Cells overexpressing CD31 and methods of use thereof

Abstract

Provided herein are engineered immune cells overexpressing a CD31 polypeptide.

Description

Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P CELLS OVEREXPRESSING CD31 AND METHODS OF USE THEREOF CLAIM OF PRIORITY

[0001] This application claims the benefit of US Provisional Application No.63 / 516,745, filed July 31, 2023. The entire content of the foregoing application is hereby incorporated by reference. SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically as an XML file named “51624-0080WO1_SL_ST26.XML.” The XML file, created on July 31, 2024, is 37,640 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] This disclosure relates to cells overexpressing a CD31 polypeptide and methods of use thereof. BACKGROUND

[0004] The development of universal off-the-shelf cell therapy products (e.g., CAR-T) is a major goal of next-generation cell therapies. General-purpose cell therapies based on "allogeneic" cells generally have the issue of "transplantation rejection," where the transplanted cells induce Host-versus-Graft (HvG) reactions, and are cleared by the patient's immune system, especially by natural killer cells (NK cells) and T lymphocytes. This reduces the persistence and curative effects of the allogeneic cell therapies in the patient. Thus, there is a need to prevent or reduce HvG reactions and at the same time, improve the efficacy of allogeneic cell therapies. SUMMARY

[0005] The present disclosure relates to the prevention or mitigation of Host-versus-Graft (HvG) reactions related to cell therapies (e.g., CAR-T or CAR-NK therapies). Specifically, the disclosure relates to engineered cells that overexpress CD31 polypeptides for the prevention or mitigation of T cell- or NK cell-mediated killing.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0006] In one aspect, the disclosure is related to engineered cells that overexpress a CD31 polypeptide, wherein the engineered cell is an allogeneic cell isolated from a donor (e.g., for being administered to a subject).

[0007] In some embodiments, the engineered cell expresses an exogenous CD31 polypeptide.

[0008] In some embodiments, the CD31 polypeptide comprises an extracellular domain of CD31.

[0009] In some embodiments, the CD31 polypeptide comprises an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0010] In some embodiments, the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

[0011] In some embodiments, the truncated intracellular domain of CD31 comprises 2 amino acids of the intracellular domain of CD31.

[0012] In some embodiments, the extracellular domain of CD31 comprises an amino acid sequence set forth in SEQ ID NO:1 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0013] In some embodiments, the CD31 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0014] In one aspect, the present disclosure is related to engineered cells that express a CD31 polypeptide comprising an extracellular domain, wherein the extracellular domain comprises at least a portion of C2 Ig domains of CD31.

[0015] In some embodiments, the extracellular domain comprises only 1, 2, 3, 4 or 5 C2 Ig domains of CD31.

[0016] In some embodiments, the extracellular domain comprises no more than 5 C2 Ig domains of CD31.

[0017] In some embodiments, the C2 Ig domains are selected from C2 Ig domains 1, 2, 3, 4, 5 and 6 of CD31.

[0018] In some embodiments, the extracellular domain of CD31 comprises an amino acid sequence set forth in one or more of SEQ ID NOs:16-21 or a sequence that is at least 80%, 85%,Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in one or more of SEQ ID NOs: 16-21.

[0019] In some embodiments, the extracellular domain comprising a membrane proximal region of CD31.

[0020] In some embodiments, the membrane proximal region comprises a portion of C2 Ig domain 6 of CD31.

[0021] In some embodiments, the membrane proximal region comprises or consists of an amino acid sequence set forth in SEQ ID NO:12 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0022] In some embodiments, the CD31 polypeptide comprises a transmembrane domain of CD31.

[0023] In some embodiments, the transmembrane domain of CD31 comprises an amino acid sequence set forth in SEQ ID NO: 4 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0024] In some embodiments, the CD31 polypeptide comprises an intracellular domain of CD31.

[0025] In some embodiments, the intracellular domain of CD31 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 9 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 9.

[0026] In some embodiments, the intracellular domain is a truncated intracellular domain, and wherein the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

[0027] In some embodiments, the truncated intracellular domain of CD31 comprises 2 amino acids of the intracellular domain of CD31.

[0028] In some embodiments, the CD31 polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 10-12 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0029] In some embodiments, the engineered cell is selected from a group consisting of a T cell, a NK cell, a Treg cell, a B cell, a monocyte, a macrophage cell, a dendritic cell or a combination thereof.

[0030] In some embodiments, the T cell is selected from a group consisting of a natural killer T (NK-T) cell, a γδ T cell, and an αβ T cell.

[0031] In some the αβ T cell is modified to be suitable for allogeneic cell therapy.

[0032] In some embodiments, the expression of the CD31 on the engineered cell is increased about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a wild-type cell or the control cell.

[0033] In some embodiments, the engineered cell further expresses a chimeric antigen receptor (CAR), TCR, or TAC receptor.

