1 peptides in T2 cells by flow cytometry.

[0104] Fig.36Ais a graph and table showing the activation dose response using MAGE-A3 MP1 ScFv CAR and NY-ESO-1 ScFv LIR1 blocker.

[0105] Fig.36B are graphs and tables showing inhibitory dose response using MAGE-A3 MP1 ScFv CAR and NY-ESO-1 ScFv LIR1 blocker.

[0106] Fig.36C is a graph showing the concentration of the peptide from the activator MAGE peptide normalized to the constant activator MAGE peptide concentration used for each curve and plotted on the x-axis. Fig.36B Plot of x-values ​​versus NY-ESO-1 peptide concentrations. B: NY-ESO-1 LIR1 blocker, A: MAGE-A3 peptide 2ScFv CAR.

[0107] Fig.37 is a series of graphs and tables showing the varying degrees of blockade observed when the HLA-A*02 ScFv LIR1 inhibitor is used with different EGFR ScFv CAR activators.

[0108] Fig.38A is a series of fluorescence activated cell sorting (FACS) plots showing T cells expressing EGFR ScFv CAR activator receptors after incubation of T cells expressing different EGFR ScFv CARs and HLA-A*02 ScFv LIR1 inhibitors with HeLa cells expressing EGFR activator alone (target A), inhibitor target alone (target B), or activator and inhibitor targets (target AB).

[0109] Fig.38B are graphs showing quantification of activator receptor expression before exposure to target cells and after 120 hours of co-culture with target cells expressing activator ligand alone (Target A) or target cells expressing both activator and blocker ligands (Target AB).

[0110] Fig.39A is a graph showing cell surface expression of activator receptors on T cells expressing the EGFR ScFv CAR (CT-482) activator and the HLA-A*02 ScFv LIR1 inhibitor (C1765) after co-culture with HeLa cell populations expressing EGFR (target A), expressing HLA-A*02 (target B), expressing a combination of EGFR and HLA-A*02 on the same cell (target AB), mixed populations of HeLa cells expressing target A and target AB on different cells, or mixed populations of HeLa cells expressing target B and target AB on different cells.

[0111] Fig.39B is a graph showing cell surface expression of inhibitory receptors on T cells expressing the EGFR ScFv CAR (CT-482) activator and the HLA-A*02 ScFv LIR1 inhibitor (C1765) after co-culture with HeLa cell populations expressing EGFR (target A), expressing HLA-A*02 (target B), expressing a combination of EGFR and HLA-A*02 on the same cell (target AB), mixed populations of HeLa cells expressing target A and target AB on different cells, or mixed populations of HeLa cells expressing target B and target AB on different cells.

[0112] Fig.40 is a diagram of an experiment to determine whether loss of activator receptor expression by T cells is reversible.

[0113] Fig.41A is a series of plots showing that loss of activator surface expression is reversible and corresponds to T cell cytotoxicity. At the top: Percent killing of HeLa target cells by T cells is shown. At the bottom: Activator and inhibitor receptor expression as determined by FACS.

[0114] Fig.41B is a series of plots showing that loss of activator surface expression is reversible and corresponds to T cell cytotoxicity. At the top: Percent killing of HeLa target cells by T cells is shown. At the bottom: Activator and inhibitor receptor expression as determined by FACS. DETAILED DESCRIPTION

[0115] The present inventors have developed a solution to the problem of identifying suitable markers and achieving cell selectivity in treating diseases (especially cancer) with cell therapy. The main goal of the present invention is to target cells based on loss of heterozygosity ( Figure 1 ). Use of a dual receptor system in which activation and inhibitory signals are integrated at the cellular level ( Figure 2A , 2B , 3A and 3B), achieving selective targeting of tumors rather than non-tumor cells. Differences in the expression of surface proteins that are absent or lost in target cells but present in normal cells are thus converted into targeted anti-tumor cell therapy. These differences improve the targeting of cell therapy and protect normal cells from the cytotoxic effects of effector cells using adoptive cell therapy.

[0116] In some embodiments, the methods disclosed herein use two engineered receptors, the first comprising a ligand binding domain for an activator ligand and the second comprising a ligand binding domain for an inhibitor ligand, which are selectively activated in target cells using "AND NOT" Boolean logic. Figure 2A , 2B , 3A and 3B). Normal cells express both activator and inhibitor ligands, but activation of effector cells by the first receptor is blocked by binding of the second receptor containing the inhibitor LBD to the inhibitor ligand, which exerts a protective effect and dominates the activity of the first activator receptor. In contrast, in target cells that express the activator ligand but not the inhibitor ligand, binding of the activator LBD to the activator ligand results in activation of the cell. Advantages of the dual activator / inhibitor receptor strategy of the present disclosure include the ability to adjust the activator and inhibitor combination to create an effective but specific tumor-targeted adoptive cell therapy. In addition, this approach can overcome the variable effector cell to target cell ratio (E:T ratio) in the body, as well as the potential large excess of normal cells to tumor cells (e.g., 10 13 Normal cells and 10 9 Furthermore, the inventors have identified activators and inhibitors that cover a large panel of potential patients, making this a commercially viable approach.

[0117] The specificity of adoptive cell therapy to a specific cell type can be achieved by the different activities of the first receptor and the second receptor and the differential expression of the first ligand and the second ligand. The binding of the first ligand to the first receptor provides an activation signal, and the binding of the second ligand to the second receptor prevents or reduces the activation of effector cells even in the presence of the first ligand. The first ligand can be more widely expressed than the second ligand, for example, in cells targeted by adoptive cell therapy and in healthy cells (non-target cells) that are not target cells of adoptive cell therapy. In contrast, the second ligand is expressed in non-target cells, but not in target cells. Only target cells, rather than non-target cells, express the first ligand but not the second ligand, thereby activating the effector cells comprising the dual receptors of the present disclosure in the presence of these cells.

[0118] The present disclosure provides compositions and methods for targeting cells (e.g., tumor cells) based on loss of heterozygosity by using two engineered receptors. Two engineered receptors (one is an inhibitor and the other is an activator) each contain different ligand binding domains that recognize different ligands. When only the first activator ligand is present, the difference in expression of the first ligand and the second ligand is used to selectively activate effector cells expressing two receptors. Therefore, in some embodiments, the first ligand binding domain and the second ligand binding domain are on different receptor molecules; that is, a separate receptor that is not part of a single genetic construct, fusion protein, or protein complex. In some embodiments, when each receptor binds to its cognate ligand, one receptor activates the cell and the other receptor inhibits the cell. In some embodiments, the receptor comprising the second inhibitor ligand binding domain dominates signal transduction, so that if the target cell expresses two targets, the result is to inhibit effector cells. Only when there is no inhibitory target in the cell, the first activator ligand induces activation of effector cells by the receptor comprising the first activator ligand binding domain.

[0119] Any widely expressed cell surface molecule (such as cell adhesion molecules, cell-cell signaling molecules, extracellular domains, molecules related to chemotaxis, glycoproteins, G protein coupled receptors, transmembrane, neurotransmitter receptors or voltage-gated ion channels or any of these peptide antigens) can be used as the first ligand. As another example, the first ligand can be transferrin receptor (TFRC). Any cell surface molecule not expressed on the target cell surface can be used as the second ligand. In those embodiments where the engineered receptor is used for adoptive cell therapy to treat cancer and the target cell is a cancer cell, the second ligand can be selected based on the loss of heterozygosity of the second ligand in the cancer cell. For example, due to mutations that cause loss of heterozygosity, the exemplary genes that often lose their expression in cancer cells include HLA class I alleles, minor histocompatibility antigens (MiHA) and Y chromosome genes.

[0120] The present disclosure also provides vectors and polynucleotides encoding the engineered receptors described herein.

[0121] The present disclosure also provides methods of making populations of immune cells comprising the engineered receptors described herein, and methods of using the same to treat disorders.

[0122] definition

[0123] Before describing the present disclosure in greater detail, it may be helpful to understand the present disclosure by providing definitions of certain terms that will be used herein.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For purposes of this disclosure, the following terms are defined below. Other definitions are also set forth throughout this disclosure.

[0125] The articles "a / an" and the grammatical objects of the articles are used herein to refer to one or more than one (i.e., at least one or one or more). For example, "an element" refers to one element or one or more elements.

[0126] The use of alternatives (eg, "or") should be understood to mean one, two, or any combination of the alternatives.

[0127] The term "and / or" should be understood to mean one or both alternatives.

[0128] Throughout this specification, unless the context requires otherwise, the words "comprise / comprises / comprising" will be understood to mean the inclusion of a specified step, or element, or group of steps or elements, but not the exclusion of any other step, or element, or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. "Consisting essentially of" means including any element listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified for the listed elements in this disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but there are no other elements that substantially affect the activity or action of the listed elements.

[0129] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "another embodiment," or "other embodiments," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the various occurrences of the aforementioned phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that the affirmative recitation of a feature in one embodiment is used as a basis for excluding the feature in a particular embodiment.

[0130] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, size, dimension, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, size, dimension, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, size, dimension, amount, weight, or length range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% about a reference quantity, level, value, number, frequency, percentage, size, dimension, amount, weight, or length.

[0131] As used herein, the term "isolated" refers to material that is substantially or essentially free from components that normally accompany it in its native state. In certain embodiments, the terms "obtained" or "derived from" are used synonymously with isolated.

[0132] The terms "subject", "patient" and "individual" are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro are also contemplated. "Subject", "patient" or "individual" as used herein include any animal that exhibits pain that can be treated using the vectors, compositions and methods contemplated herein. Suitable subjects (e.g., patients) include experimental animals (such as mice, rats, rabbits or guinea pigs), farm animals and domestic animals or pets (such as cats or dogs). Non-human primates are included, and optionally human patients.

[0133] As used herein, "treatment" or "treating" includes any beneficial or desired effect, and can include even minimal amelioration of symptoms. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition, or its associated symptoms.

[0134] As used herein, "prevent" and similar words, such as "prevented," "preventing," and the like, indicate methods for preventing, inhibiting, or reducing the likelihood of disease symptoms. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of disease symptoms. As used herein, "preventing" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease prior to onset or recurrence.

[0135] As used herein, the term "amount" refers to an "effective amount" or "effective amount" of a virus to achieve beneficial or desired prophylactic or therapeutic results, including clinical results.

[0136] A "prophylactically effective amount" refers to an amount of virus effective to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic amount is less than a therapeutically effective amount because a prophylactic dose is used in subjects prior to disease or at an early stage of disease.

[0137] A "therapeutically effective amount" of a virus or cell may vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the virus or cell to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the virus or cell outweigh the beneficial effects of the treatment. The term "therapeutically effective amount" includes an amount effective for "treating" a subject (e.g., a patient).

[0138] The amount of an "increase" or "elevation" of a physiological response (e.g., electrophysiological activity or cellular activity) is generally a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimal points between 1 and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.) in the level of activity in untreated cells.

[0139] The amount of a "reduction" or "reduction" in a physiological response (e.g., electrophysiological activity or cellular activity) is generally a "statistically significant" amount and can include a 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimal points between 1 and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.) reduction in the level of activity in untreated cells.

[0140] By "maintain," or "retain," or "maintain," or "no change," or "no substantial change," or "no substantial reduction," we generally mean a physiological response that is comparable to the response elicited by a vehicle or control molecule / composition. A comparable response is one that is not significantly different or measurably different from a reference response.

[0141] In general, "sequence identity" or "sequence homology" refers to the exact correspondence of nucleotides to nucleotides or amino acids to amino acids of two polynucleotides or polypeptide sequences, respectively. Typically, the 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 with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". Whether it is a nucleic acid or an amino acid sequence, the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. For example, the advanced BLAST computer program (including version 2.2.9) available from the National Institutes of Health can also be used to compare sequence information to determine the percent identity. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and 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). In brief, the BLAST program defines identity as the number of identical alignment symbols (usually nucleotides or amino acids) divided by the total number of shorter symbols in the two sequences. The program can be used to determine the percent identity over the entire length of the protein being compared. For example, in the blastp program, default parameters are provided to optimize searches with short query sequences. The program also allows the use of SEG filters to mask query sequence segments determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The expected degree of sequence identity ranges from about 80% to 100%, and integer values ​​therebetween. Typically, the percent identity between a disclosed sequence and a claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0142] As used herein, the term "exogenous" refers to any molecule originating from outside an organism, including nucleic acids, proteins or peptides, small molecule compounds, etc. In contrast, the term "endogenous" refers to any molecule originating from within an organism (ie, produced naturally by the organism).

[0143] As used herein, the term "MOI" refers to the multiplicity of infection, that is, the ratio of agent (eg, viral particles) to infected target (eg, cells).

[0144] All publications and patents mentioned herein are hereby incorporated by reference in their entirety, as if each individual publication or patent was specifically and individually indicated not to be incorporated by reference. In the event of a conflict, the present application (including any definitions herein) will control. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0145] In this specification, unless otherwise specified, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, and, where appropriate, its fractions (such as one tenth and one hundredth of an integer). When immediately preceding a number or value, the term "about" refers to plus or minus 10% of a number or value range.

[0146] As used herein, "target cell" refers to a cell targeted by adoptive cell therapy. For example, a target cell can be a cancer cell, which can be killed by a transplanted T cell of adoptive cell therapy. The target cells of the present disclosure express an activator ligand as described herein, and do not express an inhibitor ligand.

[0147] Activator

[0148] The present disclosure provides a first ligand (activator) and a first engineered receptor comprising a first ligand binding domain that binds to the first activator ligand.

[0149] The present disclosure provides a first engineered receptor comprising an extracellular region, the extracellular region comprising a first ligand binding domain capable of specifically binding to a first ligand, the first ligand activating or promoting activation of the receptor, which promotes activation of effector cells expressing the receptor. The present disclosure also provides a second engineered receptor comprising a second ligand binding domain capable of binding to a second ligand, wherein even in the presence of a first receptor bound to the first ligand, the binding of the second ligand binding domain to the second ligand inhibits or reduces activation of effector cells.

[0150] As used herein, "activator" or "activator ligand" refers to a first activator ligand binding domain (LBD) that binds to an engineered receptor (such as a CAR or TCR) disclosed herein, thereby mediating the first ligand for activation of T cells expressing the engineered receptor. Activators are expressed by target cells (e.g., cancer cells) and may also be more widely expressed than just target cells. For example, activators may be expressed on some or all types of normal non-target cells.

[0151] In some embodiments, the first ligand is a peptide ligand from any activator target disclosed herein. In some embodiments, the first ligand is a peptide antigen complexed with a major histocompatibility (MHC) class I complex (peptide MHC or pMHC), such as an MHC complex comprising a human leukocyte antigen A*02 allele (HLA-A*02).

[0152] A target cell-specific first activator ligand comprising a peptide antigen complexed with a pMHC containing any of the human leukocyte antigens (HLA) HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G is contemplated to be within the scope of the present disclosure. In some embodiments, the first ligand comprises a pMHC containing HLA-A. The HLA-A 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 unchanged. There are thousands of HLA-A gene variants, all of which fall within the scope of the present disclosure. In some embodiments, MHC-I comprises a human leukocyte antigen A*02 allele (HLA-A*02).

[0153] In some embodiments, the first activator ligand comprises a pMHC comprising HLA- B. Hundreds of forms (alleles) of the HLA-B gene are known, each of which is provided with a specific number (eg, HLA-B*27).

[0154] In some embodiments, the first activator ligand comprises a pMHC comprising HLA-C. HLA-C belongs to the HLA class I heavy chain paralogue. The class I molecule is a heterodimer composed of a heavy chain and a light chain (beta-2 microglobulin). More than one hundred HLA-C alleles are known in the art.

[0155] In some embodiments, the first activator ligand comprises a pMHC comprising HLA-A. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-B. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-C. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-E. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-F. In some embodiments, the first activator ligand comprises a pMHC comprising HLA-G.

[0156] In some embodiments, the first activator ligand comprises HLA-A. In some embodiments, the first activator ligand comprises HLA-B. In some embodiments, the first activator ligand comprises HLA-C. In some embodiments, the first activator ligand comprises HLA-E. In some embodiments, the first activator ligand comprises HLA-F. In some embodiments, the first activator ligand comprises HLA-G. In some embodiments, the first activator ligand comprises HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

[0157] In some embodiments, the first activator ligand binding domain comprises a ScFv domain.

[0158] In some embodiments, the first activator ligand binding domain comprises only a Vβ ligand binding domain.

[0159] In some embodiments, the first activator ligand binding domain comprises an antigen binding domain isolated or derived from a T cell receptor (TCR).For example, the first agonist ligand binding domain comprises the TCR α and β chain variable domains.

[0160] In some embodiments, the first activator ligand and the second inhibitor ligand are different.

[0161] In some embodiments, the first activator ligand is expressed by target cells and is not expressed by non-target cells (ie, normal cells that are not targeted by adoptive cell therapy). In some embodiments, the target cells are cancer cells and the non-target cells are non-cancerous cells.

[0162] In some embodiments, the activator ligand has high cell surface expression on the target cell. This high cell surface expression confers the ability to deliver a large activation signal. Methods for measuring cell surface expression will be known to those of ordinary skill in the art and include, but are not limited to, immunohistochemistry using appropriate antibodies to the activator ligand followed by microscopy or fluorescence activated cell sorting (FACS).

[0163] In some embodiments, the activator ligand is encoded by a gene with an essential cell function. Essential cell functions are functions required for cell survival, including protein and lipid synthesis, cell division, replication, respiration, metabolism, ion transport, and providing structural support for tissues. Selecting an activator ligand encoded by a gene with an essential cell function prevents the loss of the activator ligand due to aneuploidy in cancer cells, and makes the gene encoding the activator ligand less likely to undergo mutagenesis during cancer evolution. In some embodiments, the activator ligand is encoded by a gene with haploinsufficient (i.e., the loss of a copy of the gene encoding the activator ligand is not tolerated by the cell and causes cell death or an unfavorable mutant phenotype).

[0164] In some embodiments, the activator ligand is present on all target cells. In some embodiments, the target cells are cancer cells.

[0165] 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.

[0166] In some embodiments, the activator ligand is present on all cells (a ubiquitous activator ligand). The activator ligand may be expressed on all cells if, for example, a second inhibitor ligand is also expressed on all cells except the target cells.

[0167] In some embodiments, the first activator ligand is expressed by a plurality of target cells and a plurality of non-target cells. In some embodiments, the plurality of non-target cells express both the first activator ligand and the second inhibitor ligand.

[0168] In some embodiments, and the first activator ligand and the second inhibitor ligand are present on a plurality of non-target target cells at a ratio of the first ligand to the second ligand of about 1:100 to about 100:1. In some embodiments, and the first activator ligand and the second inhibitor ligand are present on a plurality of non-target target cells at a ratio of the first ligand to the second ligand of about 1:50 to about 50:1. In some embodiments, and the first activator ligand and the second inhibitor ligand are present on a plurality of non-target target cells at a ratio of the first ligand to the second ligand of about 1:25 to about 25:1. In some embodiments, and the first activator ligand and the second inhibitor ligand are present on a plurality of non-target target cells at a ratio of the first ligand to the second ligand of about 1:10 to about 10:1. In some embodiments, and the first activator ligand and the second inhibitor ligand are present on a plurality of non-target target cells at a ratio of the first ligand to the second ligand of about 1:5 to about 5:1. In some embodiments, and the first activator ligand and the second inhibitor ligand are present on a plurality of non-target target cells at 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 inhibitor ligand are present on the plurality of non-target target cells at 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 inhibitor ligand are present on the plurality of non-target target cells at a ratio of about 1:1.

[0169] The first activator ligand is recognized by a first ligand binding domain (sometimes referred to herein as an activator LBD).

[0170] Exemplary activator ligands include ligands selected from the group consisting of: cell adhesion molecules, cell-cell signaling molecules, extracellular domains, molecules involved in chemotaxis, glycoproteins, G protein-coupled receptors, transmembrane proteins, neurotransmitter receptors, and voltage-gated ion channels. In some embodiments, the first activator ligand is a transferrin receptor (TFRC) or a peptide antigen thereof. The human transferrin receptor is described in NCBI Record No. AAA61153.1, the contents of which are incorporated herein by reference. In some embodiments, TFRC is encoded by the following sequence:

[0171]

[0172] In some embodiments, the activator ligand is a tumor-specific antigen (TSA). In some embodiments, the tumor-specific antigen is mesothelin (MSLN), CEA cell adhesion molecule 5 (CEACAM5 or CEA), epidermal growth factor receptor (EGFR) or its peptide antigen. In some embodiments, TSA is MSLN, CEA, EGFR, Delta-like typical Notch ligand 4 (DLL4), cell surface-associated mucin 16 (MUC 16, also known as CA125), ganglioside GD2 (GD2), receptor tyrosine kinase-like orphan receptor 1 (ROR1), erb-b2 receptor tyrosine kinase 2 (HER2 / NEU) or its peptide antigen. Exemplary mouse and humanized ScFv antigen binding domains targeting TSA are shown in Table 1 below:

[0173] Table 1. Exemplary ScFv antigen binding domains targeting tumor specific antigens (TSA)

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] In some embodiments, the activator ligand is MSLN or a peptide antigen thereof, and the activator ligand binding domain comprises an MSLN binding domain. In some embodiments, the MSLN ligand binding domain comprises an ScFv domain. In some embodiments, the MSLN ligand binding domain comprises a sequence of SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, or SEQ ID NO: 92. In some embodiments, the MSLN ligand binding domain comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, or SEQ ID NO: 92. In some embodiments, the MSLN ligand binding domain is encoded by a sequence comprising SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, or SEQ ID NO: 93. In some embodiments, the MSLN ligand binding domain is encoded by a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the sequence of SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, or SEQ ID NO:93.

[0180] In some embodiments, the activator ligand is CEA or its peptide antigen, and the activator ligand binding domain comprises a CEA binding domain. In some embodiments, the CEA ligand binding domain comprises a ScFv domain. In some embodiments, the CEA ligand binding domain comprises a sequence of SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 282, SEQ ID NO: 284 or SEQ ID NO: 286. In some embodiments, the CEA ligand binding domain comprises a sequence that is at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 282, SEQ ID NO: 284 or SEQ ID NO: 286. In some embodiments, the CEA ligand binding domain is encoded by a sequence comprising SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287. In some embodiments, the CEA ligand binding domain is encoded by a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a sequence of SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287.