[0034] In some embodiments, the CAR specifically targets an antigen.

[0035] In some embodiments, the antigen is a tumor antigen.

[0036] In some embodiments, the tumor antigen selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF- R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta-like 3 (DLL3), CD70, CS-1, c-Met, Glycolipid F77, PD-L1, and PD-L2.

[0037] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 3 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0038] In one aspect, the present disclosure is related to compositions comprising any one of the engineered cells described herein, and a pharmaceutically acceptable carrier.

[0039] In one aspect, the present disclosure is related to methods of making any one of the engineered cells described herein, comprising introducing a vector expressing the CD31 polypeptide into a cell, thereby making the engineered cell.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0040] In some embodiments, the vector is a retroviral vector.

[0041] In one aspect, the present disclosure is related to methods of treating a disease or disorder in a subject, the method comprising administering to the subject, an effective amount of any one of the engineered cells described herein, or any one of the compositions described herein.

[0042] In some embodiments, the disease or disorder is cancer, autoimmune disease, or infection.

[0043] In one aspect, the present disclosure is related to methods of inhibiting immune clearance of an engineered cell in a subject, comprising administering any one of the engineered cells described herein to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell.

[0044] In some embodiments, the immune clearance of the engineered cell is through T cell- mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

[0045] In one aspect, the present disclosure is related to methods of increasing the in vivo expansion of an engineered cell in a subject, comprising administering any one of the engineered cells described herein to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell.

[0046] In one aspect, the present disclosure is related to methods of reducing Host-versus- Graft (HvG) reactions in a subject, comprising administering any one of the engineered cells described herein to a subject in need thereof, thereby reducing HvG reactions in the subject.

[0047] In some embodiments, the subject has undergone or will receive a cell or organ transplantation. In some embodiments, the cell transplantation is a stem cell transplantation.

[0048] In some embodiments, the engineered cell is administered before, during and / or after the cell or organ transplantation.

[0049] In some embodiments, the engineered cell is a CAR-T cell.

[0050] In some embodiments, the subject is a human subject.

[0051] In one aspect, the present disclosure is related to CD31 polypeptides comprising or consisting of an extracellular domain, wherein the extracellular domain comprises at least a portion of C2 Ig domains of CD31.

[0052] In some embodiments, the extracellular domain comprises only 1, 2, 3, 4 or 5 C2 Ig domains of CD31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0053] In some embodiments, the extracellular domain comprises no more than 5 C2 Ig domains of CD31.

[0054] In some embodiments, the C2 Ig domains are selected from C2 Ig domains 1, 2, 3, 4, 5 and 6 of CD31.

[0055] In some embodiments, the extracellular domain of CD31 comprises an amino acid sequence set forth in one or more of SEQ ID NOs:16-21 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in one or more of SEQ ID NOs: 16-21.

[0056] In some embodiments, the extracellular domain comprising a membrane proximal region of CD31.

[0057] In some embodiments, the membrane proximal region comprises a portion of C2 Ig domain 6 of CD31.

[0058] In some embodiments, the membrane proximal region comprises or consists of an amino acid sequence set forth in SEQ ID NO:12 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0059] In some embodiments, the CD31 polypeptide comprises a transmembrane domain of CD31.

[0060] In some embodiments, the transmembrane domain of CD31 comprises an amino acid sequence set forth in SEQ ID NO: 4 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0061] In some embodiments, the CD31 polypeptide comprises an intracellular domain of CD31.

[0062] In some embodiments, the intracellular domain of CD31 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 9 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 9.

[0063] In some embodiments, the intracellular domain is a truncated intracellular domain, and wherein the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0064] In some embodiments, the truncated intracellular domain of CD31 comprises 2 amino acids of the intracellular domain of CD31.

[0065] In some embodiments, the CD31 polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 10-12 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.

[0066] In some embodiments, the CD31 polypeptide comprises a truncated intracellular domain of CD31 and one of the following: (1) a CD31 extracellular domain comprising amino acids of SEQ ID NO: 1 and a K89A mutation and a N25Q mutation; (2) a CD31 extracellular domain comprising an amino acid sequence of SEQ ID NO: 12 and a CD8 hinge region; (3) a CD31 extracellular domain comprising amino acids of SEQ ID NOs: 16-17 and a CD28 hinge and transmembrane domain; (4) a CD31 extracellular domain comprising the amino acid of SEQ ID NO: 12 and a transmembrane domain of CD31 and an intracellular domain of CD31; and (5) a CD31 extracellular domain comprising the amino acid of SEQ ID NO: 12 and a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0067] In one aspect, the present disclosure is related to nucleic acids comprising one or more nucleic acid sequences encoding any one of CD31 polypeptides described herein; and optionally an engineered receptor.