[0181] In some embodiments, the activator ligand is CEA or its peptide antigen, and the activator ligand binding domain comprises a CEA binding domain. In some embodiments, the CEA ligand binding domain comprises CDR-H1 of EFGMN (SEQ ID NO: 294), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296) or WDFAHYFQTMDY (SEQ ID NO: 297), CDR-L1 of KASQNVGTNVA (SEQ ID NO: 298) or KASAAVGTYVA (SEQ ID NO: 299), CDR-L2 of SASYRYS (SEQ ID NO: 300) or SASYRKR (SEQ ID NO: 301), and CDR-L3 of HQYYTYPLFT (SEQ ID NO: 302), or a sequence having at least 85% or at least 95% identity thereto. In some embodiments, the CEA ScFv comprises CDR-H1 of EFGMN (SEQ ID NO:294), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO:295), CDR-H3 of WDFAYYVEAMDY (SEQ ID NO:296) or WDFAHYFQTMDY (SEQ ID NO:297), CDR-L1 of KASQNVGTNVA (SEQ ID NO:298) or KASAAVGTYVA (SEQ ID NO:299), CDR-L2 of SASYRYS (SEQ ID NO:300) or SASYRKR (SEQ ID NO:301), and CDR-L3 of HQYYTYPLFT (SEQ ID NO:302). In some embodiments, the CEA binding domain comprises CDR-H1 of EFGMN (SEQ ID NO:294), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO:295), CDR-H3 of WDFAYYVEAMDY (SEQ ID NO:296), CDR-L1 of KASQNVGTNVA (SEQ ID NO:298), CDR-L2 of SASYRYS (SEQ ID NO:300), and CDR-L3 of HQYYTYPLFT (SEQ ID NO:302).In some embodiments, the CEA ScFv comprises CDR-H1 of EFGMN (SEQ ID NO:294), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO:295), CDR-H3 of WDFAYYVEAMDY (SEQ ID NO:296), CDR-L1 of KASAAVGTYVA (SEQ ID NO:299), CDR-L2 of SASYRKR, and CDR-L3 of HQYYTYPLFT (SEQ ID NO:302). In some embodiments, the CEA binding domain comprises CDR-H1 of EFGMN (SEQ ID NO:294), CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO:295), CDR-H3 of WDFAHYFQTMDY (SEQ ID NO:297), CDR-L1 of KASAAVGTYVA (SEQ ID NO:299), CDR-L2 of SASYRKR, and CDR-L3 of HQYYTYPLFT (SEQ ID NO:302).

[0182] In some embodiments, the activator ligand is CEA or its peptide antigen, and the activator receptor is CEA CAR. In some embodiments, CEA CAR comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 288, SEQ ID NO: 290, or SEQ ID NO: 292. In some embodiments, CEA CAR comprises SEQ ID NO: 288, SEQ ID NO: 290, or SEQ ID NO: 292 or is substantially composed thereof. In some embodiments, CEA CAR is encoded by a sequence comprising SEQ ID NO: 289, SEQ ID NO: 291, or SEQ ID NO: 293 or is substantially composed thereof. In some embodiments, CEA CAR is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 289, SEQ ID NO: 291, or SEQ ID NO: 293.

[0183] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR binding domain. In some embodiments, the EGFR ligand binding domain comprises a ScFv domain. In some embodiments, the EGFR ligand binding domain comprises a sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, or SEQ ID NO: 391. In some embodiments, the EGFR ligand binding domain comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, or SEQ ID NO: 391. In some embodiments, the EGFR ligand binding domain is encoded by a sequence comprising SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, or SEQ ID NO: 119. In some embodiments, the EGFR ligand binding domain is encoded by a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the sequence of SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, or SEQ ID NO:119.

[0184] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR ligand binding domain. In some embodiments, the EGFR binding domain comprises a VH and / or VL domain selected from the group disclosed in Table 2, or a sequence having at least 90% identity thereto. In some embodiments, the EGFR ligand binding domain comprises a VH domain selected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, and SEQ ID NO: 130. In some embodiments, the EGFR ligand binding domain comprises a VH selected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, and SEQ ID NO: 130, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the EGFR ligand binding domain comprises a VL domain selected from the group consisting of SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, and SEQ ID NO: 131. In some embodiments, the EGFR ligand binding domain comprises a VH selected from the group consisting of SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, and SEQ ID NO: 131, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto.

[0185] Table 2. EGFR variable heavy chain (VH) and variable light chain (VL) domains

[0186]

[0187]

[0188] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain is an EGFR ligand binding domain. In some embodiments, the EGFR binding domain comprises a complementary determining region (CDR) selected from a group of CDRs disclosed in Table 3. In some embodiments, the EGFR ligand binding domain comprises a CDR having at least 95% sequence identity with the CDRs disclosed in Table 3. In some embodiments, the EGFR ligand binding domain comprises a CDR selected from SEQ ID NO: 131-166. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 1 (CDR H1) selected from the group consisting of SEQ ID NO: 132-137. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 2 (CDR H2) selected from the group consisting of SEQ ID NO: 138-143. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 3 (CDR H3) selected from the group consisting of SEQ ID NO: 144-149. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 1 (CDR L1) selected from the group consisting of SEQ ID NOs: 150-155. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 2 (CDR L2) selected from the group consisting of SEQ ID NOs: 156-160. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 3 (CDR L3) selected from the group consisting of SEQ ID NOs: 161-166. In some embodiments, the EGFR ligand binding domain comprises a CDR H1 selected from SEQ ID NOs: 132-137, a CDR H2 selected from SEQ ID NOs: 138-143, a CDR H3 selected from SEQ ID NOs: 144-149, a CDR L1 selected from SEQ ID NOs: 150-155, a CDR L2 selected from SEQ ID NOs: 156-160, and a CDR L3 selected from SEQ ID NOs: 156-160.

[0189] Table 3. EGFR antigen binding domain CDRs.

[0190]

[0191] In some embodiments, the activator ligand is a pan-HLA ligand, and the activator binding domain is a pan-HLA binding domain, i.e., a binding domain that binds and recognizes an antigenic determinant shared between the products of the HLA A, B, and C loci. Various single variable domains known in the art or disclosed herein are suitable for use in the embodiments. Such scFvs include, for example, but are not limited to, the following mouse and humanized pan-HLA scFv antibodies. An exemplary pan-HLA ligand is W6 / 32, which recognizes a conformational epitope and reacts with the HLA class I α3 and α2 domains.

[0192] Table 4. Pan-HLA ScFv binding domains derived from W6 / 32

[0193]

[0194]

[0195] In some embodiments, the activator ligand is a pan-HLA ligand, and the activator ligand binding domain comprises a pan-HLA ligand binding domain. In some embodiments, the pan-HLA ligand binding domain comprises a ScFv domain. In some embodiments, the pan-HLA ligand binding domain comprises a sequence of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177. In some embodiments, the pan-HLA ligand binding domain comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177. In some embodiments, the pan-HLA ligand binding domain is encoded by a sequence comprising SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, or SEQ ID NO: 178. In some embodiments, the pan-HLA ligand binding domain is encoded by a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the sequence of SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, or SEQ ID NO: 178.

[0196] In some embodiments, the activator ligand is a CD19 molecule (CD19) or a peptide antigen thereof, and the activator ligand binding domain comprises a CD19 ligand binding domain. In some embodiments, the CD19 ligand binding domain comprises a ScFv domain. In some embodiments, the CD19 ligand binding domain comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 275 or SEQ ID NO: 277. In some embodiments, the CD-19 ligand binding domain comprises a sequence of SEQ ID NO: 275 or SEQ ID NO: 277. In some embodiments, the CD19 ligand binding domain is encoded by a sequence comprising SEQ ID NO: 276 or SEQ ID NO: 278. In some embodiments, the CD19 ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a sequence of SEQ ID NO: 276 or SEQ ID NO: 278.

[0197] In some embodiments, the activator ligand is a CD19 molecule (CD19) or a peptide antigen thereof, and the activator receptor is a CAR. In some embodiments, the CD19 CAR comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 279 or SEQ ID NO: 281. In some embodiments, the CD19 CAR comprises SEQ ID NO: 279 or SEQ ID NO: 281 or is substantially composed thereof. In some embodiments, the CD19 CAR is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a sequence of SEQ ID NO: 280 or SEQ ID NO: 390. In some embodiments, the CD19 CAR is encoded by a sequence comprising SEQ ID NO: 280 or SEQ ID NO: 390 or consisting essentially of it. It will be appreciated by those of ordinary skill that the first activator ligand binding domain for the first receptor can be isolated or derived from any source known in the art, including but not limited to T cell receptors, chimeric antigen receptors, and antibody binding domains recognized in the art. For example, the first ligand binding domain can be derived from any antibody disclosed in Table 5 and binds to a first ligand selected from the antigens described in Table 5. Thus, immune cells comprising the described dual receptor system can be used to treat any disease or condition described in Table 5. It will be apparent to those of skill in the art to select an appropriate first activator receptor ligand binding domain to treat any cancer described herein.

[0198] Table 5. Exemplary Antibodies

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] Inhibitors

[0206] The present disclosure provides a second ligand (inhibitor) and a second engineered receptor comprising a second ligand binding domain that binds to the inhibitor ligand.

[0207] The present disclosure provides a second engineered receptor comprising an extracellular region, wherein the extracellular region comprises a second ligand binding domain capable of specifically binding to a second ligand, wherein the second ligand inhibits activation of effector cells expressing the first receptor and the second receptor, wherein the effector cell is activated by binding of the first ligand to the first engineered receptor.

[0208] As used herein, an "inhibitor" or "inhibitor ligand" (sometimes referred to as a "blocker") 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 the activation of immune cells expressing the engineered receptor. The inhibitor is not expressed by target cells. Inhibitor ligands are also expressed in a variety 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 the activation of effector cells by a variety of mechanisms. For example, the binding of an inhibitor ligand to an inhibitor LBD can block the signaling that occurs when an activator ligand binds to an activator LBD, which in the absence of an inhibitor would result in activation of immune cells expressing the engineered receptor described herein.

[0209] Alternatively or in addition, the binding of the inhibitor ligand to the second engineered receptor can result in the loss of cell surface expression of the first activator receptor from the surface of the immune cell comprising the dual receptor system described herein. Without wishing to be bound by theory, it is believed that the engagement of the activator ligand and the inhibitor ligand in the immune cell on normal cells results in the removal of the inhibitor receptor from the activator receptor molecule near the surface of the immune cell. This process desensitizes the immune cell locally, reversibly increasing its activation threshold. Immune cells that only engage with the activator ligand on the target cell result in a local activation signal that is not hindered by the signal from the second inhibitory receptor. This local activation increases until the release of cytotoxic particles results in selective cell death of the target cell. However, regulating the expression level of the surface receptor may not be the only mechanism by which the blocker receptor inhibits the activation of the first activator receptor by the immune cell. Without wishing to be bound by theory, other mechanisms may play a role, including but not limited to crosstalk between the activator and blocker receptor signaling pathways.

[0210] In some embodiments, the second ligand is not expressed by the target cell and is expressed by a non-target cell. In some embodiments, the target cell is a cancer cell and the non-target cell is a non-cancer cell.

[0211] In some embodiments, the second inhibitor ligand binding domain comprises a ScFv domain.

[0212] In some embodiments, the second inhibitor ligand binding domain comprises only a Vβ ligand binding domain.

[0213] In some embodiments, the second inhibitor ligand binding domain comprises an antigen binding domain isolated or derived from a T cell receptor (TCR).For example, the second inhibitor ligand binding domain comprises the TCR α and β chain variable domains.

[0214] Inhibitor Targets

[0215] In some embodiments, the inhibitor ligand comprises a gene or peptide antigen thereof with highly uniform surface expression across tissues. Without wishing to be bound by theory, highly uniform surface expression across tissues allows the inhibitor ligand to deliver a large, even inhibitory signal. Alternatively or in addition, the expression of the activator and inhibitor targets may be correlated, i.e., both are expressed at similar levels on non-target cells.

[0216] In some embodiments, the second inhibitor ligand is a peptide ligand. In some embodiments, the second inhibitor ligand is a peptide antigen complexed with a major histocompatibility (MHC) class I complex (peptide MHC or pMHC). Inhibitor ligands comprising a peptide antigen complexed with a pMHC containing any of HLA-A, HLA-B or HLA-C are contemplated within the scope of the present disclosure.

[0217] In some embodiments, the inhibitor ligand is encoded by a gene that is absent or polymorphic in many tumors.

[0218] Methods for distinguishing differential expression of inhibitor ligands between target cells and non-target cells will be apparent to those skilled in the art. For example, the presence or absence of inhibitor ligands in non-target cells and target cells can be determined by immunohistochemistry with antibodies that bind to the inhibitor ligands, followed by microscopy or FACS, RNA expression profiling of target cells and non-target cells, or DNA sequencing of non-target cells and target cells to determine whether the genomic locus of the inhibitor ligand contains a mutation in the target cells or non-target cells.

[0219] Allele loss due to loss of heterozygosity (LOH)

[0220] Homozygous deletions in primary tumors are rare and infrequent, making it impossible to generate target B candidates. For example, in an analysis of 2218 primary tumors of 21 human cancer types, the top four candidates were cyclin-dependent kinase inhibitor 2A (CDKN2A), RB transcriptional co-repressor 1 (RB1), phosphatase and tensin homolog (PTEN), and N3PB2. However, CDKN2A (P16) is missing in only 5% homozygous deletions in 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, the loss of a single copy of a gene in cancer cells due to the loss of a hemizygous state occurs much more frequently.

[0221] In some embodiments, the second inhibitor ligand comprises an allele of a gene lost in the target cell due to 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 may result in the loss of one copy of one or more genes in the cancer cell.

[0222] As used herein, "loss of heterozygosity (LOH)" refers to a genetic alteration that occurs at high frequency in cancer whereby one of two alleles is deleted, leaving a single monoallelic (hemizygous) locus.

[0223] HLA class I alleles

[0224] In some embodiments, the second inhibitor ligand comprises an HLA class I allele. Major histocompatibility complex (MHC) class I is a protein complex that displays antigens to cells of the immune system, triggering an immune response. Human leukocyte antigens (HLA) corresponding to MHC class I include HLA-A, HLA-B, and HLA-C.

[0225] In some embodiments, the second inhibitor ligand comprises an HLA class I allele. In some embodiments, the second inhibitor ligand comprises an HLA class I allele lost in the target cell by 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 main 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 unchanged. There are thousands of HLA-A variants, all of which fall within the scope of the present disclosure.

[0226] In some embodiments, the second inhibitor ligand comprises an HLA-B allele. The HLA-B gene has many possible variations (alleles). Hundreds of forms (alleles) of the HLA-B gene are known, each of which is provided with a specific number (e.g., HLA-B27).

[0227] In some embodiments, the second inhibitor ligand comprises an HLA-C allele. HLA-C belongs to the HLA class I heavy chain paralogue. The class I molecule is a heterodimer composed of a heavy chain and a light chain (beta-2 microglobulin). More than 100 HLA-C alleles have been described.

[0228] In some embodiments, the HLA class I allele has widespread or ubiquitous RNA expression.

[0229] In some embodiments, the HLA class I allele has a known or typically high minor allele frequency.

[0230] In some embodiments, the HLA class I allele does not require a peptide-MHC antigen, such as when the HLA class I allele is recognized by a pan-HLA ligand binding domain.

[0231] In some embodiments, the second inhibitor ligand comprises an HLA-A allele. In some embodiments, the HLA-A allele comprises HLA-A*02. Various single variable domains known in the art or disclosed herein that bind and recognize HLA-A*02 are suitable for use in the embodiments. Such scFvs include, for example, but are not limited to, the following mouse and humanized scFv antibodies that bind to HLA-A*02 in a peptide-independent manner, as shown in Table 6 below (complementarity determining regions are underlined):

[0232] Table 6. HLA-A*02 ScFv binding domains

[0233]

[0234]

[0235]

[0236] Exemplary heavy and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3, respectively) of the HLA-A*02 ligand binding domain are shown in Table 7 below.

[0237] Table 7. CDRs corresponding to the HLA-A*02 antigen binding domain

[0238]

[0239] In some embodiments, the scFv comprises a complementarity determining region (CDR) of any one of SEQ ID NOs: 41-52. In some embodiments, the scFv comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 41-52. In some embodiments, the scFv comprises a sequence that is identical to any one of SEQ ID NOs: 41-52. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs of any one of SEQ ID NOs: 53-64, and wherein the light chain of the antibody comprises the light chain CDRs of any one of SEQ ID NOs: 53-64. In some embodiments, the heavy chain of the antibody comprises a sequence that is at least 95% identical to the heavy chain portion of any one of SEQ ID NOs: 53-64, and wherein the light chain of the antibody comprises a sequence that is at least 95% identical to the light chain portion of any one of SEQ ID NOs: 53-64.

[0240] In some embodiments, the heavy chain of the antibody comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOs: 53-64, and wherein the light chain of the antibody comprises a sequence identical to the light chain portion of any one of SEQ ID NOs: 53-64.

[0241] In some embodiments, the ScFv comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to any one of SEQ ID NOs: 53-64.

[0242] In some embodiments, the second inhibitory ligand is HLA-A*02, and the inhibitory ligand binding domain comprises an HLA-A*02 ligand binding domain. In some embodiments, the second ligand binding domain binds to HLA-A*02 independently of a peptide in a pMHC complex comprising HLA-A*02. In some embodiments, the HLA-A*02 ligand binding domain comprises a ScFv domain. In some embodiments, the HLA-A*02 ligand binding domain comprises the sequence of any one of SEQ ID NOs: 53-64. 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 the sequence of any one of SEQ ID NOs: 53-64. In some embodiments, the HLA-A*02 ligand binding domain is encoded by a sequence comprising any one of SEQ ID NOs: 179-190. In some embodiments, the HLA-A*02 ligand binding domain is encoded by a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the sequence of any one of SEQ ID NOs: 179-190.

[0243] Minor histocompatibility antigens

[0244] In some embodiments, the second inhibitor ligand comprises a minor histocompatibility antigen (MiHA). In some embodiments, the second inhibitor ligand comprises an allele of MiHA that is lost in the target cell by LOH.

[0245] MiHA is a peptide derived from a protein containing non-synonymous differences between alleles and displayed by a common HLA allele. The non-synonymous differences can be caused by SNP, deletion, frameshift mutation or insertion in the coding sequence of the gene encoding MiHA. The length of exemplary MiHA can be about 9-12 amino acids, and can bind MHC class I and MHC class II proteins. The combination of TCR and the MHC complex displaying MiHA can activate T cells. The heredity and immunological properties of MiHA will be known to those of ordinary skill in the art. Candidate MiHA is a known peptide presented by a known HLA class I allele, which is known to elicit T cell responses in the clinic (e.g., in graft-versus-host disease or transplant rejection), and allows patient selection by simple SNP genotyping.

[0246] In some embodiments, MiHA has widespread or ubiquitous RNA expression.

[0247] In some embodiments, the MiHA has a high minor allele frequency.

[0248] In some embodiments, the MiHA comprises a peptide derived from a Y chromosome gene.

[0249] In some embodiments, the second inhibitor ligand comprises a MiHA selected from the group of MiHAs disclosed in Tables 8 and 9.

[0250] Exemplary but non-limiting examples of MiHAs contemplated within the scope of the present invention are disclosed below in Table 8. The columns in Table 8 indicate from left to right the name of the MiHA, the gene from which it is derived, the sequence of the MHC class I variant and peptide variants [indicated in brackets A / B variants] that can display the MiHA).

[0251] Table 8. HLA class I autosomal MiHA.

[0252]

[0253]

[0254]

[0255]

[0256]

[0257] Exemplary but non-limiting examples of MiHAs contemplated within the scope of the present invention are disclosed below in Table 9. The columns in Table 9 indicate from left to right the name of the MiHA, the gene from which it is derived, the sequence of the MHC class I variant and peptide variants [indicated in brackets A / B variants] that can display the MiHA).

[0258] Table 9. HLA class I Y-linked MiHA

[0259]

[0260] In some embodiments, MiHA comprises HA- 1. HA-1 is a peptide antigen having the sequence of VL[H / R]DDLLEA (SEQ ID NO: 273) and is derived from the Rho-type GTPase activating protein 45 (HA-1) gene.

[0261] Exemplary ligand binding domains that selectively bind to the HA-1 variant H peptide (VLHDDLLEA (SEQ ID NO: 191)) are shown below in Table 10. The TCRα and TCRβ sequences in SEQ ID NO: 193 are separated by a P2A self-cleaving polypeptide of the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 192) with an N-terminal GSG linker.

[0262] Table 10. Ftcr HA-1 (H) inhibitory receptor sequences

[0263]

[0264]

[0265] In some embodiments, the second inhibitory ligand comprises HA-1 (H). In some embodiments, the second inhibitory ligand binding domain is separated from or derived from TCR. In some embodiments, the second inhibitory ligand binding domain comprises TCR alpha and TCR beta variable domains. In some embodiments, 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: 193. In some embodiments, the TCR alpha and TCR beta variable domains separated by a self-cleaving polypeptide sequence comprise SEQ ID NO: 193 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: 194 or a sequence having at least 80% identity, at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the TCR alpha variable domain comprises SEQ ID NO: 199, or a sequence at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the TCR beta variable domain comprises SEQ ID NO: 200, or a sequence at least 90%, at least 95%, or at least 99% identical thereto.

[0266] Loss of Y chromosome antigens

[0267] In some embodiments, the second inhibitor ligand comprises a Y chromosome gene, i.e., a peptide encoded by a gene on the Y chromosome. In some embodiments, the second inhibitor ligand comprises a peptide encoded by a Y chromosome gene that is lost in the target cell by loss of the Y chromosome (LoY). For example, about one-third of the characteristic MiHAs are from the Y chromosome. The Y chromosome contains more than 200 protein-coding genes, all of which are contemplated to be within the scope of the present disclosure.

[0268] As used herein, "loss of the Y" or "LoY" refers to a genetic alteration that occurs at a high frequency in tumors whereby one copy of part or all of the Y chromosome is deleted, resulting in the loss of genes encoded by the Y chromosome.

[0269] It is known that Y chromosome loss occurs in certain cancers. For example, 40% of somatic loss of Y chromosomes has been reported in renal clear cell carcinoma (Arseneault et al., Sci. Rep. 7: 44876 (2017)). Similarly, clonal loss of Y chromosomes has been reported in 5 subjects of 31 male breast cancer subjects (Wong et al., Oncotarget 6 (42): 44927-40 (2015)). The loss of Y chromosomes 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)). In addition, in 4 patients of 7 male patients with gastric cancer, Y chromosome loss was associated with X chromosome disomy (Saal et al., Virchows Arch B Cell Pathol (1993)). Therefore, Y chromosome genes can be lost in a variety of cancers and can be used as inhibitor ligands together with the engineered receptors targeting cancer cells disclosed herein.

[0270] Antigen binding domain

[0271] The present disclosure provides a first ligand binding domain and a second ligand binding domain, wherein the first ligand binding domain activates a first engineered receptor, thereby activating an immune cell expressing the first engineered receptor, and the second ligand binding domain activates a second engineered receptor, and the second engineered receptor inhibits the activation of immune cells expressing the second engineered receptor even in the presence of the first engineered receptor bound to the first ligand.

[0272] Any type of ligand binding domain that can modulate receptor activity in a ligand-dependent manner is contemplated within the scope of the present disclosure. In some embodiments, the ligand binding domain is an antigen binding domain. Exemplary antigen binding domains particularly include ScFv, SdAb, Vβ domains only, and TCR antigen binding domains derived from TCR α and β chain variable domains.

[0273] In some embodiments, the first activator LBD comprises an antigen binding domain. In some embodiments, the second inhibitor LBD comprises an antigen binding domain. Any type of antigen binding domain is contemplated to be within the scope of the present disclosure.

[0274] For example, the first activator LBD and / or the second inhibitor LBD can comprise an antigen binding domain, which can be expressed as part of a continuous polypeptide chain, such as a single domain antibody fragment (sdAb) or a heavy chain antibody HCAb, a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some aspects, the first activator LBD and / or the second inhibitor LBD comprises an antigen binding domain comprising an antibody fragment. In further aspects, the activator LBD comprises an antibody fragment comprising a scFv or a sdAb. In further aspects, the inhibitor LBD comprises an antibody fragment comprising a scFv or a sdAb.

[0275] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen.Antibodies can be complete immunoglobulins or fragments thereof of polyclonal or monoclonal origin, and can be derived from natural or recombinant sources.

[0276] The term "antibody fragment" or "antibody binding domain" refers to at least a portion of an antibody or a recombinant variant thereof that contains an antigen binding domain (i.e., the antigen-determining variable region of an intact antibody) sufficient to confer recognition and specific binding to a target such as an antigen and its defined epitope on the antibody fragment. 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") (VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.

[0277] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected by a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0278] A "heavy chain variable region" or "VH" with respect to an antibody (or "VHH" in the case of a single domain antibody such as a Nanobody) refers to the fragment of the heavy chain that contains the three CDRs inserted between flanking extensions called framework regions, which are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0279] Unless otherwise indicated, as used herein, a scFv may have the VL and VH variable regions in either order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0280] 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 ("κ") and lambda ("λ") light chains refer to the two major antibody light chain isotypes.

[0281] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. The term should also be interpreted as meaning an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0282] The terms "Vβ domain", "Vβ domain only", "β chain variable domain", or "single variable domain TCR (svd-TCR)" refer to an antigen binding domain consisting essentially of a single T cell receptor (TCR) β variable domain that specifically binds to an antigen in the absence of a second TCR variable domain. In some embodiments, the first activator LBD comprises or consists essentially of only the Vβ domain. In some embodiments, the second inhibitor LBD comprises or consists essentially of only the Vβ domain.

[0283] In some embodiments, only Vβ domains can include additional elements except TCR variable domains, including additional amino acid sequences, additional protein domains (covalently associated, non-covalently associated or covalently and non-covalently associated with TCR variable domains), fusions or non-covalent associates of TCR variable domains with other types of macromolecules (e.g., polynucleotides, polysaccharides, lipids or combinations thereof), fusions or non-covalent associates of TCR variable domains with one or more small molecules, compounds or ligands, or combinations thereof. As described, any additional elements can be combined, provided that the TCR variable domains are configured to specifically bind epitopes in the absence of a second TCR variable domain.

[0284] In other embodiments, only the Vβ domain as described herein acts independently of the α chain lacking the Vα segment.For example, in some embodiments, the one or more Vβ domains alone are fused with transmembrane domain proteins (e.g., CD3ζ and CD28) and intracellular domain proteins (e.g., CD3ζ, CD28, and / or 4-1BB) that can activate T cells in response to antigens.

[0285] In some embodiments, only the Vβ domain engages the antigen using the complementarity determining regions (CDRs).Each Vβ domain contains three complementarity determining regions (CDR1, CDR2, and CDR3).

[0286] In some embodiments, the first Vβ domain-only comprises a TCR Vβ domain or an antigen-binding fragment thereof.

[0287] In humans, the TCR variable regions of α and γ chains are each encoded by V and J segments, but the variable regions of β and δ chains are each encoded by D segments in addition. There are multiple variable (V), diversity (D) and connection (J) gene segments (such as 52 Vβ gene segments, 2 Dβ gene segments and 13 Jβ gene segments) (Janeway et al. (editor), 2001, Immunobiology: TheImmune System in Health and Disease. The 5th edition, New York, Figure 4.13), which can be used to recognize the enzyme RAG-1 and RAG-2 of the recombination signal sequence (RSS) adjacent to the coding sequence of V, D and J gene segments to reorganize with different V (D) J arrangements. RSS is composed of conservative heptamers and nonamers separated by a spacer of 12 or 23bp. At the 3' side of each V segment, at the 5' and 3' sides of each D segment and at the 5' side of each J segment, RSS is found. During recombination, RAG-1 and RAG-2 cause the formation of DNA hairpins at the coding ends of the ligation products (coding ligation products) and remove the RSS and the intervening sequence between them (signal ligation products). The variable region is further diversified at the junction by deletion of a variable number of coding terminal nucleotides, random addition of nucleotides by terminal deoxynucleotidyl transferase (TdT), and palindromic nucleotides generated by template-mediated filling-in of asymmetrically cleaved coding hairpins.

[0288] Patent application WO 2009 / 129247 (incorporated herein by reference in its entirety) discloses an in vitro system (called the HuTarg system) utilizing V(D)J recombination to generate de novo antibodies in vitro. By using TCR-specific V, D and J elements, the variable region of only the Vβ domain is produced using the same system as in patent application WO 2017 / 091905 (incorporated herein by reference in its entirety). In a natural in vivo system, the nucleic acid sequence encoding CDR1 and CDR2 is contained in the V (α, β, γ or δ) gene segment, and the sequence encoding CDR3 is composed of part of the V and J segments (for Vα or Vγ) or by part of the V segment, the entire D segment and a part of the J segment (for Vβ or Vδ), but due to the action of TdT and other recombination and DNA repair enzymes, there are random insertions and deletions of nucleotides at the VJ and VDJ recombination junctions. The recombinant T cell receptor gene comprises alternating framework (FR) and CDR sequences, as does the resulting T cell receptor expressed thereby (i.e., FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4). Using in vitro V(D)J recombination (i.e., VJ or VDJ recombination), randomized insertions and deletions can be added in or near CDR1, CDR2 and / or CDR3 (i.e., not just CDR3), additional insertions can be added before and / or after CDR1, CDR2 and / or CDR3 using flanking sequences in the recombination substrate, and additional deletions can be made by deleting sequences in the recombination substrate in or near CDR1, CDR2 and / or CDR3.

[0289] In some embodiments, a TCR Vβ chain is identified that specifically binds to an epitope in the absence of a TCR Vα chain. Exemplary CDR3 amino acid sequences that bind to an epitope in the absence of a TCR Vα chain are listed in Table 11 below.

[0290] Table 11. CDR3 amino acid sequences of identified Vβ domains

[0291]

[0292]

[0293] In some embodiments, only the Vβ domain specifically binds to the epitope in the absence of a second TCR variable domain, and is composed of an optional N-terminal and / or C-terminal amino acid sequence (of any length or sequence) flanking the variable domain defined by the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 region. FR1, FR2, FR3, and FR4 can be obtained from natural Vα, Vβ, Vγ, or Vδ domains or encoded by natural Vα, Vβ, Vγ, or Vδ gene segments, but optionally include independently at the C-terminus of FR1, the N-terminus of FR2, the C-terminus of FR2, the N-terminus of FR3, the C-terminus of FR3, and the N-terminus of FR4. One or more amino acids (e.g., 0, 1, 2, 3, 4, 5, or more than 5 amino acids) are deleted or inserted. CDR1, CDR2 and CDR3 can be obtained from natural Vα, Vβ, Vγ or Vδ domains or encoded by natural Vα, Vβ, Vγ or Vδ gene segments, but one or more of CDR1, CDR2 and CDR3 independently contain insertions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 amino acids) and / or deletions (e.g., 0, 1, 2, 3, 4, 5 or more than 5 amino acids) at the C-terminus, N-terminus or anywhere within the CDR sequence. In some embodiments, CDR1 contains insertions or deletions of amino acids at the N-terminus, C-terminus or internally, wherein at least 50% (or optionally 60%, 70% or 80%) of the natural CDR amino acid residues are retained. In some embodiments, CDR2 contains an insertion or deletion of amino acids at the N-terminus, C-terminus or internally, wherein at least 50% (or optionally 60%, 70% or 80%) of the natural CDR amino acid residues are retained. In some embodiments, CDR3 contains an insertion or deletion of amino acids at the N-terminus, C-terminus or internally, wherein at least 50% (or optionally 60%, 70% or 80%) of the natural CDR amino acid residues are retained. Insertion and / or deletion can be due to an in vitro V (D) J recombination method or by TdT and recombinase and DNA repair enzymes (e.g., Artemis nuclease, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end joining factor 1 (NHEJ1), PAXX and DNA polymerase λ and μ in vitro action One or more) is produced. Insertion and / or deletion (which includes substitution) can be further caused by the insertion and / or deletion of the CDR nucleic acid sequence of the in vitro V (D) J recombination substrate. Only the Vβ domain may also comprise a TCR constant region or a portion thereof. Only the Vβ domain may be fused and / or complexed with another protein domain. Double strand breaks may be introduced into the DNA prior to in vitro use of the above-mentioned recombinases and DNA repair enzymes.Only the Vβ domain can be (or can be incorporated into) a fusion protein. As used herein, the term "fusion protein" means a protein encoded by at least one nucleic acid coding sequence, which is composed of a fusion of two or more coding sequences from separate genes, whether the organisms of those genes are the same or different.

[0294] In some embodiments, the first activator LBD comprises a ScFv domain and the second inhibitor LBD comprises only a Vβ domain. In some embodiments, the first activator LBD comprises only a Vβ domain and the second inhibitor LBD comprises a ScFv domain. In some embodiments, both the first activator LBD and the second inhibitor LBD are ScFv domains. In some embodiments, both the first activator LBD and the second inhibitor LBD are only Vβ domains.

[0295] Additional antigen binding domains for use with the activator and / or inhibitor receptors of the present disclosure are described in Table 12 below. In Table 12, the names of the constructs are described as ScFv inhibitor name [B] / ScFv activator name [A]. In some embodiments, the first ligand binding domain or the second ligand binding domain comprises the sequence of any one of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, or SEQ ID NO: 224, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto.

[0296] Table 12. Additional antigen binding domain sequences

[0297]

[0298]

[0299]

[0300] Engineered receptors

[0301] The present disclosure provides a first engineered receptor comprising a first activator ligand binding domain described herein and a second engineered receptor comprising a second inhibitor ligand binding domain.

[0302] Chimeric Antigen Receptor (CAR)

[0303] In some embodiments, the first engineered receptor or the second engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the first engineered receptor and the second engineered receptor are chimeric antigen receptors. All CAR architectures are contemplated to be within the scope of the present disclosure.

[0304] Extracellular domain

[0305] In some embodiments, the first ligand binding domain or the second ligand binding domain is fused to the extracellular domain of the CAR.

[0306] Hinge area

[0307] In some embodiments, the CAR of the present disclosure comprises an extracellular hinge region. The incorporation of the hinge region can affect the cytokine production of CAR-T cells and improve the in vivo expansion of CAR-T cells. Exemplary hinges can be separated from or derived from IgD and CD8 domains, such as IgG1.

[0308] In some embodiments, the hinge is isolated or derived from CD8α or CD28. In some embodiments, the CD8α hinge comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 1). In some embodiments, the CD8α hinge comprises SEQ ID NO: 1. In some embodiments, the CD8α hinge consists essentially of SEQ ID NO: 1. In some embodiments, the CD8α hinge is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence:

[0309] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 2).

[0310] In some embodiments, the CD8α hinge is encoded by SEQ ID NO:2.

[0311] In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 3). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 3. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the sequence of TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 4).

[0312] In some embodiments, the CD28 hinge is encoded by SEQ ID NO:4.

[0313] Transmembrane domain

[0314] The CAR of the present disclosure can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In some embodiments, a transmembrane domain naturally associated with a domain in CAR is used. For example, a CAR comprising a CD28 costimulatory domain can also use a CD28 transmembrane domain. In some cases, the transmembrane domain can be selected or modified by amino acid replacement to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0315] Transmembrane domain can be derived from natural origin or from synthetic origin.In the case where the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein.The membrane-spanning region can be separated from or derived from α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from immunoglobulins such as IgG4 (i.e. at least comprising their membrane-spanning regions). Alternatively, the membrane-spanning domain can be synthetic, in which case it will mainly include hydrophobic residues (such as leucine and valine).In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic membrane-spanning domain.Optionally, a short oligopeptide or polypeptide linker (preferably between 2 and 10 amino acids in length) can be connected between the membrane-spanning domain and the cytoplasmic signaling domain of CAR. Glycine-serine doublets provide particularly suitable linkers.

[0316] In some embodiments of the CAR of the present disclosure, 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 the same sequence as FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 5). In some embodiments, the CD28 transmembrane domain comprises SEQ ID NO: 5 or is substantially composed thereof. 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 the same sequence as the following:

[0317]

[0318] In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO:6.

[0319] In some embodiments of the CAR of the present disclosure, CAR comprises an IL-2Rβ transmembrane domain. In some embodiments, the IL-2Rβ 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 the same sequence as IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 7). In some embodiments, the IL-2Rβ transmembrane domain comprises SEQ ID NO: 7 or is substantially composed thereof. In some embodiments, the IL-2Rβ 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 the same sequence as the following:

[0320]

[0321] In some embodiments, the IL-2Rβ transmembrane domain is encoded by SEQ ID NO:8.

[0322] Cytoplasmic domain

[0323] The cytoplasmic domain or additional intracellular signaling domain of the CAR of the present invention is responsible for activating at least one normal effector function of the immune cell in which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. Regulating the effector function of T cells, for example, includes inhibiting or downregulating the induction or proliferation of effector T cells. Therefore, the term "intracellular signaling domain" refers to a part of a protein that transduces effector function signals and instructs cells to perform specialized functions. Although the entire intracellular signaling domain can generally be used, in many cases, the entire domain does not have to be used. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of a complete chain, as 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. Therefore, the term intracellular signaling domain is intended to include any truncated portion of one or more intracellular signaling domains sufficient to transduce effector function signals.

[0324] Examples of intracellular signaling domains for use in the CARs of the present disclosure include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability.

[0325] Therefore, the intracellular domain of CAR of the present disclosure includes at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures that there is T cell receptor (TCR) signaling necessary for the effector function of activating CAR T cells. In some embodiments, the at least one cytoplasmic activation is CD247 molecule (CD3 ζ) activation domain, stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain or 12kDa DNAX activation protein (DAP12) activation domain. In some embodiments, the CD3 ζ activation domain includes and RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 9) sequence has at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or the same amino acid sequence.

[0326] In some embodiments, the CD3 zeta activation domain comprises or consists essentially of SEQ ID NO: 9. In some embodiments, the CD3 zeta activation domain is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to:

[0327]

[0328] In some embodiments, the CD3 zeta activation domain is encoded by SEQ ID NO:10.

[0329] It is known that the signal generated by the TCR alone is generally insufficient to fully activate T cells and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0330] The primary cytoplasm signaling sequence regulates the primary activation of the TCR complex in an irritating manner or in an inhibitory manner. The primary cytoplasm signaling sequence that works in an irritating manner can contain a signaling motif (which is referred to as an activation motif or ITAM based on immunoreceptor tyrosine). In some embodiments, ITAM contains a tyrosine (YxxL) (SEQ ID NO: 21) separated by any two other amino acids from leucine or isoleucine.

[0331] 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.

[0332] In some embodiments, the cytoplasmic domain is a CD3 zeta activation domain. In some embodiments, the CD3 zeta activation domain comprises a single ITAM. In some embodiments, the CD3 zeta activation domain comprises two ITAMs. In some embodiments, the CD3 zeta activation domain comprises three ITAMs.

[0333] In some embodiments, the CD3 zeta activation domain comprising a single ITAM comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 11). In some embodiments, the CD3 zeta activation domain comprises SEQ ID NO: 11. In some embodiments, the CD3 zeta activation domain comprising a single ITAM consists essentially of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 11). In some embodiments, the CD3 zeta activation domain comprising a single ITAM is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence:

[0334]

[0335] In some embodiments, the CD3 zeta activation domain is encoded by SEQ ID NO:12.

[0336] Additional examples of ITAMs containing primary cytoplasmic signaling sequences that can be used in the CAR of the present disclosure include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, 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 CD3 ζ.

[0337] Costimulatory domain

[0338] In some embodiments, the cytoplasmic domain of the CAR can be designed to include the CD3 ζ signaling domain itself or in combination with any other desired cytoplasmic domain useful in the context of the CAR disclosed herein. For example, the cytoplasmic domain of the CAR can include a CD3 ζ chain portion and a co-stimulatory domain. The co-stimulatory domain refers to a portion of the CAR that includes the intracellular domain of the co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for the effective response of lymphocytes to antigens. Examples of such molecules include costimulatory domains selected from the group consisting 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 receptor superfamily member 4 (OX40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death protein 1 (PD-1), T cell inducible co-stimulatory molecule (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) co-stimulatory domain, or their functional fragments.

[0339] The cytoplasmic domains in the cytoplasmic signaling part of the CAR disclosed herein can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker (e.g., a length between 2 and 10 amino acids) can form a connection. Glycine-serine doublets provide examples of suitable linkers.

[0340] In some embodiments, the intracellular domain of the CAR of the present disclosure comprises at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is separated from or derived from CD28. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or the same as the following sequence:

[0341]

[0342] In some embodiments, the CD28 costimulatory domain comprises or consists essentially of SEQ ID NO: 13. In some embodiments, the CD28 costimulatory domain is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to:

[0343]

[0344] In some embodiments, the CD28 costimulatory domain is encoded by SEQ ID NO:14.

[0345] In some embodiments, the intracellular domain of the CAR of the present disclosure includes an interleukin-2 receptor β chain (IL-2Rβ or IL-2R-β) cytoplasmic domain. In some embodiments, the IL-2Rβ domain is truncated. In some embodiments, the IL-2Rβ cytoplasmic domain includes one or more STAT5 recruitment motifs. In some embodiments, CAR includes one or more STAT5 recruitment motifs outside the IL-2Rβ cytoplasmic domain.

[0346] In some embodiments, the IL-2Rβ intracellular domain comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to:

[0347]

[0348] In some embodiments, the IL2R-β intracellular domain comprises or consists essentially of SEQ ID NO: 15. In some embodiments, the IL-2R-β intracellular domain is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to:

[0349]

[0350] In some embodiments, the IL-2R-β intracellular domain is encoded by SEQ ID NO:16.

[0351] In one embodiment, the IL-2R-β cytoplasmic domain comprises one or more STAT5 recruitment motifs. Exemplary STAT5 recruitment motifs are provided by the following references: Passerini et al. (2008) STAT5-signaling cytokinesregulate 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 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.

[0352] In some embodiments, the STAT5 recruitment motif consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 17).

[0353] Inhibitory domain

[0354] In some embodiments, for example, in the second engineered receptor of the present disclosure providing an inhibitory signal, the inhibitory signal is transmitted by the intracellular domain of the receptor. In some embodiments, the engineered receptor comprises an inhibitory intracellular domain. In some embodiments, the second engineered receptor is a CAR (inhibitory CAR) comprising an inhibitory intracellular domain.

[0355] 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 separated from 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 a key role in attenuating or terminating T cell responses.

[0356] The inhibitory domain can be isolated from human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and CD200 receptor 1.

[0357] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region, or a combination thereof. In some embodiments, the inhibitory domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory domain comprising ITIM can be separated from or derived from immune checkpoint inhibitors, such as CTLA-4 and PD-1.

[0358] The inhibitory domain can 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.

[0359] 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, which can induce TRAIL-dependent apoptosis in target cells after cell-cell contact is established. Physiologically, the TRAIL signaling system has been shown to be essential for immune surveillance, for shaping the immune system by regulating the number of T helper 1 and T helper 2 cells and "unhelper" CD8+ T cells, and for suppressing spontaneous tumor formation.

[0360] In some embodiments, the inhibitory domain comprises an intracellular domain isolated or derived from a CD200 receptor. The cell surface glycoprotein CD200 receptor 1 (Uniprot reference: Q8TD46) represents another example of an inhibitory intracellular domain of the invention. This inhibitory receptor for the CD200 / OX2 cell surface glycoprotein limits inflammation by inhibiting the expression of proinflammatory molecules, including TNF-α, interferons, and inducible nitric oxide synthase (iNOS), in response to selected stimuli.

[0361] In some embodiments, the engineered receptor comprises an inhibitory domain isolated or derived from killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin-like receptor B1 (LIR1, also known as LIR-1 and LILRB1), programmed cell death protein 1 (PD-1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin-like receptor K1 (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, ZAP70 SH2 domain (e.g., one or both of the N- and C-terminal SH2 domains), or ZAP70 KI_K369A (kinase-inactive ZAP70).

[0362] In some embodiments, the inhibitory domain is isolated or derived from a human protein.

[0363] In some embodiments, the second inhibitory receptor comprises a cytoplasmic domain and a transmembrane domain separated 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, separated or derived from the same protein (e.g., an ITIM-containing protein). In some embodiments, the second inhibitory receptor comprises a hinge region separated or derived from the same protein (e.g., an ITIM-containing protein) as the intracellular domain and / or transmembrane domain.

[0364] In some embodiments, the second inhibitory engineered receptor comprises an inhibitory domain. In some embodiments, the second inhibitory engineered 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 with the intracellular domain of CAR. In some embodiments, the inhibitory intracellular domain is fused with the transmembrane domain of CAR.

[0365] T cell receptor (TCR)

[0366] In some embodiments, the first engineered receptor or the second engineered receptor is a T cell receptor (TCR). In some embodiments, the first engineered receptor and the second engineered receptor are T cell receptors (TCR).

[0367] As used herein, "TCR" (sometimes also referred to as "TCR complex" or "TCR / CD3 complex") refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more invariant CD3 chains (ζ, γ, δ, and ε) (sometimes referred to as subunits). The TCR alpha and beta chains can be disulfide-linked to bind to the peptide-MHC complex as heterodimers. Once the TCR alpha / β heterodimer engages the peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing primary stimulation signals. In an exemplary TCR complex, the TCR alpha and TCR beta polypeptides form heterodimers, CD3 ε and CD3 δ form heterodimers, CD3 ε and CD3 γ form heterodimers, and two CD3 ζ form homodimers.

[0368] Extracellular domain

[0369] The present disclosure provides a first engineered receptor comprising a first extracellular ligand binding domain and a second engineered receptor comprising a second extracellular ligand binding domain. The first engineered receptor, the second engineered receptor, or both can be a TCR. Any suitable ligand binding domain can be fused to the extracellular domain, hinge domain, or transmembrane domain of the engineered TCR described herein.

[0370] In some embodiments, the first ligand binding domain and / or the second ligand binding domain are fused to the extracellular domain of the TCR subunit. The TCR subunit can be TCRα, TCRβ, CD3δ, CD3ε or CD3γ. In some embodiments, both the first ligand binding domain and the second ligand binding domain are fused to the same TCR subunit in different TCR receptors. In some embodiments, the first ligand binding domain and the second ligand binding domain are fused to different TCR subunits in different TCR receptors. In some embodiments, the first activator ligand binding domain is fused to the first TCR subunit in the first engineered receptor, and the second inhibitor ligand binding domain is fused to the second TCR subunit in the second engineered receptor. In some embodiments, the first and second TCR subunits are different subunits. In some embodiments, the first and second TCR subunits are the same subunits. For example, the first ligand binding domain can be fused to TCRα, and the second ligand binding domain can be fused to TCRβ. As a further example, the first ligand binding domain is fused to TCRβ and the second ligand binding domain is fused to TCRα.