[0068] In one aspect, the present disclosure is related to vectors comprising any one of the nucleic acids described herein.

[0069] In one aspect, the present disclosure is related to engineered cells comprising any one of the CD31 polypeptides described herein; any one of the nucleic acids described herein; or any one of the vectors described herein.

[0070] In one aspect, the disclosure is related to an engineered cell that overexpresses a CD31 polypeptide, wherein the engineered cell is an allogeneic cell isolated from a donor (e.g., for being administered to a subject).

[0071] In some embodiments, the engineered cell expresses an exogenous CD31 polypeptide.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0072] In some embodiments, the CD31 polypeptide comprises an extracellular domain of CD31.

[0073] In some embodiments, the CD31 polypeptide comprises an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0074] In some embodiments, the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

[0075] In some embodiments, the extracellular domain of CD31 comprises an amino acid sequence set forth in SEQ ID NO:1 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0076] In some embodiments, the CD31 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0077] In some embodiments, the engineered cell is selected from a group consisting of a T cell, a NK cell, a Treg cell, a B cell, a monocyte, a macrophage cell, a dendritic cell or a combination thereof.

[0078] In some embodiments, the T cell is selected from a group consisting of a natural killer T (NK-T) cell, a γδ T cell, and an αβ T cell which is modified to be suitable for allogeneic cell therapy.

[0079] In some embodiments, the engineered cell is a cell line.

[0080] In some embodiments, the expression of the CD31 on the engineered cell is increased about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a wild-type cell or the control cell.

[0081] In some embodiments, the engineered cell further expresses a chimeric antigen receptor (CAR), TCR, or TAC receptor.

[0082] In some embodiments, the CAR specifically targets an antigen.

[0083] In some embodiments, the antigen is a tumor antigen.

[0084] In some embodiments, the antigen is a tumor antigen selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholineAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta-like 3 (DLL3), CD70, CS-1, c-Met, Glycolipid F77, PD-L1, and PD-L2.

[0085] In some embodiments, the CAR comprises an amino acid sequence set forth in SEQ ID NO: 3 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0086] In one aspect, the disclosure is related to a composition comprising any one of the engineered cells described herein, and a pharmaceutically acceptable carrier.

[0087] In one aspect, the disclosure is related to a method of making any one of the engineered cells described herein, comprising introducing a vector expressing the CD31 polypeptide into a cell, thereby making the engineered cell.

[0088] In some embodiments, the vector is a retroviral vector.

[0089] In one aspect, the disclosure is related to a method of treating a disease or disorder in a subject, the method comprising administering to the subject, an effective amount of any one of the engineered cells described herein, or the composition described herein.

[0090] In some embodiments, the disease or disorder is cancer, autoimmune disease, or infection.

[0091] In one aspect, the disclosure is related to a method of inhibiting immune clearance of an engineered cell in a subject, comprising: (a) introducing a vector expressing an exogenous CD31 polypeptide into the engineered cell, wherein the exogenous CD31 polypeptide comprises an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31, thereby overexpressing the CD31 or a portion thereof; (b) administering the engineered cell from step (a) to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell.

[0092] In some embodiments, the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

[0093] In some embodiments, the truncated intracellular domain of CD31 comprises 2 amino acids (KC) of the intracellular domain of CD31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0094] In some embodiments, the CD31 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0095] In some embodiments, the engineered cell is a CAR-T cell.

[0096] In some embodiments, the immune clearance of the engineered cell is through T cell- mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

[0097] In one aspect, the disclosure is related to a method of increasing the in vivo expansion of an engineered cell in a subject, comprising: (a) introducing a vector expressing an exogenous CD31 polypeptide into the engineered cell, wherein the exogenous CD31 polypeptide comprises an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31, thereby overexpressing the CD31 or a portion thereof; (b) administering the engineered cell from step (a) to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell.

[0098] In some embodiments, the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

[0099] In some embodiments, the truncated intracellular domain of CD31 comprises 2 amino acids (KC) of the intracellular domain of CD31.

[0100] In some embodiments, the CD31 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0101] In some embodiments, the subject is a human subject.

[0102] In one aspect, the disclosure is related to a CD31 polypeptide comprising an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0103] In some embodiments, the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

[0104] In some embodiments, the truncated intracellular domain of CD31 comprises 2 amino acids (KC) of the intracellular domain of CD31.

[0105] In some embodiments, the CD31 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0106] In one aspect, the disclosure is related to a nucleic acid, comprising one or more nucleic acid sequences encoding any one of the CD31 polypeptides described herein; and optionally an engineered receptor.

[0107] In one aspect, the disclosure is related to a vector comprising the nucleic acid described herein.

[0108] Other features and advantages of the disclosure will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS

[0109] FIGs. 1A-1C show that CD31tICD (CD31 with truncated intracellular domain) protects T2 cells from NK Cell and TCR-T cell-mediated killing. FIG.1A shows the expression of RV-CD31tICD-BFP in T2 cells 3 days after transduction. FIG.1B shows NK92 cell-mediated killing on T2 cells or CD31tICD-overexpressing T2 cells 4 hours after co-culture. The effector NK92 to target cell ratio ranged from 10:1 to 0.625:1. FIG. 1C shows E7-NCI TCR-T cell- mediated killing on T2 cells or CD31tICD-overexpressing T2 cells after overnight co-culture. The effector TCR-T cells to target cell ratio ranged from 10:1 to 0.625:1.

[0110] FIGs. 2A-2B show overexpression of CD31tICD and other candidate genes in γδT cells. FIG.2A shows Donor 4 (D4) and Donor 22 (D22) γδT cell purity on Day 9. FIG.2B shows the transduction efficiency of CD31tICD and other candidate genes in D4 and D22 γδT cell.

[0111] FIGs. 3A-3D show that overexpression of CD31tICD and other candidate genes promoted γδT cell expansion. FIGs.3A-3B show D4 γδT and D22 γδT cell count on day9 with or without the overexpression of CD31tICD and other candidate genes. FIGs.3C-3D show D4 γδT and D22 γδT cell expansion on day9 with or without the overexpression of CD31tICD and other candidate genes.

[0112] FIGs.4A-4B show the killing capability of D4 BCMA-CAR or D22 BCMA-CAR γδT cells on H929 with or without the expression of CD31tICD; the effector to target ratio ranged from 10:1 to 0.3:1. BCMA-CAR γδT cell function is not affected by the expression of CD31tICD.

[0113] FIGs.5A-5E show that CD31tICD protected γδT cells from NK cell-mediated killing. FIGs.5A-5B shows the killing capability of 5 different donor NK cells on D4 and D22 γδT cells. 5 different donor derived NK cells (D15, D17, D19, D32, D0081) were co-cultured with two different donors expanded γδT cells (D4 and D22) overnight at different E:T ratio. NK cells wereAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P effector cells. γδT cells were target cells. FIG.5C shows the B2M KO efficiency in D4 and D22 γδT cells. FIG.5D shows the killing capability of NK cells on D4 γδT cells with or without the expression of CD31tICD after 4hr of co-culture. T2 tumor cells and B2M KO D4 γδT cells were used as control. The effector to target ratio ranged from 10:1 to 0.3:1. FIG.5E shows the killing capability of NK cells on Donor4 γδT cells with or without the expression of CD31tICD after overnight co-culture. T2 tumor cells, B2M KO D4 γδT cells and D4-CD47 were used as control. The effector to target ratio ranged from 10:1 to 0.3:1.

[0114] FIG. 6 shows that CD31tICD protected γδT cells from TCR-T cell-mediated killing. The killing capability of E7 NCI TCR-T cells on D22 γδT cells with or without the expression of CD31tICD after overnight co-culture was shown. T2 tumor cells, B2M KO D4 γδT cells and D22- CD47 were used as control. The effector to target ratio ranged from 10:1 to 0.3:1.

[0115] FIG. 7 is a schematic illustration of CD31 full length, truncated intracellular domain and CD31v3 engineered variants. CD31fl: CD31 full length; CD31tICD: CD31 with a truncated intracellular domain; CD31v3-1: membrane proximal region + full-length transmembrane domain + full-length intracellular domain; CD31v3-2: membrane proximal region + full-length transmembrane domain + truncated intracellular domain; and CD31v3-3: only contains membrane proximal region.

[0116] FIGs.8A-8B shows BCMA CAR expression rates and MFI values of various BCMA CAR g9d2 T cells for functional assays. FIG.8A shows that HLA-A2-negative PBMC donor cells were activated / expanded in g9d2 T cell media and transduced with BCMA CAR retroviruses at MOI=1. At setting up of the 3-way mixed lymphocyte reaction assays, the transduction rates of various BCMA CAR g9d2 T cells were normalized to round 55%. FIG.8B shows the MFI values of various BCMA CAR g9d2 T cells after BCMA CAR percentages were normalized round 55%. UTD: un-transduced g9d2 T cells. Vγ9: anti-human TCR Vγ9 antibody. BCMA CAR+CD47 is used as a control. The dashed line shows the MFI value of BCMA CAR alone.