[0371] In some embodiments, the first activator LBD comprises a ScFv domain and the second inhibitor LBD comprises only a Vβ domain. In some embodiments, the first activator LBD comprises only a Vβ domain and the second inhibitor LBD comprises a ScFv domain. In some embodiments, both the first activator LBD and the second inhibitor LBD are ScFv domains. In some embodiments, both the first activator LBD and the second inhibitor LBD are only Vβ domains.

[0372] In some embodiments, the first engineered TCR of the present disclosure comprises an extracellular domain containing only a Vβ domain, a transmembrane domain, and an intracellular domain. In some embodiments, the intracellular domain comprises one or more exogenous domains.

[0373] In some embodiments, the first engineered TCR of the present disclosure comprises an extracellular domain comprising a ScFv domain, a transmembrane domain, and an intracellular domain. In some embodiments, the intracellular domain comprises one or more exogenous domains.

[0374] In some embodiments, a second engineered TCR of the present disclosure comprises an extracellular domain containing only a Vβ domain, a transmembrane domain, and an inhibitory intracellular domain.

[0375] In some embodiments, the second engineered TCR of the present disclosure comprises an extracellular domain comprising a ScFv domain, a transmembrane domain, and an inhibitory intracellular domain.

[0376] TCR subunits include TCRα, TCRβ, CD3ζ, CD3δ, CD3γ and CD3ε. Any one or more of TCRα, TCRβ chain, CD3γ, CD3δ or CD3ε or their fragments or derivatives can be fused with one or more domains capable of providing the stimulation signal of the present disclosure, thereby enhancing TCR function and activity. Any one or more of TCRα, TCRβ chain, CD3γ, CD3δ or CD3ε or their fragments or derivatives can be fused with the inhibitory intracellular domain of the present disclosure.

[0377] In some embodiments, such as those in which the first engineered receptor or the second engineered receptor comprises a first polypeptide and a second polypeptide, the antigen binding domain is isolated or derived from a T cell receptor (TCR) extracellular domain or an antibody.

[0378] In some embodiments, the first engineered receptor and the second engineered receptor comprise a first antigen binding domain and a second antigen binding domain.The one or more antigen binding domains of the engineered receptors can be provided on the same or different polypeptide as the intracellular domain.

[0379] In some embodiments, the antigen binding domain of the first engineered receptor and / or the second engineered receptor comprises a single chain variable fragment (scFv).

[0380] In some embodiments, the first engineered receptor and / or the second engineered receptor comprises a second polypeptide. The disclosure provides a receptor with two polypeptides, each of which has a portion of a ligand binding domain (e.g., a homologue of a heterodimer LDB (such as a homologue of a LBD based on TCRα / β or Fab)). The disclosure also provides a receptor with two polypeptides, each of which has a portion of a ligand binding domain (e.g., a homologue of a heterodimer LDB (such as a LBD based on TCRα / β or Fab)) and a portion of a ligand binding domain fused to a hinge or transmembrane domain, while another portion of the ligand binding domain does not have an intracellular domain. Additional variations include receptors in which each polypeptide has a hinge domain and each polypeptide has a hinge and a transmembrane domain. In some embodiments, the hinge domain does not exist. In other embodiments, the hinge domain is a membrane-proximal extracellular region (MPER), such as a LILRB1 D3D4 domain.

[0381] In some embodiments, such as those in which the first engineered receptor and / or the second engineered receptor comprises at least two polypeptides, the first polypeptide comprises a first chain of an antibody and the second polypeptide comprises a second chain of the antibody.

[0382] In some embodiments, the receptor comprises a Fab fragment of an antibody. In embodiments, the first polypeptide comprises an antigen binding fragment and an intracellular domain of an antibody heavy chain, and the second polypeptide comprises an antigen binding fragment of an antibody light chain. In some embodiments, the first polypeptide comprises an antigen binding fragment and an intracellular domain of an antibody light chain, and the second polypeptide comprises an antigen binding fragment of an antibody heavy chain.

[0383] In some embodiments, the first engineered receptor and / or the second engineered receptor comprises an extracellular fragment of a T cell receptor (TCR). In some embodiments, the first polypeptide comprises an antigen binding fragment and an intracellular domain of a TCR alpha chain, and the second polypeptide comprises an antigen binding fragment of a TCR beta chain. In some embodiments, the first polypeptide comprises an antigen binding fragment and an intracellular domain of a TCR beta chain, and the second polypeptide comprises an antigen binding fragment of a TCR alpha chain.

[0384] TCR containing only the Vβ domain

[0385] Certain embodiments of the present disclosure relate to engineered TCRs comprising a TCR variable domain that specifically binds an antigen in the absence of a second TCR variable domain (only the Vβ domain).

[0386] In some embodiments, the engineered TCR comprises additional elements in addition to the TCR variable domain, including additional amino acid sequences, additional protein domains (covalently associated, non-covalently associated or covalently and non-covalently associated with the TCR variable domain), fusions or non-covalent associates of the TCR variable domain with other types of macromolecules (e.g., polynucleotides, polysaccharides, lipids or combinations thereof), fusions or non-covalent associates of the TCR variable domain with one or more small molecules, compounds or ligands, or combinations thereof. As described, any additional elements may be combined provided that the TCR variable domain is configured to specifically bind to an epitope in the absence of a second TCR variable domain.

[0387] As described herein, the engineered TCR comprising only Vβ domains can include a single TCR chain (such as α, β, γ or δ chain), or it can include a single TCR variable domain (such as α, β, γ or δ chain). If the engineered TCR is a single TCR chain, the TCR chain includes a transmembrane domain, a constant domain (or C domain) and a variable domain (or V domain), and does not include the second TCR variable domain. Therefore, the engineered TCR can include TCR α chain, TCR β chain, TCR γ chain or TCR δ chain or be composed of it. Engineered TCR can be a membrane-bound protein. Alternatively, the engineered TCR can be a membrane-associated protein.

[0388] In some embodiments, an engineered TCR as described herein utilizes a surrogate α chain lacking a Vα segment that forms an activation-competent TCR in complex with six CD3 subunits.

[0389] In other embodiments, the engineered TCR as described herein works independently of the alternative α chain lacking the Vα segment. For example, in some embodiments, the one or more engineered TCRs are fused with transmembrane domain proteins (e.g., CD3ζ and CD28) and intracellular domain proteins (e.g., CD3ζ, CD28, and / or 4-1BB) that can activate T cells in response to antigens.

[0390] In some embodiments, the engineered TCR comprises one or more single TCR chains fused to only the Vβ domains described herein. For example, the engineered TCR can comprise or be substantially composed of a single αTCR chain, a single βTCR chain, a single γTCR chain or a single δTCR chain fused to only one or more Vβ domains.

[0391] In some embodiments, the engineered TCR uses the complementarity determining regions (CDRs) to engage antigens. Each engineered TCR contains three complementarity determining regions (CDR1, CDR2, and CDR3).

[0392] The first ligand and / or the second ligand only Vβ binding domain can be a human TCR variable domain. Alternatively, the first and / or the second only Vβ domain can be a non-human TCR variable domain. The first and / or the second only Vβ domain can be a mammalian TCR variable domain. The first and / or the second only Vβ domain can be a vertebrate TCR variable domain.

[0393] In an embodiment, wherein only the Vβ domain is incorporated into a fusion protein (e.g., a fusion protein comprising a TCR subunit and optionally an additional stimulatory intracellular domain). A fusion protein may comprise only the Vβ domain and one or more other protein domains.

[0394] Transmembrane domain

[0395] The present disclosure provides a first fusion protein comprising a first activator LBD and a second fusion protein comprising a second inhibitor LBD and an inhibitor intracellular domain. In some embodiments, the first fusion protein and the second fusion protein comprise a transmembrane domain.

[0396] The present disclosure provides polypeptides comprising a transmembrane domain and an intracellular domain capable of providing a stimulatory signal or an inhibitory signal. In some embodiments, the engineered TCR comprises multiple intracellular domains capable of providing a stimulatory signal.

[0397] As used herein, "transmembrane domain" refers to a domain of a protein that spans a cell membrane. A transmembrane domain is typically composed primarily of nonpolar amino acids and may traverse the lipid bilayer once or several times. A transmembrane domain typically contains an alpha helix, a configuration that maximizes internal hydrogen bonding.

[0398] Transmembrane domains isolated or derived from any source are contemplated to be within the scope of the fusion proteins of the present disclosure.

[0399] In some embodiments, the membrane-spanning domain is a membrane-spanning domain associated with a domain in other domains of the fusion protein, or a membrane-spanning domain separated or derived from a protein identical to a domain in other domains of the fusion protein. In some embodiments, the membrane-spanning domain and the second intracellular domain are from the same protein, such as TCR complex subunits, such as TCRα, TCRβ, CD3δ, CD3ε or CD3γ. In some embodiments, the extracellular domain (svd-TCR), the membrane-spanning domain and the second intracellular domain are from the same protein, such as TCR complex subunits, such as TCRα, TCRβ, CD3δ, CD3ε or CD3γ. In other embodiments, the extracellular domain (comprising one or more ligand binding domains, such as only Vβ domains and ScFv domains), the membrane-spanning domain and the intracellular domain are from different proteins. For example, in some embodiments, the engineered svd-TCR includes CD28 membrane-spanning domains and CD28, 4-1BB and CD3ζ intracellular domains.

[0400] The transmembrane domain may be derived from a natural source or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0401] In some embodiments, whenever the TCR complex has bound to a target, the transmembrane domain is capable of conducting a signal to the intracellular domain. The transmembrane domain particularly used in the present invention may include at least, for example, the transmembrane region of the α, β or ζ chain of TCR, CD3δ, CD3ε or CD3γ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0402] In some embodiments, the transmembrane domain can be attached to the extracellular region of the fusion protein, such as the antigen binding domain of the TCR α or β chain, by a hinge (e.g., a hinge from a human protein). For example, in one embodiment, the hinge can be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge or a CD8a hinge.

[0403] In some embodiments, the hinge is isolated or derived from CD8α or CD28. In some embodiments, the CD8α hinge comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 1). In some embodiments, the CD8α hinge comprises SEQ ID NO: 1. In some embodiments, the CD8α hinge consists essentially of SEQ ID NO: 1. In some embodiments, the CD8α hinge is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence:

[0404] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 2).

[0405] In some embodiments, the CD8α hinge is encoded by SEQ ID NO:2.

[0406] In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 3). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 3. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the following sequence:

[0407] TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 4).

[0408] In some embodiments, the CD28 hinge is encoded by SEQ ID NO:4.

[0409] 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 the same identity as the following sequence: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 26). In some embodiments, the TCR alpha transmembrane domain comprises SEQ ID NO: 26 or consists essentially of it. In some embodiments, the TCR alpha transmembrane domain is encoded by the following sequence:

[0410] GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG (SEQ ID NO: 27).

[0411] In some embodiments, the transmembrane domain comprises a TCRβ transmembrane domain. In some embodiments, the TCRβ 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 the same identity as the following sequence: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 28). In some embodiments, the TCRβ transmembrane domain comprises SEQID NO: 28 or consists essentially of it. In some embodiments, the TCRβ transmembrane domain is encoded by the following sequence:

[0412] ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG (SEQ ID NO: 20).

[0413] In some embodiments, the transmembrane domain comprises a CD3 zeta transmembrane domain. In some embodiments, the CD3 zeta transmembrane domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the following sequence: LCYLLDGILFIYGVILTALFL (SEQ ID NO: 29). In some embodiments, the CD3 zeta transmembrane domain comprises or consists essentially of SEQ ID NO: 29.

[0414] The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane region 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).

[0415] In some embodiments, transmembrane domains can 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, thereby minimizing interactions with other members of the receptor complex.

[0416] When present, the transmembrane domain can be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain can be a membrane anchoring domain. Without limitation, a natural or artificial transmembrane domain can comprise a hydrophobic alpha helix of about 20 amino acids, usually with a positive charge on the transmembrane segment flank. The transmembrane domain can have one transmembrane segment or more than one transmembrane segment. The prediction of transmembrane domains / segments can be performed using publicly available prediction tools (e.g., TMHMM, Krogh et al. Journal of Molecular Biology 2001; 305(3): 567-580; or TMpred, Hofmann and Stoffel Biol. Chem. Hoppe-Seyler 1993; 347: 166). Non-limiting examples of membrane anchoring systems include the platelet-derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (post-translationally added to the signal sequence), and the like.

[0417] Intracellular domain

[0418] The present disclosure provides fusion proteins comprising an intracellular domain.The term "intracellular domain" as used herein refers to the intracellular portion of a protein.

[0419] In some embodiments, the intracellular domain comprises one or more domains capable of providing a stimulatory signal to the transmembrane domain. In some embodiments, the intracellular domain comprises a first intracellular domain capable of providing a stimulatory signal and a second intracellular domain capable of providing a stimulatory signal. In other embodiments, the intracellular domain comprises 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, a 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 T cell receptor-associated protein kinase 70 ζ chain (ZAP70) domain, a linker (LAT) domain for activated T cells, a lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) α, TCRβ, CD3δ, CD3γ and CD3ε intracellular domains.

[0420] In some embodiments, the intracellular domain comprises at least one intracellular signaling domain. The intracellular signaling domain generates a signal that promotes cellular function (e.g., immune effector function of a TCR-containing cell (e.g., a T cell expressing a TCR)). In some embodiments, the intracellular domain of the fusion protein of the present disclosure comprises at least one intracellular signaling domain. For example, the intracellular domain of CD3γ, δ, or ε comprises a signaling domain.

[0421] In some embodiments, the extracellular domain, transmembrane domain, and intracellular domain are isolated or derived from the same protein, such as T cell receptor (TCR) α, TCR β, CD3 δ, CD3 γ, or CD3 ε.

[0422] Examples of intracellular domains for use in the fusion proteins of the present disclosure include cytoplasmic sequences of TCRα, TCRβ, CD3ζ and 4-1BB, and intracellular signaling co-receptors that function together to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capabilities.

[0423] 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 stimulation or antigen-dependent stimulation.

[0424] The intracellular signaling domain is generally responsible for activating at least one normal effector function of the immune cell into which the fusion protein has been introduced. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be a cytolytic activity or an auxiliary activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function.

[0425] Although the entire intracellular signaling domain can be used in some cases, it is not necessary to use the entire intracellular signaling domain in many cases. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the complete chain as long as it transduces an effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0426] In some embodiments, the intracellular domain comprises a CD3 delta intracellular domain. In some embodiments, the CD3 delta intracellular domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to:

[0427] GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKGGSRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 30).

[0428] In some embodiments, the CD3 delta intracellular domain comprises or consists essentially of SEQ ID NO: 30. In some embodiments, the CD3 delta intracellular domain is encoded by the following sequence:

[0429]

[0430] In some embodiments, the intracellular domain comprises a CD3 epsilon intracellular domain. In some embodiments, the CD3 epsilon intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or the same as the following sequence: KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGGSRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 32). In some embodiments, the CD3 epsilon intracellular domain comprises SEQ ID NO: 32 or consists essentially of it. In some embodiments, the CD3 epsilon intracellular domain is encoded by the following sequence:

[0431]

[0432] In some embodiments, the intracellular domain comprises a CD3γ intracellular domain. In some embodiments, the CD3γ intracellular domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the following sequence:

[0433] GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNGGSRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 33).

[0434] In some embodiments, the CD3γ intracellular domain comprises or consists essentially of SEQ ID NO: 33. In some embodiments, the CD3γ intracellular domain is encoded by the following sequence:

[0435]

[0436] In some embodiments, the intracellular domain comprises a CD3 zeta intracellular domain. In some embodiments, the CD3 zeta intracellular domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the following sequence:

[0437] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 9) or a subsequence thereof.

[0438] In some embodiments, the CD3 zeta intracellular domain comprises or consists essentially of SEQ ID NO:9.

[0439] 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 a TCCAGC sequence.

[0440] In some embodiments, the intracellular domain comprises a TCRβ intracellular domain. In some embodiments, the TCRβ intracellular domain comprises an amino acid sequence that is at least 80% identical, at least 90% identical, or identical to the following sequence: MAMVKRKDSR (SEQ ID NO: 35). In some embodiments, the TCRβ intracellular domain comprises SEQ ID NO: 35 or consists essentially of it. In some embodiments, the TCRβ intracellular domain is encoded by the following sequence:

[0441] ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO: 36).

[0442] 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 δ, CD3 γ and CD3 ε 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 δ, CD3 γ and CD3 ε intracellular domains, and two additional stimulatory intracellular domains.

[0443] Exemplary costimulatory intracellular signaling domains include those derived from proteins responsible for costimulatory signals or antigen-independent stimulation.

[0444] The term "costimulatory molecule" refers to a cognate binding partner on a T cell, which specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of a T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors. Costimulatory molecules and their ligands are necessary for an effective immune response. Costimulatory molecules include, but are not limited to, MHC 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).

[0445] A "costimulatory domain" (sometimes referred to as a "costimulatory intracellular signaling domain") can be an intracellular portion of a costimulatory protein. A costimulatory domain can be a domain that transduces a costimulatory signal of a costimulatory protein. Costimulatory proteins can represent the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activated NK cell 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, specifically binding to a ligand of CD83, CD4, etc. A costimulatory domain can include a complete intracellular portion of a molecule derived therefrom or a complete native intracellular signaling domain, or a functional fragment thereof.

[0446] In some embodiments, the stimulatory domain includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain includes a CD28 or 4-1BB co-stimulatory domain. CD28 and 4-1BB are fully characterized co-stimulatory molecules necessary for complete T cell activation and known to enhance T cell effector functions. For example, CD28 and 4-1BB have been used in chimeric antigen receptors (CAR) to promote cytokine release, cytolytic function and the persistence of the first generation CAR containing only CD3ζ signaling domains. Similarly, the inclusion of co-stimulatory domains (such as CD28 and 4-1BB domains) in engineered TCR can increase T cell effector functions, and specifically allow co-stimulation in the absence of co-stimulatory ligands, which are usually down-regulated on the surface of tumor cells.

[0447] In some embodiments, the stimulatory domain comprises a CD28 intracellular domain. In some embodiments, the CD28 intracellular domain comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to the following sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 37). In some embodiments, the CD28 intracellular domain comprises or consists essentially of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 37). In some embodiments, the CD28 intracellular domain is encoded by a nucleotide sequence comprising:

[0448] AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC (SEQ ID NO: 38).

[0449] In some embodiments, the stimulatory domain comprises a 4-1BB intracellular domain. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or the same as the following sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 39). In some embodiments, the 4-1BB intracellular domain comprises KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 39) or is substantially composed thereof. In some embodiments, the 4-1BB intracellular domain is encoded by a nucleotide sequence comprising: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGGCCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 40).

[0450] Inhibitory domain

[0451] The present invention provides inhibitory intracellular domains that can be fused to the transmembrane domain or intracellular domain of any TCR subunit to generate an inhibitory TCR.

[0452] 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 separated from 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 a key role in attenuating or terminating T cell responses.

[0453] The inhibitory domain can be isolated from human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and CD200 receptor 1.

[0454] In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, or a combination thereof. In some embodiments, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region, or a combination thereof. In some embodiments, the inhibitory domain comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the inhibitory domain comprising ITIM can be separated from or derived from immune checkpoint inhibitors, such as CTLA-4 and PD-1.

[0455] The inhibitory domain can 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.

[0456] 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, which can induce TRAIL-dependent apoptosis in target cells after cell-cell contact is established. Physiologically, the TRAIL signaling system has been shown to be essential for immune surveillance, for shaping the immune system by regulating the number of T helper 1 and T helper 2 cells and "unhelper" CD8+ T cells, and for suppressing spontaneous tumor formation.

[0457] In some embodiments, the inhibitory domain comprises an intracellular domain isolated or derived from a CD200 receptor. The cell surface glycoprotein CD200 receptor 1 (Uniprot reference: Q8TD46) represents another example of an inhibitory intracellular domain of the invention. This inhibitory receptor for the CD200 / OX2 cell surface glycoprotein limits inflammation by inhibiting the expression of proinflammatory molecules, including TNF-α, interferons, and inducible nitric oxide synthase (iNOS), in response to selected stimuli.

[0458] In some embodiments, the engineered receptor comprises an inhibitory domain isolated or derived from a killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), a killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), a leukocyte immunoglobulin-like receptor B1 (LIR1), programmed cell death protein 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 ZAP70KI_K369A (kinase-inactive ZAP70).

[0459] In some embodiments, the inhibitory domain is isolated or derived from a human protein.

[0460] In some embodiments, the second inhibitory receptor comprises a cytoplasmic domain and a transmembrane domain separated 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, separated or derived from the same protein (e.g., an ITIM-containing protein). In some embodiments, the second inhibitory receptor comprises a hinge region separated or derived from the same protein (e.g., an ITIM-containing protein) as the intracellular domain and / or transmembrane domain.

[0461] In some embodiments, the second engineered receptor is a TCR (inhibitory TCR) comprising an inhibitory domain. In some embodiments, the inhibitory TCR comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory intracellular domain is fused with an intracellular domain of TCRα, TCRβ, CD3δ, CD3γ or CD3ε or a portion of TCR thereof. In some embodiments, the inhibitory intracellular domain is fused with a transmembrane domain of TCRα, TCRβ, CD3δ, CD3γ or CD3ε.

[0462] In some embodiments, the second engineered receptor is a TCR comprising an inhibitory domain (inhibitory TCR). In some embodiments, the inhibitory domain is isolated or derived from LILRB1.

[0463] LILRB1 inhibitory receptor

[0464] The present disclosure provides a second inhibitory receptor comprising a LILRB1 inhibitory domain and optionally a LILRB1 transmembrane and / or hinge domain or a functional variant thereof. Inclusion of a LILRB1 transmembrane domain and / or a LILRB1 hinge domain in an inhibitory receptor can increase the inhibitory signal generated by the inhibitory receptor compared to a reference inhibitory receptor having another transmembrane domain or another hinge domain. The second inhibitory receptor comprising a LILRB1 inhibitory domain can be a CAR or a TCR, as described herein. As described herein, any suitable ligand binding domain can be fused to a second inhibitory receptor based on LILRB1.

[0465] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) (also known as leukocyte immunoglobulin-like receptor B1), as well as ILT2, LIR1, MIR7, PIRB, CD85J, ILT-2 LIR-1, MIR-7 and PIR-B are members of the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1 protein belongs to the subfamily B class of LIR receptors. These receptors contain two to four extracellular immunoglobulin domains, a transmembrane domain and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The LILRB1 receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen presenting cells and transduces negative signals that inhibit stimulation of the immune response. LILRB1 is believed to regulate inflammatory responses as well as cytotoxicity, and plays a role in limiting autoreactivity. There are multiple transcript variants encoding different subtypes of LILRB1, all of which are considered to be within the scope of the present disclosure.

[0466] 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, the one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to the sequence or subsequence of SEQ ID NO: 65. In some embodiments, the one or more domains of LILRB1 comprise an amino acid sequence that is identical to the sequence or subsequence of SEQ ID NO: 65. In some embodiments, the one or more domains of LILRB1 consist of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to the sequence or subsequence of SEQ ID NO: 65. In some embodiments, the one or more domains of LILRB1 consist of an amino acid sequence that is identical to the sequence or subsequence of SEQ ID NO: 65.

[0467] In some embodiments of a receptor having one or more domains isolated or derived from LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to the sequence or a subsequence of SEQ ID NO:66.