[0117] FIG.9 shows representative flow cytometry data showing that BCMA CAR+CD31v3 g9d2 T cells persist better in 3-way mixed lymphocyte reaction assays (3-w MLR). Allogeneic HLA-A2-positive or autologous HLA-A2-negative PBMC samples were labelled with cell trace violet (CVT) and then were mixed with the g9d2 T cells and NCI-H929 tumor cells. The ratio of PBMC-CVT:g9d2 T cells:NCI-H929 cells is 200 or 400:1:1 at the 3-w MLR assay set up. Half media were replenished every other day. The cells in each sample were counted and evaluated byAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P flow cytometry on day 4 and 8 or 9. HLA-A2: anti-human HLA-A2 antibody. PBMC-CVT: PBMC labelled with CVT. Allo1, 2, and 3 are allogeneic HLA-A2-positive PBMC 1, 2, and 3. Auto is autologous HLA-A2-negative PBMC sample. The dashed-line box highlights CD31 variants.

[0118] FIGs. 10A-10B show that HLA-A2-negative BCMA CAR+CD31v3-2 and 3 g9d2 T cells persist better in 3-way mixed lymphocyte reaction assays (3-w MLR) at two different ET ratios. In FIG.10A, half media were replenished every other day. The cells in each sample were counted and analyzed by flow cytometry on day 4 and day 9. HLA-A2-negative BCMA CAR+ cell numbers were calculated with live cell count and flow cytometry data. Representative of 2 donor pairs of allogeneic PBMC:BCMA CAR g9d2 T:NCI-H929 is 200:1:1. In FIG. 10B, half media were replenished every other day and the cells in each sample were counted and analyzed by flow cytometry on day 4 and day 8. HLA-A2-negative BCMA CAR g9d2 T cell numbers were calculated with live cell count and flow cytometry data. Representative of 6 donor pairs of allogeneic PBMC: BCMA CAR g9d2 T: NCI-H929 is 400:1:1. The dashed-lines indicate the level of BCMA-CAR g9d2 T cells on day 8 or 9.

[0119] FIGs. 11A-11B show that BCMA CAR+CD31v3-2 and 3 g9d2 T cells have comparable tumor killing capabilities and persist better. In FIG. 11A, BCMA CAR+CD31v3-2 and 3 g9d2 T cells showed similar killing capabilities of NCI-H929 cells to BCMA CAR and BCMA CAR+CD47 controls. In FIG.11B, BCMA CAR+CD31v3-2 and 3 g9d2 T cells persisted better than BCMA CAR and BCMA CAR+CD47 controls. NCI-H929 tumor cells and BCMA CAR g9d2 T cells were cocultured at ET ratio 1:1. The same number of NCI-H929 cells used at round 1 were continuously added into the coculture to rechallenge the g9d2 T cells every 24h after round 1 till round 5. Half media was replenished every other day. Cell count and flow cytometry analysis were performed at round 1, 3 and 5 tumor cell challenge. Representative of 2 tumor rechallenge experiments. The tumor rechallenge experiments were performed two times. The figure shows representative results.

[0120] FIG.12 lists some sequences described in the present disclosure. DETAILED DESCRIPTION

[0121] Cell therapies based on "allogeneic" cells generally have the issues of "transplantation rejection." Usually, the transplanted cells can induce Host-versus-Graft (HvG) reactions, and theseAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P cells are killed by the subject's immune system, e.g., by natural killer cells (NK cells) and T lymphocytes. This may reduce the persistence and efficacy of the allogeneic cell products.

[0122] CD31 is a transmembrane glycoprotein and contains both adhesive and signaling function. The present disclosure demonstrated that the overexpression of CD31 prevented both NK cell- and TCR-T cell-mediated killing in T2 tumor cells and human primary type II γδ T cells. The overexpression of CD31 can also promote the expansion and survival of type II γδ T cells. Therefore, the cells and methods described herein have various applications in allogeneic cell therapy and transplantation.

[0123] The present disclosure is based on, in part, that engineered allogeneic immune cells that overexpress a CD31 polypeptide can reduce NK cell- and TCR-T cell-mediated killing, and can be used in allogeneic cell therapies. In one aspect, the disclosure provides engineered immune cells overexpressing a CD31 polypeptide.

[0124] In one aspect, the disclosure provides engineered cells that overexpress a CD31 polypeptide. The engineered cells can be allogeneic cell isolated from a donor.

[0125] In one aspect, the disclosure provides compositions comprising the engineered cell described herein. In one aspect, the disclosure provides methods of making the engineered cell described herein.

[0126] In one aspect, the disclosure provides methods of treating a disease or disorder in a subject by administering an effective amount of the engineered cell described herein.

[0127] In one aspect, the disclosure provides methods of inhibiting immune clearance of an engineered cell by overexpressing a CD31 polypeptide on the engineered cell.

[0128] In one aspect, the disclosure provides methods of increasing the in vivo expansion of an engineered cell by overexpressing a CD31 polypeptide on the engineered cell.

[0129] In one aspect, the disclosure provides engineered cell that expresses a non-native CD31 polypeptide, and also engineered cell that overexpresses a CD31 polypeptide.

[0130] As used herein, the term “derived from” when made in reference to a domain or protein described herein refers to a domain or protein that is obtained from the relevant the domain or protein with or without additional modifications (e.g., by recombinant expression or de novo synthesis). The term encompasses domains with naturally occurring sequences and sequences with mutations. A domain derived from a particular protein can have a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the relevant functional portion of the particularAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P protein. A domain derived from a particular protein can be from a natural or a synthetic source. For example, an intracellular domain derived from CD31 can have a sequence that is identical to the intracellular domain of CD31, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of the intracellular domain of CD31.

[0131] As used herein, the term “CD31 polypeptide” refers to a polypeptide derived from a wild-type CD31 or a functional variant thereof. The CD31 may be a wild-type CD31 (e.g., human CD31). The CD31 may have one or more mutations (e.g., insertions, deletions, or substitutions). The CD31 may be a human CD31. The CD31 polypeptide may include an extracellular domain of CD31, a transmembrane of CD31 and / or an intracellular domain of CD31. The CD31 may be a truncated CD31. The CD31 may include an extracellular domain of CD31, a transmembrane of CD31 and a truncated intracellular domain of CD31. The CD31 polypeptide may include a hinge region, a transmembrane domain, and / or an intracellular domain from a different molecule. For example, the CD31 polypeptide may include a hinge region from CD8 or CD28. The CD31 polypeptide may include a transmembrane domain from CD8 or CD28. The hinge region, transmembrane domain or intracellular domain may be full-length or a portion thereof.

[0132] As used herein, the terms “extracellular domain” or “extracellular region” are used interchangeably herein to refer to the portion of a receptor that is outside the cell membrane. The extracellular domain can be entire portion of a receptor that is outside the cell membrane, or just a part thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). The extracellular domain can be derived from the extracellular domain of a wild- type receptor or a functional variant thereof. The extracellular domain may have one or more mutations, including e.g., insertions, deletions, or substitutions. An extracellular domain of CD31 may include the full length of the extracellular domain of, e.g., a wild-type human CD31. In some embodiments, an extracellular domain of CD31 includes a fragment (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion) of the full length of the extracellular domain of, e.g., a wild-type human CD31. CD31 is a type I transmembrane molecule with six C2 Ig domains (see, e.g., Clare M. Isacke, Michael A. Horton, CD31: Platelet endothelial cell adhesion molecule-1, PECAM-1, Editor(s): Clare M. Isacke, Michael A. Horton, In Factsbook, The Adhesion Molecule FactsBook (Second Edition), Academic Press, 2000, Pages 94-96, ISBN 9780123565051). The first two Ig domains, with contributions from domains 3, 5 and 6, mediate the homophilic interaction, whereas domain 2 is the main domain interacting with integrins andAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P glycosaminoglycans. Alternative splicing and tyrosine phosphorylation within the cytoplasmic domain modulates the configuration of the ligand binding domain, suggesting a mechanism for regulating ligand specificity. As used herein, “C2 Ig domain” may also be referred to as “immunoglobulin-like domain,” “Ig-related domain,” or “Ig-like C2-type” (see, e.g., Newton, et al., Journal of Biological Chemistry, Volume 272, Issue 33, 1997, Pages 20555-20563; Fawcett et al., The Journal of Cell Biology, Volume 128, Number 6, March 19951229-124; and the UniProt website).

[0133] For example, in wild-type human CD31, C2 Ig domain 1 may have an amino acid sequence of SEQ ID NO: 16; C2 Ig domain 2 may have an amino acid sequence of SEQ ID NO: 17; C2 Ig domain 3 may have an amino acid sequence of SEQ ID NO: 18; C2 Ig domain 4 may have an amino acid sequence of SEQ ID NO: 19; C2 Ig domain 5 may have an amino acid sequence of SEQ ID NO: 20; and C2 Ig domain 6 may have an amino acid sequence of SEQ ID NO: 21. An extracellular domain of CD31 may include one or more of the C2 Ig domains (e.g., 1, 2, 3, 4, 5 or 6 C2 Ig domains) or any fragment(s) of the C2 Ig domains (e.g., a fragment of C2 Ig domain 6 having an amino acid sequence of SEQ ID NO: 21) of the extracellular domain of CD31.

[0134] An extracellular domain of CD31 may include a membrane proximal region. As used herein, a membrane proximal region of CD31 is a region of the extracellular domain of CD31 that is proximal to the cell membrane. The membrane proximal region of CD31 may be N-terminal to the transmembrane domain of CD31. Membrane proximal region of CD31 may have a length of about 1-50, 1-40, 1-30, 1-20, 1-10, 10-50, 10-40, 10-30, 10-20 or 20-30 amino acids. The membrane proximal region of CD31 may have a length of about 23 amino acids. Membrane proximal region of CD31 may include an amino acid sequence of that is about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12.