[0468] In some embodiments of the receptor having one or more domains of LILRB1, the one or more domains of LILRB1 are encoded by a polynucleotide sequence that is identical to the sequence or a subsequence of SEQ ID NO:66.

[0469] In various embodiments, an inhibitory receptor comprising a polypeptide is provided, wherein the polypeptide comprises one or more of the following: 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 inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69) and SIYATL (SEQ ID NO: 70).

[0470] As used herein, "immunoreceptor tyrosine-based inhibitory motif" or "ITIM" refers to a conserved sequence of amino acids having the consensus sequence S / I / V / LxYxxI / V / L (SEQ ID NO: 274), etc., present in the cytoplasmic tails of many inhibitory receptors of the immune system. After an inhibitory receptor possessing an ITIM interacts with its ligand, the ITIM motif is phosphorylated, allowing the inhibitory receptor to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2 or the inositol phosphatase known as SHIP.

[0471] 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 3 ITIMs, at least 4 ITIMs, at least 5 ITIMs, or at least 6 ITIMs. In some embodiments, the intracellular domain has 1, 2, 3, 4, 5, or 6 ITIMs.

[0472] In some embodiments, the polypeptide comprises an intracellular domain comprising at least one ITIM selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69), and SIYATL (SEQ ID NO:70).

[0473] In another specific embodiment, the polypeptide comprises an intracellular domain comprising at least two immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69) and SIYATL (SEQ ID NO:70).

[0474] In some embodiments, the intracellular domain comprises two ITIMs, NLYAAV (SEQ ID NO: 67) and VTYAEV (SEQ ID NO: 68). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO: 71. In some embodiments, the intracellular domain comprises or consists essentially of a sequence that is identical to SEQ ID NO: 71.

[0475] In some embodiments, the intracellular domain comprises two ITIMs, VTYAEV (SEQ ID NO: 68) and VTYAQL (SEQ ID NO: 69). In some embodiments, the intracellular domain comprises a sequence that is at least 95% identical to SEQ ID NO: 72. In some embodiments, the intracellular domain comprises or consists essentially of a sequence that is identical to SEQ ID NO: 72.

[0476] In some embodiments, the intracellular domain comprises two ITIMs, VTYAQL (SEQ ID NO: 69) and SIYATL (SEQ ID NO: 70). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 73. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 73.

[0477] In some embodiments, the intracellular domain comprises the following ITIMs: NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), and VTYAQL (SEQ ID NO: 69). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 74. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 74.

[0478] In some embodiments, the intracellular domain comprises the following ITIMs: VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70). In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 75. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 75.

[0479] In some embodiments, the intracellular domain comprises the following ITIMs: NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69), and SIYATL (SEQ ID NO: 70). In embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NO: 76. In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to SEQ ID NO: 76.

[0480] In some embodiments, the intracellular domain comprises a sequence at least 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 81).In some embodiments, the intracellular domain comprises or consists essentially of a sequence identical to the LILRB1 intracellular domain (SEQ ID NO: 81).

[0481] The LILRB1 intracellular domain or a functional variant thereof disclosed herein may 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 a functional variant thereof has 2, 3, 4, 5 or 6 ITIMs.

[0482] In specific embodiments, the intracellular domain comprises two, three, four, five or six immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69) and SIYATL (SEQ ID NO:70).

[0483] In a specific embodiment, the intracellular domain comprises an intracellular domain of at least three immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69) and SIYATL (SEQ ID NO:70).

[0484] In a specific embodiment, the intracellular domain comprises three immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69) and SIYATL (SEQ ID NO:70).

[0485] In a specific embodiment, the intracellular domain comprises four immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69) and SIYATL (SEQ ID NO:70).

[0486] In a specific embodiment, the intracellular domain comprises five immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69) and SIYATL (SEQ ID NO:70).

[0487] In a specific embodiment, the intracellular domain comprises six immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69) and SIYATL (SEQ ID NO:70).

[0488] In a specific embodiment, the intracellular domain comprises at least 7 immunoreceptor tyrosine-based inhibition motifs (ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ ID NO:67), VTYAEV (SEQ ID NO:68), VTYAQL (SEQ ID NO:69) and SIYATL (SEQ ID NO:70).

[0489] The LILRB1 protein has four immunoglobulin (Ig)-like domains called D1, D2, D3, and D4. In some embodiments, the LILRB1 hinge domain comprises the 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% or identical to SEQ ID NO: 77. In some embodiments, the LILRB1 D3D4 domain comprises or consists essentially of SEQ ID NO: 77.

[0490] 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 or identical to SEQ ID NO:84, SEQ ID NO:77 or SEQ ID NO:78. 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:84, SEQ ID NO:77 or SEQ ID NO:78.

[0491] In some embodiments, the LILRB1 hinge domain comprises a sequence identical to SEQ ID NO:84, SEQ ID NO:77, or SEQ ID NO:78.

[0492] In some embodiments, the LILRB1 hinge domain consists essentially of a sequence identical to SEQ ID NO:84, SEQ ID NO:77, or SEQ ID NO:78.

[0493] In some embodiments, the transmembrane domain is a LILRB1 transmembrane domain or a functional variant thereof. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is 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:85. In some embodiments, the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO:85. In some embodiments, the LILRB1 transmembrane domain comprises a sequence that is identical to SEQ ID NO:85. In an embodiment, the LILRB1 transmembrane domain consists essentially of a sequence that is identical to SEQ ID NO:85.

[0494] In some embodiments, the transmembrane domain can be attached to the extracellular region of the second inhibitory receptor, such as an antigen binding domain or a ligand binding domain, by a hinge (e.g., a hinge from a human protein). For example, in some embodiments, the hinge can be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge, a CD8a hinge, or a LILRB1 hinge.

[0495] In some embodiments, the second inhibitory receptor comprises an inhibitory domain. In some embodiments, the second inhibitory receptor comprises an inhibitory intracellular domain and / or an inhibitory transmembrane domain. In some embodiments, the inhibitory domain is isolated or derived from LILR1B.

[0496] Combination of inhibitory receptors containing LILRB1 domains

[0497] In some embodiments, the LILRB1-based inhibitory receptors of the present disclosure comprise more than one LILRB1 domain or a functional equivalent thereof. For example, in some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and an intracellular domain, or a LILRB1 hinge domain, a transmembrane domain, and an intracellular domain.

[0498] In certain embodiments, the inhibitory receptor 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: 79. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 79. In some embodiments, the polypeptide comprises a sequence that is identical to SEQ ID NO: 79.

[0499] In another embodiment, the inhibitory receptor comprises: a LILRB1 transmembrane domain or a functional variant thereof, and a LILRB1 intracellular domain and / or an intracellular domain comprising at least one immunoreceptor tyrosine-based inhibition motif (ITIM), wherein the ITIM is selected from NLYAAV (SEQ ID NO: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69) and SIYATL (SEQ ID NO: 70). 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: 67), VTYAEV (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69) and SIYATL (SEQ ID NO: 70).

[0500] In some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and an intracellular domain. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or identical to SEQ ID NO: 80. In some embodiments, the polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 80. In some embodiments, the polypeptide comprises a sequence that is identical to SEQ ID NO: 80. In some embodiments, the inhibitory receptor comprises a LILRB1 transmembrane domain and an intracellular domain of SEQ ID NO: 80 fused to an extracellular ligand binding domain. In some embodiments, the inhibitory receptor comprises a first polypeptide containing SEQ ID NO: 80 fused to a TCR alpha variable domain and a second polypeptide containing SEQ ID NO: 80 fused to a TCR beta variable domain.

[0501] In a preferred embodiment, the inhibitory receptor 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), wherein each ITIM is independently selected from LYAAV (SEQ ID NO: 67), VTYAE (SEQ ID NO: 68), VTYAQL (SEQ ID NO: 69) and SIYATL (SEQ ID NO: 11).

[0502] In some embodiments, the inhibitory receptor comprises a sequence that is at least 95% identical to SEQ ID NO:82 or SEQ ID NO:83, or at least 99% identical to SEQ ID NO:82 or SEQ ID NO:83, or identical to SEQ ID NO:82 or SEQ ID NO:83.

[0503] In some embodiments, the polypeptide comprises a sequence that is at least 99% identical to SEQ ID NO:79, or at least 99% identical to SEQ ID NO:79, or identical to SEQ ID NO:79.

[0504] In some embodiments, the polypeptide comprises a sequence that is at least 99% identical to SEQ ID NO:80, or at least 99% identical to SEQ ID NO:80, or identical to SEQ ID NO:80.

[0505] Table 13. Polypeptide sequences of illustrative inhibitory receptors based on LILRB1

[0506]

[0507]

[0508] Connectors

[0509] In some embodiments, the engineered receptor comprises a linker connecting two domains of the engineered receptor. Provided herein are linkers that, in some embodiments, can be used to connect the domains of the engineered receptors described herein.

[0510] The terms "linker" and "flexible polypeptide linker" as used in the context of connecting protein domains (e.g., an intracellular domain or a domain within a scFv) refer to a peptide linker composed of amino acids (such as glycine and / or serine residues), used alone or in combination, that connects two domains together.

[0511] Any linker can be used, and many fusion protein linker formats are known. For example, the linker can be flexible or rigid. Chen et al. (Adv Drug Deliv Rev. 2013; 65(10): 1357-1369) provide non-limiting examples of rigid and flexible linkers.

[0512] The antigen binding domains described herein can be linked to each other in a random or specific order.

[0513] The antigen binding domains described herein may be linked to each other in any orientation from N to C terminus.

[0514] Optionally, a short oligo- or polypeptide linker, for example between 2 and 40 amino acids in length (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids), may form the connection between the domains.

[0515] In some embodiments, the linker is a peptide of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 amino acid residues. Non-limiting examples of amino acids present in the linker include Gly, Ser, Glu, Gin, Ala, Leu, Iso, Lys, Arg, Pro, etc. In some embodiments, the linker is [(Gly)n1Ser]n2, wherein n1 and n2 can be any number (e.g., n1 and n2 can be independently 1, 2, 4, 5, 6, 7, 8, 9, 10 or greater than 10). In some embodiments, n1 is 4.

[0516] In some embodiments, the flexible polypeptide linker is a Gly / Ser linker and comprises an amino acid sequence (Gly-Gly-Ser), (Gly-Gly-Gly-Ser, SEQ ID NO: 231), or (Gly-Gly-Gly-Gly-Ser, SEQ ID NO: 226) which may be repeated n times, wherein n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, and n=10. In some embodiments, the flexible polypeptide linker includes, but is not limited to, GGS, GGGGS (SEQ ID NO: 226), GGGGS GGGGS (SEQ ID NO: 227), GGGGS GGGGS GGGGS (SEQ ID NO: 228), GGGGSGGGGS GGGGS GG (SEQ ID NO: 229), or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 230).

[0517] In some embodiments, the linker includes multiple repeats of (Gly Gly Ser), (Gly Ser), or (Gly Gly Gly Ser (SEQ ID NO: 231)). The linkers described in WO2012 / 138475 (incorporated herein by reference) are also included within the scope of the present invention.

[0518] In some embodiments, the linker sequence comprises a long linker (LL) sequence. In some embodiments, the long linker sequence comprises GGGGS (SEQ ID NO: 226) repeated four times. In some embodiments, GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 230) is used to connect the intracellular domain in the TCR alpha fusion protein of the present disclosure.

[0519] In some embodiments, the long linker sequence comprises three repetitions of GGGGS (SEQ ID NO: 226). In some embodiments, GGGGS GGGGS GGGGS (SEQ ID NO: 228) is used to link the intracellular domains in the TCRβ fusion proteins of the present disclosure.

[0520] In some embodiments, the linker sequence comprises a short linker (SL) sequence. In some embodiments, the short linker sequence comprises GGGGS (SEQ ID NO: 226).

[0521] In some embodiments, glycine-serine doublets may be used as suitable linkers.

[0522] In some embodiments, the domains are fused directly to each other via peptide bonds without the use of a linker.

[0523] Determination

[0524] Provided herein are assays that can be used to measure the activity of the engineered receptors of the disclosure.

[0525] The activity of engineered receptors can be measured using cell lines engineered to express receptor activity reporters (such as luciferase reporters). Exemplary cell lines include Jurkat T cells, but any suitable cell line known in the art can be used. For example, Jurkat cells expressing luciferase reporters under the control of the NFAT promoter can be used as effector cells. The expression of luciferase by this cell line reflects the signal transduction mediated by TCR.

[0526] Reporter cells can be transfected with each of the various fusion protein constructs, combinations of fusion protein constructs, or controls described herein.

[0527] The expression of the fusion protein in the reporter cells can be confirmed by detecting the expression of the fusion protein using a fluorescently labeled MHC tetramer (eg, Alexa Fluor 647 labeled NY-ESO-1-MHC tetramer).

[0528] In order to measure the activity of engineered receptors, target cells are loaded with antigens before being exposed to effector cells comprising reporters and engineered receptors. For example, target cells can be loaded with antigens at least 12, 14, 16, 18, 20, 22 or 24 hours before being exposed 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 antigens at serial dilution concentrations, such as NY-ESO-1 peptides. Effector cells can then be co-cultured with target cells for a suitable time period, such as 6 hours. Luciferase is then measured by luminescence readings after co-cultivation. Luciferase luminescence can be normalized to maximum and minimum intensities to allow comparison of the activation peptide concentrations of each engineered receptor construct.

[0529] Provided herein are methods for determining the relative EC50 of the engineered receptors disclosed herein. As used herein, "EC50" refers to the concentration of an inhibitor or agent at which the response (or binding) is reduced by half. The EC50 of the engineered receptor disclosed herein refers to the concentration of antigen at which the binding of the engineered receptor to the antigen is reduced by half. The binding of the antigen or probe to the engineered receptor can be measured by staining with a labeled peptide or a labeled peptide-MHC complex (e.g., MHC:NY-ESO-1pMHC complex conjugated to a fluorophore). EC50 can be obtained by nonlinear regression curve fitting of the reporter signal and peptide titration. Probe binding and EC50 can be normalized to the level of a benchmark TCR (e.g., NY-ESO-1 (clone 1G4)) without a fusion protein.

[0530] Polynucleotide

[0531] The present disclosure provides polynucleotides encoding the sequences of the engineered receptors described herein.

[0532] In some embodiments, the sequence of the first fusion protein and / or the second fusion protein is operably linked to a promoter. In some embodiments, the sequence encoding the first fusion protein is operably linked to a first promoter, and the sequence encoding the second fusion protein is operably linked to a second promoter.

[0533] The present disclosure provides vectors comprising the polynucleotides described herein.

[0534] The present disclosure provides a vector encoding one or more coding sequences of any engineered receptor described herein. In some embodiments, the sequence of the first fusion protein and / or the second fusion protein is operably connected to a promoter. In some embodiments, the sequence encoding the first fusion protein is operably connected to the first promoter, and the sequence encoding the second fusion protein is operably connected to the second promoter.

[0535] In some embodiments, the first engineered receptor is encoded by a first vector and the second engineered receptor is encoded by a second vector. In some embodiments, both engineered receptors are encoded by a single vector.

[0536] In some embodiments, the first receptor and the second receptor are encoded by a single vector. The method of using a single vector to encode multiple polypeptides will be known to those of ordinary skill in the art, and in particular includes encoding multiple polypeptides under the control of different promoters, or if a single promoter is used to control the transcription of multiple polypeptides, using a sequence encoding an internal ribosome entry site (IRES) and / or a self-cleaving peptide. Exemplary self-cleaving peptides include T2A, P2A, E2A and F2A self-cleaving peptides. In some embodiments, the T2A self-cleaving peptide includes the sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 271). In some embodiments, the P2A self-cleaving peptide includes the sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 192). In some embodiments, the E2A self-cleaving peptide includes the sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 272). In some embodiments, the F2A self-cleaving peptide includes the sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 273).

[0537] In some embodiments, the vector is an expression vector, ie, used to express the fusion protein in a suitable cell.

[0538] 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 transmission in daughter cells. Lentiviral vectors have the added advantage over vectors derived from oncorretroviruses (such as murine leukemia viruses) because they can transduce non-proliferating cells (such as hepatocytes). They also have the added advantage of low immunogenicity.

[0539] The expression of natural or synthetic nucleic acids encoding fusion proteins is usually 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 replication and integration in eukaryotic organisms. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0540] The polynucleotide encoding the fusion protein can be cloned into many 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.

[0541] In addition, expression vectors can be provided to cells (such as immune cells) in the form of viral vectors. Viral vector technology is well known in the art, and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally speaking, suitable vectors contain at least one in vivo replication function starting point, promoter sequence, convenient restriction endonuclease site and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193).

[0542] Multiple virus-based systems have been developed to transfer genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0543] Other promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these are located in the region of 30-110 base pairs (bp) upstream of the start site, but recently it has been shown that many promoters also contain functional elements downstream of the start site. The intervals between promoter elements are often flexible, so when the elements are reversed or moved relative to each other, promoter function is retained. In the thymidine kinase (tk) promoter, the intervals between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it seems that individual elements can work collaboratively or independently to activate transcription.

[0544] 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 the high-level expression of any polynucleotide sequence operably connected thereto. Another example of a suitable promoter is elongation growth factor 1 alpha (EF-1 alpha). 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. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as a part of the present invention. The use of inducible promoters provides a molecular switch that can open the expression of its operably connected polynucleotide sequence when such expression is desired, or closes the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0545] In order to evaluate the expression of fusion protein, the expression vector to be introduced into the cell can also contain a selectable marker gene or a reporter gene or both, to facilitate identification and selection of expressing cells from a cell population sought to be transfected or infected by a viral vector. In other aspects, a selectable marker can be carried on a separate DNA fragment and used in a cotransfection procedure. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to be able to be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo, etc.

[0546] Reporter gene is used to identify cells of potential transfection or transduction and to evaluate the function of regulatory sequences. Generally, reporter gene is not present in recipient organism or tissue or is not expressed by recipient organism or tissue and encodes a gene of a polypeptide whose expression is performed by some easily detectable characteristics (e.g., enzymatic activity). After DNA is introduced into recipient cells, the expression of reporter gene is determined at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared or commercially available using known techniques. Generally, the minimum 5′ flanking region showing the highest reporter gene expression level in the construct is identified as a promoter. Such promoter region can be connected to a reporter gene and used to evaluate the ability of the transcription driven by the regulating promoter of the medicament.

[0547] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical or biological means.

[0548] 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.

[0549] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors and especially retroviral vectors have become the most widely used methods for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from slow viruses, 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.

[0550] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0551] Regardless of the method used to introduce exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the invention, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0552] Immune cells

[0553] Provided herein are immune cells comprising the polynucleotides, vectors, fusion proteins, and engineered receptors described herein.

[0554] As used herein, the term "immune cell" refers to a cell that participates in the innate or adaptive (acquired) immune system. Exemplary innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, natural killer (NK) cells, polymorphonuclear leukocytes (such as neutrophils, eosinophils and basophils) and mononuclear cells (such as monocytes, macrophages and mast cells). Immune cells that work in acquired immunity include lymphocytes such as T cells and B cells.

[0555] As used herein, "T cell" refers to a type of lymphocyte derived from a bone marrow precursor that develops in the thymus. There are several different types of T cells that develop after migrating 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 ordinary technicians based on the expression of their markers. Methods for distinguishing between T cell types will be apparent to ordinary technicians.

[0556] In some embodiments, the engineered immune cells express the first receptor and the second receptor at a first receptor to second receptor ratio of about 100: 1 to 1: 100. In some embodiments, the engineered immune cells express the first receptor and the second receptor at a first receptor to second receptor ratio of about 50: 1 to 1: 50. In some embodiments, the engineered immune cells express the first receptor and the second receptor at a first receptor to second receptor ratio of about 10: 1 to 1: 10. In some embodiments, the engineered immune cells express the first receptor and the second receptor at a first receptor to second receptor ratio of about 5: 1 to 1: 5. In some embodiments, the engineered immune cells express the first receptor and the second receptor at a first receptor to second receptor ratio of about 3: 1 to 1: 3. In some embodiments, the engineered immune cells express the first receptor and the second receptor at a first receptor to second receptor ratio of about 2: 1 to 1: 2. In some embodiments, the engineered immune cells express the first receptor and the second receptor at a ratio of about 1: 1.

[0557] In some embodiments, the engineered immune cell comprising an engineered receptor of the present disclosure is a T cell. In some embodiments, the T cell is an effector T cell or a regulatory T cell.

[0558] The method of transforming immune cell (such as T cell) group with the carrier of the present disclosure is obvious to those of ordinary skill in the art.For example, CD3+ T cells can be separated from PBMC using CD3+ T cell negative separation kit (Miltenyi) according to the manufacturer's instructions.In the presence of CD3 / 28Dynabeads (1:1 cell to bead ratio) and 300 units / mL of IL-2 (Miltenyi), in the X-Vivo15 culture medium supplemented with 5% human A / B serum and 1% Pen / strep, T cells are cultured at a density of 1x 10^6 cells / mL.After 2 days, viral vectors such as lentiviral vectors can be used to transduce T cells using methods known in the art.In some embodiments, viral vectors are transduced with a multiplicity of infection (MOI) of 5.Then cells are cultured for another 5 days in a combination of IL-2 or other cytokines such as IL-7 / 15 / 21 before enrichment.It will be obvious to those of ordinary skill in the art to separate and culture other immune cell (such as B cell) groups or other T cell groups. Although this method outlines potential approaches, it should be noted that these methods are rapidly evolving. For example, excellent viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of growth to produce a >99% CD3+ highly transduced cell population.

[0559] Methods of activating and culturing T cell populations comprising the engineered TCRs, CARs, fusion proteins, or vectors encoding fusion proteins of the present disclosure will be apparent to those of ordinary skill in the art.

[0560] Whether before or after the T cells are genetically modified to express an engineered TCR, T cells can generally be activated and expanded using methods such as those described in 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; 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.

[0561] In some embodiments, the T cells of the present disclosure are expanded and activated in vitro. Generally, the T cells of the present disclosure are expanded in vitro by contacting with a surface to which an agent stimulating CD3 / TCR complex-related signals and a ligand stimulating a co-stimulatory molecule on the surface of the T cell is attached. In particular, a T cell population can be stimulated as described herein, such as by contact with an anti-CD3 antibody. In order to co-stimulate auxiliary molecules on the surface of T cells, a ligand that binds to the auxiliary molecule is used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. In order to stimulate the proliferation of CD4+ T cells or CD8+ T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, France) (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).

[0562] In some embodiments, the primary stimulation signal and costimulatory signal of T cell can be provided by different schemes.For example, the agent providing each signal can be in solution or coupled to the surface.When coupled to the surface, the agent can be coupled to the same surface (that is, in "cis" form) or to the surface separated (that is, in "trans" form).Alternately, a kind of agent can be coupled to the surface and another agent in solution.In some embodiments, the agent providing the costimulatory signal is combined with the cell surface, and the agent providing the primary activation signal is in solution or coupled to the surface.In certain embodiments, both agents can be in solution.In another embodiment, the agent can be in soluble form, and then cross-linked to the surface, such as cells or antibodies expressing Fc receptors or other binding agents that will be combined with the agent.Regarding this, see, for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for activating and amplifying artificial antigen presenting cells (aAPC) of T cells of the present invention.