[0135] As used herein, the terms “intracellular domain”, “intracellular region” or “cytoplasmic region” are used interchangeably herein to refer to the portion of a receptor that is inside the cell. The intracellular domain can be entire portion of a receptor that is inside the cell, or just a part thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). The intracellular domain can be derived from the intracellular domain of a wild-type receptor or a functional variant thereof. The intracellular domain may have one or more mutations, including e.g., insertions, deletions, or substitutions.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0136] As used herein, the terms “transmembrane domain” or “transmembrane region” are used interchangeably herein to refer to the portion of a receptor that is embedded in the cell membrane. The transmembrane region can be entire portion of a receptor that is embedded in the cell membrane, or just a part thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). The transmembrane region can be derived from the transmembrane region of a wild-type receptor or a functional variant thereof. The transmembrane region may have one or more mutations, including e.g., insertions, deletions, or substitutions.

[0137] As used herein, a “vector” is any construct capable of delivering one or more nucleic acids of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more nucleic acids of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the nucleic acid of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the nucleic acid of interest such that the nucleic acid of interest will be translated in the host cell introduced with the expression vector.

[0138] As used herein, the term “chimeric antigen receptor” or “CAR” as used herein refers to genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells. Some CARs are also known as “artificial T-cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” A CAR may comprise an extracellular ligand binding domain or an extracellular antigen binding domain specific for one or more ligands or antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain. “CAR-T cell” refers to a T cell that expresses a CAR.

[0139] As used herein, the term “T-cell receptor” or “TCR” as used herein refers to an endogenous or modified T-cell receptor comprising an extracellular antigen binding domain that binds to a specific antigenic peptide bound in an MHC molecule. The TCR may comprise a TCRα polypeptide chain and a TCRβ polypeptide chain. The TCR may comprise a TCRγ polypeptide chain and a TCRδ polypeptide chain. The TCR may specifically bind a tumor antigen. “TCR-T” refers to a T cell that expresses a recombinant TCR. Expression of a heterologous antigen receptor, such as a heterologous TCR or CAR, can alter the immunogenic specificity of the T cells so thatAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P they recognize or display improved recognition for one or more tumor antigens that are present on the surface of the cancer cells of an individual with cancer.

[0140] As used herein, the term “antigen binding domain” or “extracellular antigen binding domain” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. An antigen binding fragment may comprise at least one variable domain (e.g., a variable domain of a heavy chain, single domain antibody or VHH). Non- limiting examples of antibody fragments include, e.g., Fab, Fab’, F(ab’)2, and Fv fragments.

[0141] As used herein, the term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen(s), cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

[0142] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided. Veterinary and non-veterinary applications areAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P contemplated by the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

[0143] As used herein, the term “donor” represents an organism from which a biological sample is produced from, for example, a human from whom cells can be obtained. The organism includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “donor” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. A “donor” can also refer to more than one donor, for example one or more humans or non-human animals or non-human mammals. However, advantageously, the donor is a mammal such as a human. The donor can be a cancer patient that is to be treated with a population of cells generated by the methods described herein (i.e., an autologous donor), or can be an individual who donates a sample that, upon generation of the population of cells generated by the methods described herein, will be used to treat a different individual or cancer patient (i.e., an allogeneic donor).

[0144] As used herein, the term “overexpress” generally refers to any amount greater than an expression level exhibited by a reference standard. The terms “overexpress,” “overexpressing,” “overexpressed” and “overexpression” in the present disclosure refer an expression of a gene product or a polypeptide at a level greater than the expression of the same gene product or polypeptide prior to a genetic alteration of the host cell or in a comparable host which has not been genetically altered at defined conditions. If a host cell does not comprise a given gene product, it is possible to introduce the gene product into the host cell for expression; in this case, any detectable expression is encompassed by the term “overexpression.”

[0145] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated byAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P reference in their entirety. In case of conflict, the present specification, including definitions, will control. HvG Reactions Against Engineered Immune Cells

[0146] Adoptive immunotherapy with T cells or NK cells that were engineered by transient or stable gene transfer to express a chimeric antigen receptor (CAR) is highly effective treatment for advanced chemotherapy- and radiotherapy-refractory malignancies in hematology and oncology. One of the greatest obstacles for the application of adoptive immunotherapy is host versus graft (HvG) reactions against allo-products, due to an immune response of the patient’s immune system against the transferred cells (e.g., CAR-T cells). The HvG reactions significantly reduce the persistence and efficacy of the cell therapies. Further, HvG reactions can cause side effects (e.g., fever) and safety concerns.

[0147] The highly polymorphic HLA molecules in the population are the main cause of T cell- mediated HvG. Since β2-microglobulin (B2M) is essential for the stable existence of HLA class I molecules on the cell membrane, knocking out B2M can remove HLA class I molecules from the cell surface, thereby effectively mitigate T cell-mediated HvG responses. Loss of B2M, however, significantly increases the sensitivity of allogeneic cells to NK cell mediated cytotoxicity, predisposing them to be cleared by NK cells.