[0563] In some embodiments, two agents are immobilized on beads, either on the same bead (i.e., "cis") or on separate beads (i.e., "trans"). For example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and the two agents are co-immobilized to the same bead with equal molecular weight. In one embodiment, a 1:1 ratio of each antibody bound to the beads is used for CD4+T cell expansion and T cell growth. In some embodiments, the ratio of CD3:CD28 antibodies bound to beads is in the range of 100:1 to 1:100 and all integer values ​​therebetween. In one aspect of the invention, there are more anti-CD28 antibodies 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 beads is greater than 2:1.

[0564] The ratio of particles to cells in the range of 1:500 to 500:1 and any integer value therebetween can be used to stimulate T cells or other target cells. As can be readily appreciated by those of ordinary skill in the art, the ratio of particles to cells may depend on the particle size relative to the target cells. For example, beads of small size can only bind to a few cells, while larger beads can bind to many cells. In certain embodiments, the ratio of cells to particles in the range of 1:100 to 100:1 and any integer value therebetween and in other embodiments, the ratio of cells to particles including 1:9 to 9:1 and any integer value therebetween can also be used to stimulate T cells. In some embodiments, a ratio of cells to beads of 1:1 is used. It will be appreciated by those skilled in the art that a variety of other ratios may be suitable for use in the present invention. In particular, the ratio will vary according to particle size and cell size and type.

[0565] In another embodiment of the present invention, cells (such as T cells) are combined with the beads coated with the agent, the beads and cells are subsequently separated, and the cells are then cultured. In an alternative embodiment, the beads and cells coated with the agent are not separated before culture, but cultured together. In another embodiment, the beads and cells are first concentrated by applying a force (such as a magnetic force), resulting in an increase in the connection of cell surface markers, thereby inducing cell stimulation.

[0566] For example, cell surface proteins can be connected by allowing paramagnetic beads attached with anti-CD3 and anti-CD28 to contact T cells. In one embodiment, cells (e.g., CD4+ T cells) and beads (e.g., DYNABEADS CD3 / CD28 T paramagnetic beads in a ratio of 1:1) are combined in a buffer. Similarly, it will be readily appreciated by those of ordinary skill in the art that any cell concentration can be used. In certain embodiments, it may be desirable to significantly reduce the volume in which particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In other embodiments, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In other embodiments, a concentration of 125 or 150 million cells / ml may be used. In some embodiments, a concentration of 1 x 10 6 The cells were cultured at a density of 10 cells / mL.

[0567] In some embodiments, the mixture can be cultured for several hours (about 3 hours) to about 14 days or any hour integer value therebetween. In another embodiment, beads and T cells are cultured together for 2-3 days. Conditions suitable for T cell culture include appropriate culture medium (for example, minimum essential medium (Minimal Essential Media) or RPMI culture medium 1640 or X-vivo 15 (Lonza)), which can contain factors necessary for proliferation and viability (viability), including serum (for example, fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other additive for cell growth known to those skilled in the art. Other additives for cell growth include but are not limited to surfactant, human plasma protein powder (plasmanate) and reducing agent such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, which is supplemented with amino acids, sodium pyruvate and vitamins, is serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or a certain amount of cytokines sufficient to grow and expand T cells. In some embodiments, the culture medium includes X-VIVO-15 culture medium supplemented with 5% human A / B serum, 1% penicillin / strep and 300 units / ml IL-2 (Miltenyi).

[0568] T cells are maintained under conditions necessary to support growth, such as an appropriate temperature (eg, 37° C.) and atmosphere (eg, air plus 5% CO 2 ).

[0569] In some embodiments, the T cell comprising the engineered TCR of the present disclosure is autologous. Before amplification and genetic modification, a T cell source is obtained from a subject. Immune cells such as T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. 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 invention, any number of techniques known to those skilled in the art (such as Ficoll) can be used. TM Isolation) obtains T cells from a blood unit collected from a subject.

[0570] In some embodiments, cells from individual circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected by apheresis can be washed to remove plasma fractions, and cells are placed in appropriate buffers or media for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution lacks calcium, and may lack magnesium or may lack many (if not all) divalent cations. As will be readily understood by those of ordinary skill in the art, washing steps can be achieved by methods known to those skilled in the art (such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, BaxterCytoMate or Haemonetics Cell Saver 5) according to the manufacturer's instructions). After washing, cells can be resuspended in a variety of biocompatible buffers (e.g., PBS, PlasmaLyte A or other saline solutions with or without buffers without Ca2+, Mg2+). Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0571] In some embodiments, the monocytes are depleted by lysing the red blood cells, for example by PERCOLL TM Gradient centrifugation or by countercurrent centrifugal elutriation, separate immune cells such as T cells from peripheral blood lymphocytes. Specific subpopulations of immune cells (such as T cells, B cells or CD4+ T cells) can be further separated by positive or negative selection techniques. For example, in one embodiment, T cells are separated by incubation with anti-CD4 conjugated beads for a period of time sufficient to positively select the desired T cells.

[0572] Enrichment of immune cell populations (such as T cell populations) by negative selection can be achieved with a combination of antibodies for surface markers unique to negatively selected cells. One method is to sort and / or select cells via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies for cell surface markers present on negatively selected cells. For example, in order to enrich CD4+ cells by negative selection, monoclonal antibody mixtures typically include antibodies for CD 14, CD20, CD 11b, CD 16, HLA-DR, and CD8.

[0573] In order to separate the desired immune cell population 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 beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between cells and beads.

[0574] In some embodiments, cells can be incubated on a rotator at different speeds for different lengths of time at 2-10°C or at room temperature.

[0575] The PBMCs from which the T cells to be stimulated or from which immune cells such as T cells are isolated may also be frozen after the washing steps. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and, to a certain extent, monocytes from the cell population. After the washing steps to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and can be used in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or medium containing 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% glucose 5%, 0.45% NaCl, 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1° / min and storing in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0576] Pharmaceutical composition

[0577] The present disclosure provides a pharmaceutical composition comprising an immune cell containing the engineered receptor of the present disclosure and a pharmaceutically acceptable diluent, carrier or excipient.

[0578] Such compositions may include a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; and a preservative.

[0579] Methods of treating diseases

[0580] Provided herein are methods of treating a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising an immune cell comprising an engineered receptor of the present disclosure. The immune cell expresses two engineered receptors in the same cell.

[0581] In some embodiments, the subject in need suffers from cancer.Cancer is a disease in which abnormal cells divide uncontrollably and spread to nearby tissues.In some embodiments, cancer includes liquid tumors or solid tumors.Exemplary liquid tumors include leukemia and lymphoma.As liquid tumors, other cancers can be those occurring in, for example, blood, bone marrow and lymph nodes, and can include, for example, leukemia, myeloid leukemia, lymphocytic leukemia, lymphoma, Hodgkin's lymphoma, melanoma and multiple myeloma.Leukemia includes, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and hairy cell leukemia.Exemplary solid tumors include sarcomas and carcinomas.Cancer can actually occur in organs of the body, including blood, bone marrow, lungs, breast, colon, bones, central nervous system, pancreas, prostate and ovaries. Additional cancers that are solid tumors include, for example, prostate cancer; testicular cancer; breast cancer; brain cancer; pancreatic cancer; colon cancer; thyroid cancer; stomach cancer; lung cancer; ovarian cancer; Kaposi's sarcoma; skin cancer; squamous cell skin cancer; kidney cancer; head and neck cancer; laryngeal cancer; squamous cancers that form on the moist mucosal lining of the nose, mouth, and throat; bladder cancer; osteosarcoma; cervical cancer; endometrial cancer; esophageal cancer; liver cancer and kidney cancer. In some embodiments, the condition treated by the methods described herein is a metastasis of a melanoma cell, a prostate cancer cell, a testicular cancer cell, a breast cancer cell, a brain cancer cell, a pancreatic cancer cell, a colon cancer cell, a thyroid cancer cell, a stomach cancer cell, a lung cancer cell, an ovarian cancer cell, a Kaposi's sarcoma cell, a skin cancer cell, a kidney cancer cell, a head and neck cancer cell, a laryngeal cancer cell, a squamous cell carcinoma cell, a bladder cancer cell, an osteosarcoma cell, a cervical cancer cell, an endometrial cancer cell, an esophageal cancer cell, a liver cancer cell, or a kidney cancer cell.

[0582] Any cancer in which a plurality of cancer cells express a first activator ligand but do not express a second inhibitor ligand is contemplated to be within the scope of the present disclosure. For example, CEA-positive cancers that can be treated using the methods described herein include colorectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, lung adenocarcinoma, head and neck cancer, diffuse large B-cell carcinoma, or acute myeloid leukemia cancer.

[0583] Treating cancer can result in a decrease in tumor size. A decrease in tumor size can also be referred to as "tumor regression." Preferably, the tumor size decreases by 5% or more after treatment relative to its size before treatment; more preferably, the tumor size decreases by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by greater than 75% or more. The size of a tumor can be measured by any reproducible means of measurement. The size of a tumor can be measured as the diameter of the tumor.

[0584] Treating cancer can result in a reduction in tumor volume. Preferably, after treatment, the tumor volume is reduced by 5% or more relative to its size before treatment; more preferably, the tumor volume is reduced by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by greater than 75% or more. Tumor volume can be measured by any reproducible means of measurement.

[0585] Treating cancer results in a decrease in the number of tumors. Preferably, after treatment, the number of tumors is reduced by 5% or more relative to the number before treatment; more preferably, the number of tumors is reduced by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by greater than 75%. The number of tumors can be measured by any reproducible measurement means. The number of tumors can be measured by counting tumors visible to the naked eye or by counting tumors at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, 10x or 50x.

[0586] Treatment of cancer can result in a reduction in the number of metastatic lesions in other tissues or organs away from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or more relative to the number before treatment; more preferably, the number of metastatic lesions is reduced by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by greater than 75%. The number of metastatic lesions can be measured by any reproducible measurement means. The number of metastatic lesions can be measured by counting metastatic lesions visible to the naked eye or by counting metastatic lesions at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, 10x or 50x.

[0587] Treating cancer can result in an increase in the average survival time of the treated subject population compared to a population receiving a single carrier. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably by more than 120 days. The increase in the average survival time of a population can be measured by any reproducible means. The increase in the average survival time of a population can be measured, for example, by calculating the average survival length of the population after starting treatment with the active compound. The increase in the average survival time of a population can also be measured, for example, by calculating the average survival length of the population after completing the first round of treatment with the active compound.

[0588] Treating cancer can result in an increase in the average survival time of the treated subject population compared to an untreated subject population. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. The increase in the average survival time of a population can be measured by any reproducible means. The increase in the average survival time of a population can be measured, for example, by calculating the average survival length of the population after starting treatment with the active compound. The increase in the average survival time of a population can also be measured, for example, by calculating the average survival length of the population after completing the first round of treatment with the active compound.

[0589] Compared to a group receiving a monotherapy using a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, treating cancer can result in an increase in the average survival time of the treated subject group. Preferably, the average survival time is increased by more than 30 days; more preferably, it is increased by more than 60 days; more preferably, it is increased by more than 90 days; and most preferably, it is increased by more than 120 days. The increase in the average survival time of the group can be measured by any reproducible means. The increase in the average survival time of the group can be measured, for example, by calculating the average survival length of the group after starting treatment with the active compound. The increase in the average survival time of the group can also be measured, for example, by calculating the average survival length of the group after completing the first round of treatment with the active compound.

[0590] Compared to a group receiving a single carrier, treating cancer can result in a reduced mortality rate in the treated subject group. Compared to an untreated group, treating cancer can result in a reduced mortality rate in the treated subject group. Compared to a group receiving a monotherapy using a drug that is not a compound of the present invention or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, treating cancer can result in a reduced mortality rate in the treated subject group. Preferably, the mortality rate is reduced by more than 2%; more preferably, it is reduced by more than 5%; more preferably, it is reduced by more than 10%; and most preferably, it is reduced by more than 25%. The reduction in the mortality rate of the treated subject group can be measured by any reproducible means. The reduction in the mortality rate of the group can be measured, for example, by calculating the average number of disease-related deaths per unit time after the group begins treatment with the active compound. The reduction in the mortality rate of the group can also be measured, for example, by calculating the average number of disease-related deaths per unit time after the group completes the first round of treatment with the active compound.

[0591] Treatment of cancer may result in a reduction in tumor growth rate. Preferably, after treatment, the tumor growth rate is reduced by at least 5% relative to the value before treatment; more preferably, the tumor growth rate is reduced by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate may be measured based on the change in tumor diameter per unit time.

[0592] Treating cancer can result in a reduction in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth can be measured by any reproducible means of measurement. For example, tumor regrowth is measured by measuring an increase in tumor diameter after a previous tumor reduction after treatment. Failure of a tumor to recur after treatment has ceased indicates a reduction in tumor regrowth.

[0593] Treatment or prevention of a cell proliferative disorder can result in a reduction in the rate of cell proliferation. Preferably, after treatment, the rate of cell proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of cell proliferation can be measured by any reproducible means of measurement. For example, the rate of cell proliferation is measured by measuring the number of dividing cells per unit time in a tissue sample.

[0594] Treatment or prevention of a cell proliferative disorder may result in a decrease in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is decreased by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The proportion of proliferating cells may be measured by any reproducible means of measurement. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of non-dividing cells in a tissue sample. The proportion of proliferating cells may be equal to the mitotic index.

[0595] Treatment or prevention of a cell proliferative disorder may result in a decrease in the size of an area or zones of cell proliferation. Preferably, after treatment, the size of an area or zones of cell proliferation is reduced by at least 5% relative to its size prior to treatment; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The size of an area or zones of cell proliferation may be measured by any reproducible means of measurement. The size of an area or zones of cell proliferation may be measured as the diameter or width of an area or zones of cell proliferation.

[0596] Treatment or prevention of a cell proliferative disorder may result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% relative to their number before treatment; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. Abnormal cell appearance or morphology may be measured by any reproducible means of measurement. Abnormal cell morphology may be measured by microscopic examination, for example, using an inverted tissue culture microscope. Abnormal cell morphology may take the form of nuclear polymorphism.

[0597] Kits and Products

[0598] The present disclosure provides kits and articles of manufacture comprising polynucleotides and vectors encoding the engineered receptors described herein, and immune cells comprising the engineered receptors described herein. In some embodiments, the kit comprises items such as vials, syringes, and instructions for use.

[0599] In some embodiments, a kit comprises a polynucleotide or vector comprising a sequence encoding one or more engineered receptors of the disclosure.

[0600] In some embodiments, the kit comprises a plurality of immune cells comprising an engineered receptor as described herein. In some embodiments, the plurality of immune cells comprises a plurality of T cells.

[0601] Example

[0602] Example 1: Selection of Activator Target Ligands

[0603] The inventors investigated the GTex gene expression database (gtexportal.org / home / ) for activator ligands. Activator ligands should have the following characteristics: First, a type of activator ligand should have high surface expression, which confers the potential for delivering large activation signals. Alternatively, activators such as MiHA can have low density on the cell surface. Secondly, activator ligands can have necessary cellular functions, which prevents the alleles of activator ligands from being lost due to aneuploidy in tumor cells and makes them less likely to undergo mutagenesis during tumor evolution. Finally, activator ligands should be present on all tumor cells. If the inhibitor ligand is also expressed on all cells except target cells, the activator ligand may be expressed on all cells. Activators should also be expressed on cancer cells. When used in combination with inhibitors, activators can be, for example, widely expressed on all cells.

[0604] Figure 4A The RNA expression profile of an exemplary activator ligand, transferrin receptor (TFRC), is shown. Figure 4A As can be seen in Figure 2, TFRC expression at the RNA level is ubiquitous and relatively uniform. In addition, TFRC is an essential gene: loss-of-function homozygous TFRC mutations are embryonic lethal in mice.

[0605] Figure 4B The expression profiles of candidate blockers HLA-A and candidate activators HLA-B are shown. Figure 4B As can be seen, candidate activators and blockers HLA class I expression are tracked together, thereby alleviating the challenge of optimizing activator and blocker pairs.

[0606] Example 2: Selection of inhibitor target ligands lost in cancer cells

[0607] Loss of heterozygosity

[0608] A potential set of inhibitor ligands is a ligand lost in tumor cells due to loss of heterozygosity. In an analysis of 3131 tumor samples in 26 histological types, Beroukhim et al. found that in typical tumors, 25% of the genome was affected by single copy number changes (duplication and deletion) of the arm number, and 10% of the genome was affected by focal single copy number changes, with 2% overlap. (Beroukhim et al., Nature 463:899-905 (2010)). In addition, many LOH regions overlap between tumor types, and only 22% of the regions are unique to one tumor type. For example, Beroukhim et al. found that 80% of the amplification peaks and 78% of the deletion peaks were common to 17 of the most representative tumor types. Therefore, the alleles of LOH loss that can be selectively bound by the inhibitor LBD are potential inhibitor targets that are not expressed by target cells.

[0609] For potential inhibitor ligands lost in cancer by loss of heterozygosity, the inventors investigated the Cancer Genome Atlas Program (http: / / portals.broadinstitute.org / tcga / home). The data set all cancer data set (all_cancers) consists of 10,844 cancer samples from 33 cancer types. A type of inhibitor ligand should have the following properties: First, the inhibitor ligand should have high uniform surface expression in the tissue. This confers the ability to deliver a large, unbiased inhibitory signal. The inhibitor ligand should be absent or polymorphic in many tumors. In addition, the loss of inhibitor ligands in tumor cells should be easily distinguished by conventional methods such as antibody staining or genetic analysis. Other types of inhibitor ligands (such as MiHA) may have low surface expression.

[0610] One set of inhibitor ligands are major histocompatibility complex (MHC) alleles that are lost in cancer cells through LOH. Using these alleles as inhibitor ligands does not require a peptide MHC target (pMHC), for example, a pan-HLA-A*02 allele can be used.

[0611] Loss of the Y chromosome

[0612] The Y chromosome gene expressed by adult males is a potential inhibitor ligand lost by the Y chromosome. There are at least 60 protein-coding genes on the Y chromosome. Several Y chromosome genes are widely expressed in adult males, and may be lost in cancer by the loss of the Y chromosome. Several other widely expressed cytoplasmic proteins are pMHC inhibitor candidates (such as TMSB4Y, EIF1AY). NLGN4Y is a type I integral membrane protein widely expressed in males, and is also a candidate.

[0613] Example 3: Targeting cells lacking surface antigens using paired A and B receptors

[0614] We show that the loss-of-heterozygosity targeted system works in vitro and in mouse cancer models.

[0615] The distinction between normal cells and tumor cells depends on two functions: (i) an activator ("A") receptor that recognizes an epitope on the surface of normal cells that is also retained on the tumor; and (ii) a blocker ("B") receptor that recognizes a second surface epitope on the product of an allele that is lost from the tumor cell. In this example, we used peptide-MHC (pMHC) targets for both A and B (see Figure 5A ):

[0616] A chimeric antigen receptor comprising a scFv directed against HLA-A*02-MAGE-A3 (FLWGPRALV) pMHC as the A receptor; and

[0617] A chimeric antigen receptor comprising a scFv that binds to HLA-A*02-NY-ESO-1 (SLLMWITQC / V) as the B receptor and comprising the PD-1 intracellular domain (ICD), the CTLA-4 intracellular domain (ICD) or the LILRB1 (LIR1) intracellular domain (ICD).

[0618] Each blocker with PD-1 ICD or CTLA-4-ICD (B) receptor mediated <10x shift in activation EC50 in Jurkat cells as measured by titration of peptide loaded as stimulus on T2 cells ( Figure 5B Surprisingly, the B receptor containing the NY-ESO-1 LBD and the intracellular, transmembrane, and hinge domains of the LIR-1 (LILRB1) receptor mediated an EC50 shift of >5,000x (also Figure 5B Titration of irrelevant control HLA-A*02 binding peptides in addition to SLLMWITQC / V provided an estimate of the shift caused by competition for available HLA molecules with peptides loaded on T2 cells, with a contribution to the total shift typically <10x ( Figure 8For the EC50 shift values ​​reported here, we generally compared to the EC50 of the activator-only construct.

[0619] A total of six different scFvs grafted onto LIR-1 mediated significant shifts in EC50 for four different pMHC targets, ranging from 10x to 1,000x ( Figure 5C When fused to a standard CAR, the extent of the EC50 shift (i.e., blocking strength) correlated with the EC50 of the scFv (data not shown). LIR-1B signaling blocked A signaling from multiple A targets and scFv ( Figure 5D , Fig.26 ). Blockade is ligand-dependent ( Fig. 9 Control B receptors that have an LBD but lack an ICD or contain mutations in key elements of the ICD do not block activation of signaling through the A receptor ( Fig.10 ). Engineered T cells with A receptors and B receptors function in multiple target antigens and antigen binding domains (ie, LBD sequences).

[0620] The LIR-1 ICD also works when fused to the extracellular domain of the T cell receptor (TCR) with three different pMHC targets (see Methods). TCRs against three different pMHC targets (two from MAGE-A3 and one from HPV) were measured. In each case, the LIR-1-based B receptor shifted the activation EC50 by a large amount, ranging from 1,000x to 10,000x. The LIR-1-based B receptor with the NY-ESO-1 TCR variable domain LBD "ESO (Ftcr)" fused to it was also able to block activation by CAR or TCR. This includes the following receptor pairs:

[0621] 1. Contains binding to MAGE-A3 FLWGPRALV Activators of TCR LBD of peptide:MHC complexes (A) TCR ("MP1-TCR") blocked by B receptor containing scFv NY-ESO-1 scFv LBD ("ESO") and LIR-1 ICD, which shifted the activation EC50 by a large amount ( Figure 5E );

[0622] 2. Contains binding to MAGE-A3 MPKVAELVHFL The A TCR of the second TCR LBD of the peptide:MHC complex ("MP2-TCR") was blocked by the B receptor containing the scFv NY-ESO-1 scFv LBD ("ESO") and LIR-1 ICD, which shifted the activation EC50 by a large amount ( Figure 5E );

[0623] 3. Contains HPV binding TIHDIILECV The A TCR of the TCR LBD of the peptide:MHC complex ("HPV E6-TCR") was blocked by the B receptor containing the scFv NY-ESO-1 scFv LBD ("ESO") and LIR-1 ICD, which shifted the activation EC50 by a large amount ( Figure 5E );

[0624] 4. Contains binding to MAGE-A3 FLWGPRALV The A TCR of the TCR LBD of the peptide:MHC complex ("MP1-TCR") was blocked by the B receptor containing the NY-ESO-1 TCR LBD ("ESO(Ftcr)") and the LIR-1 ICD, and this blocker shifted the activation EC50 by a large amount ( Fig. 5F );

[0625] 5. Contains binding to MAGE-A3 FLWGPRALV A CAR with a scFv LBD of a peptide:MHC complex ("MP1-CAR") was blocked by a B receptor containing the TCR NY-ESO-1 TCR variable domain LBD ("ESO(Ftcr)") and LIR-1 ICD. This blocker shifted the activation EC50 by a large amount ( Fig. 5F ).

[0626] Confirmation of cis effect

[0627] The engineered effector cells should distinguish potential target cells that are only A+ (i.e., displaying only the activator) from those that are dual A+ and B+. To confirm that our receptor system works as expected, target-loaded beads ( ) of approximately the size of cells (d ~ 2.8 μm) were tested with engineered effector cells (Jurkat cells) that have both A and B receptors. Figure 5G ). Effector cells were indeed activated by a mixture of A+ and B+ beads, even when A+ beads accounted for only 20% of the total beads. This demonstrates that effector cells are able to recognize targets with loss of heterozygosity (represented by A+ beads) in a mixed population containing normal cells (represented by B+ beads).

[0628] Confirmation of target concentration independence

[0629] In patients, target density will vary depending on the expression levels of target A and target B. We confirmed that when the density of target A varied (data not shown) and when the density of target B varied ( Figure 5H ), the system works in both high-density and low-density target situations. Figure 5HIn this study, scFvs that bind to the B cell markers CD19 or HLA-A*02 in a peptide-independent manner were tested. These non-pMHC targets represent surface antigens that can extend to the range of 100,000 epitopes / cell. In this case, the ratio of A module to B module expression was varied using different DNA concentrations in transient transfection assays. Emax shifts of more than 10x were observed. These experiments show that the properties of dual receptor systems observed for pMHC targets are generally the same for high-density targets.

[0630] B receptor function in primary T cells

[0631] MCF7 tumor cells expressing Renilla luciferase (Biosettia) loaded with a certain titer of target peptide were used as target cells, where luciferase was used as a readout of cell viability. Primary T cells were transduced with HPV TCR ("HPVE7 TCR") as A receptor and B receptor ("ESO-LIR-1") containing anti-NY-ESO-1 scFv fused to LIR-1 hinge, transmembrane domain and ICD, or not transduced ("untransduced"). Beads coupled to HLA-A*02 tetramers bound to B receptor LBD were used to enrich transduced T cells by physical selection. In order to change the target concentration, target cells were loaded with different amounts of HPV peptides. Primary T cells were activated in a dose-dependent manner. The expression of B receptor shifted the EC50 curve by about 100x ( Fig. 6A In Jurkat cells, similar results were obtained for the anti-NY-ESO-1 CAR A receptor paired with a B receptor containing the anti-HLA-A*02 LBD and the LIR-1 hinge, transmembrane domain, and ICD at various ratios of A receptor to B receptor (achieved by transfection of various activator:blocker DNA ratios) ( Figure 6B This result was confirmed in T cells using a CD19 CAR activator paired with an HLA-A*02 blocker ( Figure 6C ). Thus, despite their complexity, heterogeneity, and donor-to-donor variability, the basic functions of the activator and blocker receptor pairs are recapitulated in primary T cells.

[0632] Example 4: Targeting loss of heterozygosity with paired A and B receptors

[0633] The HLA locus is polymorphic, with only a subset of the population possessing the HLA-A*02 allele. A ligand-binding domain of MHC that binds to the HLA*A02 allele independently of the load (a "pan-HLA-A*02" LBD) can be used to target tumors in subjects who are HLA heterozygous and have LOH of the HLA-A*02 allele in tumor cells.

[0634] The HLA-A*02-specific scFv was fused to the LIR-1 module and shown to be expressed in Jurkat cells in the presence of pMHC-dependent activators (ESO-CAR, Figure 6B ). In addition, in primary T cells expressing both the A receptor containing anti-CD19 scFv and the B receptor containing HLA-A*02 specific scFv and LIR-1LBD, the B receptor blocks the A receptor as needed ( Figure 6C ).

[0635] Raji target cells that are CD19+ and HLA-A*02 negative can be used to model tumor cells that have lost HLA-A*02 through LOH. The same cell line that stably expresses HLA-A*02 can be used as a model for normal cells. If the Raji target cells only express CD19, the Raji cell line activates Jurkat effector cells that express the CD19 CAR and the HLA-A*02 LIR-1 blocker. When the Raji target cells are transfected with a polynucleotide encoding HLA-A*02, the activation of the Jurkat effector cells is blocked ( Fig.11 ).

[0636] As described above, the A receptor that binds CD19 and the B receptor that binds HLA-A*02 function in primary T cells as well as Jurkat cells. Engineered T cells kill Raji cells expressing CD19 in the absence of HLA-A*02 expression ( Figure 6C , upper panel). Raji cells expressing both CD19 and HLA-A*02 were killed by T cells expressing only the activation module, but blocked interferon-γ (IFNg) secretion (data not shown) and cytotoxicity ( Figure 6C , middle panel) Both. Primary T cells carrying activator and blocker modules differentiate CD19+ “tumors” in mixed cultures ( Figure 6C , lower panel) and CD19+ / HLA-A*02+ “normal” cells ( Figure 6C , right).

[0637] T cell therapy based on activator and blocker mechanisms should be able to work reversibly, i.e., be able to cycle from a blocking state to an activated state and back to a blocking state. Effector cells are co-cultured with CD19+ or D19+ / HLA-A2*02+ Raji cells for multiple rounds, and the Raji cells are removed from the culture between rounds. As needed, the target cells are exposed to block-kill-block ( Fig.6D ) and the kill-block-kill program ( Fig. 6E) Both, when exposed to Raji target cells, effector cells exposed to normal cells are not activated. Effector T cells are able to cycle from a blocking state to a cytotoxic state and back, depending on the target cells they are exposed to.

[0638] Example 5: Targeted loss of heterozygosity in vivo using paired A and B receptors

[0639] In preparation for in vivo experiments, we show that an engineered CD19 / HLA-A*02 activator / blocker pair in primary T cells allows for extensive in vitro expansion using standard CD3 / CD28 stimulation ( Fig. 7A ). Thus, the cell product can be produced in sufficient quantities for use as a therapeutic agent in a patient.

[0640] CD19+ / HLA-A*02+ or CD19+ / HLA-A*02- tumor cell mouse xenografts were generated by injecting Raji target cells into the flank of immunocompromised (NGS-HLA-A2.1) mice. Figure 7B ). Raji cells were injected at two doses of 2e6 or 1e7 T cells, and tumor growth and persistence of implanted T cells were analyzed over time. In mice, only CD19+ / HLA-A*02- tumor cells were killed, and tumor controls were tracked with the number of transferred T cells, promoting survival of host mice ( Figure 7C-7E and Fig.12 ). Normal CD19+ / HLA-A*02+ cells, designed to mimic normal cells, were not affected by the treatment.

[0641] summary

[0642] We have developed a synthetic signal integration system that can utilize a large class of new cancer targets derived from LOH. The system can meet the needs of patients with LOH for cell therapy without too much experimentation. The system works robustly in Jurkat cells, primary T cells and in vivo. This system is also (i) modular and flexible, and works across CAR and TCR modalities at different target densities; (ii) when blocker and activator targets are present in cis on one surface, they are silenced, but when a few cells express only activators, they are not silenced; and (iii) reversibly switch states, meeting the needs of searching for tumor cells throughout the body.

[0643] Example 6: Method of Examples 3-5

[0644] Cell culture

[0645] Obtain the Jurkat cells encoding NFAT luciferase reporter from BPS Bioscience.All other cell lines used in this study are obtained from ATCC.In culture, Jurkat cells are maintained in RPMI culture medium supplemented with 10%FBS, 1%Pen / Strep and 0.4mg / mL G418 / Geneticin.Maintain T2, MCF7 and Raji cells according to the advice of ATCC.Prepare "normal" Raji cells by transducing Raji cells with MOI 5 with HLA-A*02 lentivirus (custom lentivirus, Alstem).HLA-A*02-positive Raji cells are sorted using FACS Melody cell sorter (BD).

[0646] Plasmid construction

[0647] NY-ESO-1 responsive inhibitory constructs were created by fusing the NY-ESO-1 scFv LBD to domains of receptors containing the hinge, transmembrane region, and / or intracellular domain of leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1, LIR-1), programmed cell death protein 1 (PDCD1, PD-1), or cytotoxic T lymphocyte protein 4 (CTLA4, CTLA-4). The gene segments were combined and inserted downstream of the human EF1ɑ promoter contained in a lentiviral expression plasmid using Golden Gate cloning.

[0648] Jurkat cell transfection

[0649] Jurkat cells were transiently transfected by Neon electroporation system (Thermo Fisher Scientific) in 100uL format according to the manufacturer's protocol using the following settings: 3 pulses, 1500V, 10 milliseconds. Each 1e6 cell was co-transfected with 1-3ug activator CAR or TCR construct and 1-3ug scFv or Ftcr blocker construct or empty vector and recovered in RPMI medium supplemented with 20% heat-inactivated FBS and 0.1% Pen / Strep.

[0650] Jurkat-NFAT-luciferase activation studies

[0651] Peptides, MAGE-A3 (MP1; FLWGPRALV), MAGE-A3 (MP2; MPKVAELVHFL), HPVE6 (TIHDIILECV), HPV E7 (YMLDLQPET), and modified NY-ESO-1 ESO (ESO; SLLMWITQV) were synthesized by Genscript. Activating peptides were serially diluted starting at 50uM. Blocker peptide NY-ESO-1 was diluted to 50uM (unless otherwise stated), added to the serial dilutions of the activating peptides, and subsequently loaded onto 1e4 T2 cells in 15uL of RPMI supplemented with 1% BSA and 0.1% Pen / Strep and incubated for 24h. The cells were incubated in 384-well low-flange white flat-bottom polystyrene TC-treated microplates. The next day, 1e4 Jurkat cells were resuspended in 15uL of RPMI supplemented with 10% heat-inactivated FBS and 0.1% Pen / Strep, added to the peptide-loaded T2 cells and co-cultured for 6 hours. Jurkat luminescence was evaluated using a one-step luciferase assay system (BPS Bioscience). The assays were performed in technical replicates.

[0652] Primary T cell transduction, expansion and enrichment

[0653] Frozen PBMCs were thawed in a 37°C water bath and cultured at 1e6 cells / mL in LymphoONE (Takara) containing 1% human serum and activated using 1:100 T cell TransAct (Miltenyi) supplemented with IL-15 (10 ng / mL) and IL-21 (10 ng / mL). After 24 hours, lentivirus was added to PBMCs at an MOI of 5. PBMCs were cultured for another 2-3 days to allow cells to expand under TransAct stimulation. After expansion, primary T cells transduced with activators and blockers were enriched using anti-PE microbeads (Miltenyi) according to the manufacturer's instructions. In brief, primary T cells were incubated with 1:100 diluted CD19-Fc (R&D Systems) at 4°C in MACS buffer (0.5% BSA + 2mM EDTA in PBS) for 30 minutes. The cells were washed 3 times in MACS buffer and incubated in secondary antibody (1:200) in MACS buffer for 30 min at 4° C. The cells were then incubated in anti-PE microbeads and passed through a LS column (Miltenyi).

[0654] In vitro cytotoxicity studies of primary T cells

[0655] For cytotoxicity studies using pMHC targets, primary T cells of enrichment were incubated for 48 hours with 2e3 MCF7 cells expressing Renilla luciferase (Biosettia) loaded with a certain titer of target peptide as described above at an effector of 3:1:target ratio. MCF7 cells expressing luciferase were quantitatively measured using the Renilla luciferase reporter assay system (Promega). For cytotoxicity studies using non-pMHC targets, primary T cells of enrichment were incubated for 6 days with 3:1 effector:target ratios with 2e3 WT Raji cells ("tumor" cells) or Raji cells ("normal" cells) transduced with HLA-A*02. WT "tumor" Raji cells or HLA-A*02 "normal" Raji cells stably expressing GFP and Renilla luciferase (Biosettia) stably express RFP, and firefly luciferase (Biosettia) is imaged together with unlabeled primary T cells using IncuCyte live cell imager. The fluorescence intensity of live Raji cells over time is quantified using IncuCyte imaging software. For reversibility studies, enriched primary T cells are similarly co-cultured with "normal" or "tumor" Raji cells for 3 days and imaged. After 3 days, CD19 negative selection is used to separate T cells from remaining Raji cells, and fresh "normal" or "tumor" Raji cells are re-inoculated as described. In separate wells, dual luciferase reporter assay system (Promega) is used to quantify live Raji cells expressing luciferase.

[0656] Mouse xenograft studies

[0657] Frozen PBMCs were thawed in a 37°C water bath and left to stand overnight in serum-free TexMACS medium (Miltenyi) before activation. PBMCs were activated at 1.5e6 cells / mL using T cell TransAct (Miltenyi) and TexMACS medium (supplemented with IL-15 (20 ng / mL) and IL-21 (20 ng / mL). After 24 hours, lentivirus was added to PBMCs at an MOI of 5. PBMCs were cultured for another 8-9 days to allow cells to expand under TransAct stimulation. After expansion, T cells were enriched for A2-LIR-1 (pMHC HLA-A*02 ScFv fused to LIR-1 hinge, TM and ICD) using anti-PE microbeads (Miltenyi) for streptavidin-PE-HLA-A*02-pMHC before in vivo injection.

[0658] Female NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A / H2-D / B2M)1Dvs / SzJ(NSG-HLA-A2 / HHD) mice aged 5-6 weeks were purchased from The Jackson Labs. Animals were acclimated to the housing environment for at least 3 days before the start of the study. Animals were injected subcutaneously in the right flank with 2e6 WT Raji cells or HLA-A*02 transduced Raji cells in a volume of 100uL. When tumors reached 70mm 3 (V = L x W x W / 2) when the average value, the animals were randomly divided into 5 groups (n = 7), and 2e6 (data not shown) or 1e7 T cells were administered through the tail vein. After T cell injection, tumor measurements were performed 3 times a week, and blood was collected for flow analysis after 10 and 17 days. After RBC lysis, the cells were stained with anti-hCD3 antibody, anti-hCD4 antibody, anti-hCD8 antibody, anti-msCD45 antibody (Biolegend).

[0659] Embodiment 7:

[0660] The ability of a blocker receptor (C1765) with an HLA-AA*02 antigen binding domain and LIR-1 ICD to block activation of Jurkat cells expressing an activator CAR (CT479) with an EGFR antigen binding domain was determined using the NFAT luciferase reporter system as previously described. Wild-type HeLa tumor cells that are EGFR+ and HLA-A*02- are used as target cells. EGFR+ / HLA-A*02-HeLa cells are also transduced with polynucleotides encoding HLA-A*02+ to generate EGFR+ / HLA-A*02+HeLa cells for use as target cells expressing both activator and blocker antigens.

[0661] like Fig.13 As shown, expression of the HLA-A*02 LIR-1 blockade in Jurkat cells expressing the EGFR CAR increased the CAR Emax compared to the CAR Emax in Jurkat cells not expressing the blockade. max Displaced >5 times.

[0662] In addition, lower blocking was observed with lower HLA-A2 expression levels on target cells. Wild-type HCT116 cells are EGFR+ and HLA-A*02. The levels of EGFR and HLA-A*02 were determined in HCT116 cells and HeLa cells transduced with polynucleotides encoding HLA-A*02 polynucleotides using anti-EGFR antibodies and anti-HLA-A*02 antibodies (BB7.2) followed by FAC sorting. Fig.14A and 14BAs shown, HCT116 cells have lower levels of the blocking HLA-A*02 antigen than transduced HeLa cells. When Jurkat cells expressing EGFR CAR and HLA-A*02 LIR-1 blocking agent were presented together with HCT116 target cells expressing EGFR and HLA-A*02 antigen, the presence of HLA-A*02 LIR-1 blocking agent inhibited the E37 expression of EGFR CAR. max The displacement is 1.8 times ( Fig. 15B In contrast, transduced HeLa cells expressing higher levels of HLA-A*02 antigen were able to mediate >5-fold EGFR CARE max Displacement( Fig.13 As a control, activation by EGFR knockout HCT116 cells was minimal ( Fig.15A ).

[0663] The blocker-to-activator ratio necessary to achieve 50% blockade using the EGFR CAR and HLA-A*02 LIR-1 blocker was determined using a bead-based system and is shown in Fig.16A and 16B middle.

[0664] To determine the EC50 of the activator antigen, the activator beads were coated with different concentrations of the activator antigen. An irrelevant protein was added to each concentration to make the total protein concentration the same, and a constant amount of beads was added to Jurkat effector cells expressing EGFRCAR ( Fig.16A ).

[0665] To determine the blocker antigen IC50, beads were incubated with an EC50 concentration (at Fig.16A The beads were coated with activator antigens (determined in ), and with different concentrations of blocking antigens. An irrelevant protein was added at each concentration so that the total protein concentration remained the same, and a constant amount of beads was added to Jurkat effector cells expressing EGFR CAR or EGFR CAR and HLA-A*02 LIR-1 blocker ( Fig. 16B ).

[0666] Example 8: LIR-1 based blockers can inhibit TCR signaling using solid tumor cell lines

[0667] Jurkat effector cells expressing MAGE-A3 activator TCR and inhibitory receptor based on NY-ESO-1 scFv LIR-1 (comprising LIR-1 hinge, TM and ICD) were assayed using A375 target cells loaded with different concentrations of activator and blocker peptides. Jurkat cell activation was assayed using the NFAT luciferase assay (see Example 6).

[0668] like Fig.17 As shown, A375 cells were loaded with 50 μM NY-ESO-1 peptide to activate TCR E max The shift was greater than 10-fold. The difference in peptide loading efficiency in A375 cells versus T2 target cells was estimated to be approximately 100x. Peptide loading may explain the apparent therapeutic window.

[0669] Example 9: Using B cell leukemia cell lines, HLA-A*02 LIR-1 based blockers can inhibit CAR signaling

[0670] NALM6 target cells were used to measure the Jurkat effector cells expressing non-pMHC high-density CD19 specific activators (CD19scFv CAR activators) in the case of co-expression or non-co-expression of inhibitory receptors (comprising LIR hinges, TIMs and ICDs) based on pMHC HLA-A*02 scFv LIR-1. NFAT luciferase was used to measure Jurkat cell activation (see Example 6), and effector cells and target cells (E:T) ratios were changed.

[0671] like Fig.18 As shown, Jurkat cell expression blocker can inhibit the E max The displacement is greater than 5 times.

[0672] Example 10: HLA-A*02 LIR-1-based blockers can inhibit CAR signaling in a dose-dependent manner

[0673] Jurkat effector cells expressing NY-ESO-1 scFv CAR and inhibitory receptors based on pMHC HLA-A*02 scFv LIR-1 were measured with T2 target cells loaded with different amounts of peptides (note that in this case, the same peptide was recognized by both activator and blocker ScFv). Jurkat cell activation was measured using NFAT luciferase assay (see Example 6). Jurkat cells were transfected with different ratios of activator and blocker DNA (i.e., 1:1, 1:2, and 1:3 activator and blocker) to change the ratio of receptors expressed by Jurkat cells.

[0674] from Fig.19 As can be seen in the figure, even when Jurkat cells were transfected with activator and blocker receptor DNA at a 1:1 ratio, the inhibitory receptor (blocker) based on MHC HLA-A*02 scFv LIR-1 was able to inhibit activation of Jurkat cells by the activator CAR. In addition, the extent of inhibitory receptor blockade of activation increased with increasing amounts of inhibitory receptor DNA compared to the activator receptor DNA used in Jurkat cell transfection.

[0675] Example 11: HLA-A*02 LIR-1 based blockers can inhibit universal (pan-HLA class I) activators with tunable strength.

[0676] The activation of Jurkat effector cells expressing a pan-HLA scFv CAR with three different scFv binding domains based on the pan-HLA antibody W6 / 32 and an inhibitory receptor based on the pMHC HLA-A*02 scFv LIR-1 was determined using HLA-A*02 positive T2 cells. Fig. 20 As can be seen in the figure, each activator scFv supports different functional signals in HLA-A*02-negative Jurkat cells. When Jurkat cells were contacted with HLA-A*02-positive T2 target cells at an E:T ratio of 1:2, the inhibitory receptor based on pMHC HLA-A*02 scFv LIR-1 was able to block the functional signals from all three pan-HLA scFv CARs. In addition, the inhibitory receptor based on pMHC HLA-A*02 scFv LIR-1 was able to inhibit the activator up to 25-fold.

[0677] Example 12: HLA-A*02 LIR-1 based inhibitory receptor can block activation by MSLN CAR activator.

[0678] The activation of Jurkat effector cells expressing the MSLN CAR activator and the pMHC HLA-A*02 ScFv LIR-1 based inhibitory receptor was determined using the NFAT luciferase assay described in Example 6.

[0679] Jurkat cells were transfected with activator:blocker DNA at a 1:4 ratio and activation was determined in a cell-free bead-based assay ( Fig.21A ). The beads were loaded with either activator antigen or activator and blocker antigens, and the ratio of beads to Jurkat cells was varied. In a cell-free bead-based assay, the pMHC HLA-A*02 ScFv LIR-1 based inhibitory receptor was able to block activation of Jurkat cells when cells were contacted with beads carrying pMHC HLA-A*02 blocker and MSLN activator in cis. The presence of pMHC HLA-A*02 blocker on the beads enabled the E of the MSLN CAR to max Displacement greater than or equal to 12X( Fig.21A ).

[0680] The activation of Jurkat cells transfected with the same activator and blocker at a 1:4 DNA ratio was determined using the chronic myeloid leukemia cell line K562 for activation. K562 expresses the activator antigen MLSN. The response of Jurkat effector cells to K562 cells transduced with HLA-A*02 to express both activator and blocker antigens (MSLN+HLA-A*02+) and to non-transduced K562 expressing the activator but not the blocker antigen (MSLN+HLA-A*02-) was determined. Fig.21B As can be seen from the figure, the expression of HLA-A*02+ by K562 cells can make MSLN CAR E max The displacement is greater than 5X.

[0681] The ability of pMHC HLA-A*02 inhibitory receptors to block activation by MSNL ScFv CAR was also determined using effector primary T cells and SiHa or HeLa target cells as described for Raji in Example 6. SiHa and HeLa cells endogenously express MSLN and are transduced to express the HLA-A*02 inhibitory receptor target. Activation of primary effector T cells was determined by observing the fold induction of IFNγ. Fig. 22 As shown, pMHC HLA-A*02 LIR-1 inhibitory receptor was able to block activation of primary T cells (greater than 10X and 5X inhibition, respectively) when primary T cells were presented with SiHa or HeLa target cells expressing HLA-A*02.

[0682] When T cells were presented with SiHa cells expressing MSLN but not HLA-A*02, the pMHC HLA-A*02 inhibitory receptor was also able to inhibit killing by T cells expressing both the MSLN ScFv CAR and the pMHC HLA-A*02 LIR-1 inhibitory receptor ( Fig.23 ).

[0683] Example 13: HLA-A*02 LIR-1 based inhibitory receptor can block activation by EGFR CAR activators.

[0684] The activation of Jurkat effector cells expressing the EGFR CAR activator and the pMHC HLA-A*02 ScFv LIR-1 based inhibitory receptor (comprising the LIR-1 hinge, transmembrane and ICD) was determined using the NFAT luciferase assay described in Example 6.

[0685] Jurkat cells were transfected with activator and blocker receptor DNA and activation was measured in a cell-free bead-based assay ( Fig.24). The beads were loaded with activator antigen, blocker antigen, or activator and inhibitor antigens, and the ratio of beads to Jurkat cells was varied. In a cell-free bead-based assay, the inhibitory receptor based on HLA-A*02 ScFv LIR-1 was able to block activation of Jurkat cells when cells were contacted with beads carrying HLA-A*02 blockers and EGFR activators in cis, but not when the HLA-A*02 blockers and EGFR activators were in trans (on different beads). The presence of HLA-A*02 blockers on the beads was able to shift the Emax of the EGFR CAR by greater than or equal to 9X ( Fig.24 ).

[0686] HeLa and SiHa cells were used as target cells to measure activation of Jurkat cells expressing EGFR CAR activators and HLA-A*02ScFv LIR-1 based inhibitory receptors. Wild-type HeLa and SiHa cell lines express EGFR but not HLA-A*02 (SiHa WT and HeLa WT) but were transduced to express the HLA-A*02 inhibitory receptor target (SiHa A02 and HeLa A02). Figures 25A-25B As can be seen, the inhibitory receptor based on HLA-A*02 ScFv LIR-1 can make EGFRE max Shift greater than 4X (using SiHa target cells) ( Fig.25A ) and greater than 5X (using HeLa target cells) ( Fig.25B ).

[0687] Example 14: Activator and blocker pairs can distinguish between KRAS alleles

[0688] MiHAs are peptides derived from proteins that contain non-synonymous differences between alleles. Using KRAS as a model for MiHA, activator and blocker pairs were able to discriminate and respond to different KRAS variants using antigen binding domains specific for KRAS G12V and KRAS G12D mutations.

[0689] Using the Jurkat-NFAT-luciferase activation studies and T2 target cells described in Example 6, the ability of KRAS ScFv or Ftcr inhibitory LIR-1 based receptors to inhibit activation mediated by activator KRAS CAR or TCR was determined.

[0690] Fig. 27 The results showed that KRAS G12V ScFV blocker could inhibit the activation of Jurkat cells by KRAS G12D TCR (C-891) and inhibit the activation of KRAS G12D E maxDisplacement 14X. Fig.28 Similar results were shown for an interactive pair (KRAS G12D ScFv blocker and KRASG12V TCR activator (C-913)), where the inhibitor was able to activate KRAS G12V E max Displacement 8X.

[0691] Fig.29 It was shown that KRAS G12V Ftcr blocker can inhibit KRAS G12D TCR. The inhibitor can make KRASG12D E max The displacement was greater than 50X. In this case, constructs with the LIR-1 transmembrane domain and intracellular domain were included on both the α and β chains of the inhibitory TCR (LIR-1 on α and β), only on the TCR α chain (LIR-1 only on α), only on the TCR β chain (LIR-1 only on β), and a version without the LIR-1 ICD was included as a control (no LIR-1). In the reciprocal experiment, the KRAS G12D Ftcr blocker was able to inhibit the KRAS G12V activator TCR, enabling the KRAS G12V E max The displacement is greater than 500X.

[0692] Finally, this effect depends on the specific ligand binding domain, as inhibitory receptors with unrelated ScFv domains have little to no effect on the activator E. max Impact Figures 31A-31B ).

[0693] Table 14 lists the KRAS ScFv and Ftcr sequences. All ScFvs are fused to the LIR-1 hinge, TM and ICD. All Ftcrs are fused to the LIR-1 TM and ICD.

[0694] Table 14. KRAS ScFv and Ftcr sequences.

[0695]

[0696]

[0697]

[0698] Table 15. Ftcr sequences fused to LIR-1 TM and without ICD as control.

[0699]

[0700] Example 15: Characterization of TCRs Recognizing MiHA-Y

[0701] The TCR α and β extracellular domains from Jb2.3 and P2A mouse TCRs were cloned into activator TCR constructs. Native mouse or human constant regions were used. b EL5 cells expressing the peptide KCSRNRQYL (SEQ ID NO: 256) were used as target cells to determine the activation of Jurkat cells transfected with MiHA-γ TCR ( Fig.32 ).from Fig.32 As can be seen, C-003121 supports robust Jurkat cell activation.

[0702] Table 16. Mouse miHA-γ TCR sequences with mouse or human constant regions

[0703]

[0704]

[0705] Example 16: Minor Histocompatibility Antigen HA-1 Inhibitory Receptor

[0706] T2 cells carrying HLA-A*02 and A*11 class I alleles were loaded with a certain titer of A*02-specific NY-ESO-1 peptide in the absence or presence of 50uM A*02-specific HA-1 blocker peptide. Activation of Jurkat effector cells expressing NY-ESO-1 TCR or NY-ESO-1 TCR and HA-1 Ftcr was determined as described above (see Example 6). Fig.33A It is shown that in the absence of a blocking peptide, the sensitivity of the NY-ESO-1 TCR is not affected by the presence of HA-1 Ftcr. In the presence of HA-1 (H) blocking peptide, NY-ESO-1 and HA-1 (H) peptides compete for the same HLA-A*02 allele, resulting in an EC50 right shift of about 30x (solid square to dashed square). However, in the presence of HA-1 Ftcr blocker, an additional right shift of about 10x activity was observed (dashed square to dashed circle). In addition, Emax was shifted downward by 1.5x (solid circle to dashed circle). Fig.33B It was shown that in the presence of a nonspecific allelic variant HA-1(R) blocker peptide, essentially no blocking was observed, indicating that the blocking was specific for a single amino acid. In general, because NY-ESO-1 is more efficiently loaded into HLA-A*02 than HA-1(R) (see Fig.35 ), only a 3-5x rightward shift was observed in the presence of HA-1(R) blocker peptide (solid to dashed lines).

[0707] The peptide sequence is as follows:

[0708] NY-ESO-1=SLLMWITQV (SEQ ID NO:265),

[0709] HA-1(H)=VLHDDLLEA(SEQ ID NO:191),

[0710] HA-1(R)=VLRDDLLEA (SEQ ID NO:266).

[0711] HA-1 Ftcr can also specifically block KRAS TCR in the presence of HA-1 (H) peptide. T2 cells carrying HLA-A*02 and A*11 class I alleles were loaded with a certain titer of A*11 specific KRAS peptide in the absence or presence of 50uM A*02 specific HA-1 blocker peptide. Activation of Jurkat effector cells expressing NY-ESO-1 TCR or NY-ESO-1 TCR and HA-1 Ftcr was determined as described above (see Example 6). Fig.34A It is shown that in the absence of a blocking peptide, the sensitivity of the KRAS TCR is not affected by the presence of HA-1 Ftcr. In the presence of the HA-1 (H) blocking peptide, the HA-1 Ftcr blocks the KRAS TCR by about 5x in terms of activity (solid circle to dashed circle). In addition, the Emax is shifted downward by 2.7x (solid circle to dashed circle). Fig.34B It is shown that in the presence of a non-specific allelic variant HA-1 (R) blocker peptide, essentially no blocking was observed, indicating that the blocking was specific to a single amino acid. In general, because KRAS and HA-1 (H) or HA-1 (R) are not loaded into the same allele, no significant right shift was observed in the presence of the blocker peptide (solid to dashed line).

[0712] Loading of T2 cells with NY-ESO, HA-1(H), and HA-1(R) peptides was compared using BB7.2 staining, which specifically recognizes the peptide-loaded HLA-A*02 class I allele product, and quantified using flow cytometry. Fig.35 The loading of HLA-A*02-specific NY-ESO-1 and HA-1(H) peptides in T2 cells was shown to be very similar. The allelic variant HA-1(R) was loaded slightly less efficiently than HA-1(H) and NY-ESO-1 peptides.

[0713] The HA-1(H)Ftcr sequence is described in Table 10, and the NY-ESO-1 and KRAS TCRs are shown in Table 17 below.

[0714] Table 17. NY-ESO-1 and KRAS TCR sequences

[0715]

[0716] Example 17: Ratios of agonist and inhibitor peptides

[0717] NFAT-luciferase signal of Jurkat cells transfected with activator MAGE-A3 CAR alone or in combination with NY-ESO-1 ScFv LIR1 blocker was measured after 6 hours of co-culture with T2 cells loaded with activator and blocker peptides. Fig.36A Shown are the responses of Jurkat cells co-cultured with T2 cells loaded with titrated amounts of the activator MAGE-A3 peptide and a fixed concentration of the blocker NY-ESO-1 peptide. Fig.36B Shown are the responses of Jurkat to T2 cells loaded with titrated amounts of the blocker NY-ESO-1 peptide and a fixed concentration of the activator MAGE-A3 peptide above the Emax concentration (approximately 0.1 μM). Fig.36C Shown are the values ​​from the curves normalized to a constant activator MAGE peptide concentration for each curve and plotted on the x-axis. Fig.36B The x value of the blocker NY-ESO-1 peptide concentration. Each curve indicates the ratio of the blocker peptide to the activator peptide required for 50% blocking (IC50). The required B: A peptide ratio is less than 1, indicating that for this pair of activator CAR and blocker, similar (or less) blocker pMHC antigens are required on the target cell to block the activator pMHC antigen. Blocking is possible at a pMHC antigen density similar to the pMHC antigen density that produces a response in the activating pMHC CAR.

[0718] Example 18: Optimization of specific receptor pairs

[0719] The EGFR ScFv CAR activator (CT-479, CT-482, CT-486, CT-487 or CT-488, such as Fig.37 T cells transfected with either EGFR ScFv CAR activator and HLA-A*02 PA2.1 ScFv LIR1 inhibitor (C1765) or with HeLa target cells were co-cultured. The wild-type HeLa cell line expresses EGFR but not HLA-A*02 but was transduced to express the HLA-A*02 inhibitory receptor target. Cells were co-cultured at a 1:1 effector to target (E:T) ratio. Fig.37 In the lower right corner, the effector cell receptor expression is indicated first, while the HeLa cell expression is in brackets. Fig.37As can be seen in Figure 3, different degrees of blockade were observed when the same HLA-A*02 PA2.1 ScFv LIR1 inhibitor was used with different EGFR activator receptors.

[0720] Example 19: Inhibitory receptors reversibly reduce surface levels of activator receptors in T cells

[0721] Primary T cells from two HLA-A*02 negative donors were transduced with EGFR ScFv CAR activators (CT-479, CT-482, CT-486, CT-487, or CT-488) and HLA-A*02PA2.1 ScFv LIR1 inhibitors (C1765). Transduced cells were enriched by FACS sorting of blocker and activator receptors or by double column purification of blocker and activator receptors. Transduced T cells were co-cultured with HeLa target cells. The wild-type HeLa cell line expresses EGFR but not HLA-A*02, but was transduced to express the HLA-A*02 inhibitory receptor target. Cells were co-cultured at a 1:1 effector to target (E:T) ratio. Surface expression of EGFR CAR activators was determined 120 hours later using labeled peptides that bind activator and blocker receptors and fluorescence-activated cell sorting. Changes in the surface levels of activators after co-culture with HeLa cells expressing both activator and blocker ligands correspond to the ability of T cells to kill target cells (compare Fig.37 and Figure 38).

[0722] T cells expressing the combination of CT-482 EGFR ScFv CAR activator and HLA-A*02 PA2.1 ScFv LIR1 inhibitor (C1765) were co-cultured with HeLa cells expressing EGFR (target A), expressing HLA-A*02 (target B), expressing a combination of EGFR and HLA-A*02 on the same cell (target AB), a mixed population of HeLa cells expressing target A and target AB on different cells, or a mixed population of HeLa cells expressing target B and target AB on different cells ( Figures 39A-39B ). T cells were cultured with HeLa target cells at a 1:1 effector to target ratio. When T cells were co-cultured with a target A plus target AB population of HeLa cells, the levels of activators decreased and then recovered ( Fig.39A ). Furthermore, activator and blocker antigens must be present together on the same cell to trigger the loss of activator surface expression on effector T cells. In contrast to activators, blocker expression was largely unchanged ( Fig.39B ).

[0723] Fig.40Shown is a schematic diagram of an experiment to determine whether the loss of activator receptor expression by T cells is reversible. T cells expressing EGFR ScFv CAR activator receptor (CT-487) and HLA-A*02 PA2.1 ScFv LIR1 (C1765) inhibitor receptors are co-cultured with HeLa target cells expressing both activator and blocker receptor targets (AB). After 3 days of co-culture, HeLa cells are removed using anti-EGFR columns, and T cells are stained for activator and inhibitor receptors, or co-cultured for 3 days with HeLa cells expressing only EGFR activator targets. After 3 days of co-culture, HeLa cells are removed again using anti-EGFR columns, and T cells are stained for activator and inhibitor receptors, or co-cultured for 3 days with HeLa cells expressing only EGFR activator targets or expressing both activator and blocker targets (AB), then dyed. The presence of activator and inhibitor receptors (stained) in T cells was determined using labeled EGFR and A2 probes, and receptor expression levels were quantified using fluorescence-activated cell sorting. The results are shown in Figures 41A-41B In. Figures 41A-41B As shown, co-culture of T cells with HeLa cells expressing both activator and inhibitor targets reduced EGFR activator staining ( Figures 41A-41B , left). When T cells were co-cultured with HeLa cells expressing activator (target A only) in round 2, expression of EGFR activator increased. Thus, activator surface loss was reversible and tracked by T cell cytotoxicity. Sequence Listing <110> A2 Biotherapeutics, Inc. <120> Cell surface receptors that respond to loss of heterozygosity <130> A2BI-009 / 03WO331656-2034 <150> 62 / 885,093 <151> 2019-08-09 <150> 63 / 005,670 <151> 2020-04-06 <160> 391 <170> PatentIn Version 3.5 <210> 1 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> CD8α hinge <400> 1 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 2 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> CD8α hinge <400> 2 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 3 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> CD28 Hinge <400> 3 Cys Thr Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys 1 5 10 15 Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser 20 25 30 Pro Leu Phe Pro Gly Pro Ser Lys Pro 35 40 <210> 4 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> CD28 Hinge <400> 4 tgtaccattg aagttatgta tcctcctcct tacctagaca atgagaagag caatggaacc 60 attatccatg tgaaagggaa acacctttgt ccaagtcccc tatttcccgg accttctaag 120 ccc 123 <210> 5 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> CD28 transmembrane domain <400> 5 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 6 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> CD28 transmembrane domain <400> 6 ttctgggtgc tggtcgttgt gggcggcgtg ctggcctgct acagcctgct ggtgacagtg 60 gccttcatca tcttttgggt g 81 <210> 7 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> IL-2Rβ transmembrane domain <400> 7 Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly 1 5 10 15 Phe Ile Ile Leu Val Tyr Leu Leu Ile 20 25 <210> 8 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> IL-2Rβ transmembrane domain <400> 8 attccgtggc tcggccacct cctcgtgggc ctcagcgggg cttttggctt catcatctta 60 gtgtacttgc tgatc 75 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3ζ activation domain <400> 9 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 10 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CD3ζ activation domain <400> 10 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gcgtagaggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggactca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 11 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> CD3ζ activation domain <400> 11 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu His Met Gln Ala Leu Pro Pro Arg 35 40 <210> 12 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> CD3ζ activation domain <400> 12 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttgcacat gcaggccctg 120 ccccctcgc 129 <210> 13 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> CD28 co-stimulatory domain <400> 13 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 14 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> CD28 co-stimulatory domain <400> 14 aggagcaagc ggagcagact gctgcacagc gactacatga acatgacccc ccggaggcct 60 ggccccaccc ggaagcacta ccagccctac gcccctccca gggatttcgc cgcctaccgg 120 agc 123 <210> 15 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> IL-2-Rβ intracellular domain <400> 15 Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn 1 5 10 15 Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly 20 25 30 Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe 35 40 45 Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu 50 55 60 Arg Asp Lys Val Thr Gln Leu Leu Pro Leu Asn Thr Asp Ala Tyr Leu 65 70 75 80 Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 85 90 <210> 16 <211> 282 <212> DNA <213> Artificial Sequence <220> <223> IL-2-Rβ intracellular domain <400> 16 aactgcagga acaccgggcc atggctgaag aaggtcctga agtgtaacac cccagacccc 60 tcgaagttct tttcccagct gagctcagag catggaggcg acgtccagaa gtggctctct 120 tcgcccttcc cctcatcgtc cttcagccct ggcggcctgg cacctgagat ctcgccacta 180 gaagtgctgg agagggacaa ggtgacgcag ctgctccccc tgaacactga tgcctacttg 240 tctctccaag aactccaggg tcaggaccca actcacttgg tg 282 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> STAT5 recruitment motif <400> 17 Tyr Leu Ser Leu 1 <210> 18 <211> 760 <212> PRT <213> Homo sapiens <400> 18 Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu 1 5 10 15 Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30 Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala 35 40 45 Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly 50 55 60 Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly 65 70 75 80 Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95 Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro 100 105 110 Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys 115 120 125 Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Ser Thr Ile 130 135 140 Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln 145 150 155 160 Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe 165 170 175 Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val 180 185 190 Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg 195 200 205 Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220 Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys 225 230 235 240 Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile 245 250 255 Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu 260 265 270 Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe 275 280 285 Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly 290 295 300 Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln 305 310 315 320 Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335 Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345 350 Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser 355 360 365 Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile 370 375 380 Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp 385 390 395 400 His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala 405 410 415 Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met 420 425 430 Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445 Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460 Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr 465 470 475 480 Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val 485 490 495 Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn 500 505 510 Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp 515 520 525 Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe 530 535 540 Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp 545 550 555 560 Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575 Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585 590 Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn 595 600 605 Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp 610 615 620 Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln 625 630 635 640 Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu 645 650 655 Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys 660 665 670 Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro 675,680,685 Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690,695,700 Gly Ser His Thr Thr Pro Ala Let Glu Asn Let Lys Let Arg Lys 705 710 715 720 Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala 725 730 735 Leu Only Thr Trp Thr Ile Gln Gly Only Asn Only Leu Ser Gly Asp 740,745,750 Val Trp Asp Ile Asp Asn Glu Phe 755,760 <210> 19 <211> 300 <212> DNA <213> CD3Please Contact Us <400> 19 aagaatagaa agccaaggc caagcctgtg acacgaggag cgggtgct...

Claims

1. An immune cell comprising: a. a first engineered receptor comprising a transmembrane region and an extracellular region, wherein the extracellular region comprises a first scFv comprising a first ligand binding domain capable of specifically binding to an epidermal growth factor receptor (EGFR) ligand; and b. a second engineered receptor, comprising a transmembrane region and an extracellular region, wherein the extracellular region comprises a second scFv comprising a second ligand binding domain capable of specifically binding to a human leukocyte antigen A*02 (HLA-A*02) allele, wherein the binding of the first ligand binding domain to the EGFR ligand activates or promotes the activation of the immune cell by the first engineered receptor, wherein the binding of the second ligand binding domain to the HLA-A*02 allele inhibits the activation of the immune cell by the first engineered receptor, and wherein the first ligand binding domain has a CDR H1 as shown in SEQ ID NO: 137, a CDR H2 as shown in SEQ ID NO: 143, a CDR H3 as shown in SEQ ID NO: 149, a CDR L1 as shown in SEQ ID NO: 155, a CDR L2 as shown in SEQ ID NO: 160, and a CDR L3 as shown in SEQ ID NO: 166, and Wherein the second ligand binding domain has CDR H1 as shown in SEQ ID NO: 41, CHR H2 as shown in SEQ ID NO: 42, CDR H3 as shown in SEQ ID NO: 43, CDR L1 as shown in SEQ ID NO: 44, CDR L2 as shown in SEQ ID NO: 45 and CDR L3 as shown in SEQ ID NO:

46.

2. An immune cell as described in claim 1, wherein the second ligand binding domain comprises SEQ ID NO: 53 or a sequence having at least 90%, at least 95% or at least 99% identity thereto.

3. The immune cell of claim 1, wherein the second engineered receptor comprises an intracellular domain isolated or derived from LILRB1.

4. The immune cell of claim 3, wherein the intracellular domain from LILRB1 comprises a sequence that is at least 95% identical to SEQ ID NO:

76.

5. The immune cell of claim 1, wherein the second engineered receptor comprises a LILRB1 transmembrane domain or a functional variant thereof.

6. The immune cell of claim 5, wherein the LILRB1 transmembrane domain or a functional variant thereof comprises a sequence that is at least 95% identical to SEQ ID NO:

85.

7. The immune cell of claim 1, wherein the second engineered receptor comprises a LILRB1 hinge domain or a functional fragment or variant thereof.

8. The immune cell of claim 7, wherein the LILRB1 hinge domain comprises a sequence that is at least 95% identical to SEQ ID NO: 84, SEQ ID NO: 77 or SEQ ID NO:

78.

9. The immune cell of claim 1, wherein the second engineered receptor comprises the LILRB1 intracellular domain and the LILRB1 transmembrane domain or a functional variant thereof.

10. The immune cell of claim 9, wherein the LILRB1 intracellular domain and the LILRB1 transmembrane domain comprise SEQ ID NO: 80 or a sequence that is at least 95% identical to SEQ ID NO:

80.

11. The immune cell of claim 1, wherein the first engineered receptor and the second engineered receptor are expressed on the surface of the immune cell at a ratio of first engineered receptor to second engineered receptor between 1:10 and 10:

1.

12. The immune cell of claim 1, wherein the first engineered receptor and the second engineered receptor are expressed on the surface of the immune cell at a ratio of first engineered receptor to second engineered receptor between 1:3 and 3:

1.

13. The immune cell of claim 1, wherein the immune cell is selected from the group consisting of: a T cell, a B cell, and a Natural Killer (NK) cell.

14. The immune cell of claim 1, wherein the immune cell is a T cell.

15. The immune cell of claim 1, wherein the immune cell is non-natural.

16. The immune cell of claim 1, wherein the immune cell is isolated.

17. A pharmaceutical composition comprising a plurality of immune cells according to any one of claims 1 to 16.

18. Use of multiple immune cells as described in any one of claims 1-16 in the preparation of adoptive cell therapy drugs for treating EGFR+ cancer, wherein the cells of the cancer express EGFR ligands, and the EGFR ligands are recognized by any one of the CDRs selected from SEQ ID NO: 137, SEQ ID NO: 143, SEQ ID NO: 149, SEQ ID NO: 155, SEQ ID NO: 160 and SEQ ID NO:

166.

19. The use of claim 18, wherein the cells of the cancer do not express the HLA-A*02 allele due to loss of heterozygosity or loss of the Y chromosome.

20. The use of claim 18, wherein non-target cells express both the EGFR ligand and the HLA-A*02 allele.

21. The use of claim 19, wherein non-target cells express both the EGFR ligand and the HLA-A*02 allele.

22. Use of the pharmaceutical composition as described in claim 17 in the preparation of an adoptive cell therapy drug for treating EGFR+ cancer, wherein the cells of the cancer express an EGFR ligand, and the EGFR ligand is recognized by any one of the CDRs selected from SEQ ID NO: 137, SEQ ID NO: 143, SEQ ID NO: 149, SEQ ID NO: 155, SEQ ID NO: 160 and SEQ ID NO:

166.

23. The use of claim 22, wherein the cells of the cancer do not express the HLA-A*02 allele due to loss of heterozygosity or loss of the Y chromosome.

24. The use of claim 22, wherein non-target cells express both the EGFR ligand and the HLA-A*02 allele.

25. The use of claim 23, wherein non-target cells express both the EGFR ligand and the HLA-A*02 allele.

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