[0148] At present, three common strategies exist for addressing HvG problems in allogeneic cell therapy (Table 1). Firstly, lymphocyte depletion drugs, including antibody drugs and bispecific chimeric antigen receptors (CAR), can be used to eliminate immune cells (especially NK cells and T cells) in the subject, thereby weakening or inhibiting the HvG response and prolong the persistence of allogeneic cell therapy products in the subject. Secondly, the allogeneic cell products could be modified by naturally occurring immunosuppressive molecules (by overexpressing, knocking down or knocking out), to reduce the activation of the subject's immune system, or to inhibit the subject's immune cells (especially NK cells and T cells), thereby reducing the rate at which allogeneic cell therapy products are cleared by the subject's immune system. Finally, according to the frequency and distribution of polymorphic HLA alleles across the population in a specific area, a variety of HLA haploidentical cells can be prepared, and a limited cell bank that covers most of the HLA types in the population can be established. Using the cellAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P bank, the immune response between HLA-incompatible donor and recipient cells can be reduced, and a longer persistence of allogeneic cell therapy products in the subject can be achieved. Table 1 Common strategies for addressing HvG reactions Strategy Immunosuppression Immune escape HLA matching Alemtuzumab (Anti-oweve , e a ove ee s a eg es a ave o v ous a su ou a e aw acks. Using lymphodepletion agents to clear or suppress the subject's immune system often increases the risk of infection, and antibody drug-mediated targeted cell clearance is expensive and often accompanied by obvious side effects. Due to a lack of clinical data, the effects of modifying naturally occurring immunosuppressive molecules (e.g., HLA-E overexpression and HLA-A / B / C knockout) are uncertain. Finally, building cell banks only partially avoids, and do not solve the HvG problem of allogeneic cell therapies.

[0150] The present disclosure provides methods of preventing, inhibiting, or reducing HvG reactions related to cell therapies (e.g., CAR-T or CAR-NK therapies). Specifically, provided herein are methods of reducing Host-versus-Graft (HvG) reactions in a subject, comprising administering any one of the engineered cells to a subject in need thereof, thereby reducing HvG reactions in the subject.

[0151] The subject may have undergone or be going to receive a cell or organ transplantation. The cell transplantation may be a stem cell transplantation. Methods of cell or organ transplantation are known in the art. Any suitable methods of cell or organ transplantation can be used in the methods described herein.

[0152] The engineered cell may be administered before, during and / or after the cell or organ transplantation. The engineered cell may be administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Suitable methods of administering the engineered cell are described herein and are known in the art.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P The CD31 Polypeptide

[0153] CD31, also known as PECAM-1, is a transmembrane glycoprotein that plays a critical role in regulating inflammation and immune responses. CD31 is expressed on endothelial cells, leukocytes, and platelets and has been implicated in various aspects of immune cell activation, migration, and adhesion. CD31 can engage in both homophilic (CD31-CD31) and heterophilic binding to other cell surface molecules. The proposed heterophilic counterreceptors of CD31 include a heparin-containing proteoglycan, the integrin αvβ3, a molecule expressed by parasitized red blood cells (RBC), and the ADP-ribosyl cyclase CD38. The T cell CD31 ligand (T-CD31L) is also a proposed heterophilic counter-receptor of CD31.

[0154] CD31 has been shown to modulate leukocyte trafficking and adhesion during inflammation by regulating integrin activation and endothelial cell junctional stability. Additionally, CD31 has been found to regulate immune cell activation and proliferation, including T cell activation and differentiation, as well as B cell activation and antibody production.

[0155] CD31 also regulates T-cell activation and tolerance in vivo by playing a unique modulatory function in the regulation of T-cell expansion, survival, and tolerance. It has been shown that CD31-mediated interactions contribute to the development of immune responses that are exquisitely T cell-dependent. Selective expression of CD31 by T cells, dendritic cells (DCs), and the endothelium might protect them against cytotoxicity by effector T cells, thus redirecting killing to CD31-expressing targets. Additionally, CD31 signals inhibit activation-induced T-cell death and induce Erk activation. These mechanisms contribute to regulating the size of clonal expansion and protecting against cytotoxicity by effector T cells. Therefore, disclosed herein are cells and methods for targeting CD31-mediated interactions as a therapeutic strategy for preventing transplant rejection and treating immune-related disorders.

[0156] A detailed review of CD31 and its functions can be found in Marelli-Berg et al., J Cell Sci (2013) 126 (11): 2343–2352; and Douaisi et al., J Immunol.2017 Mar 15; 198(6): 2310–2319, each of which is incorporated by reference in its entirety.

[0157] The present disclosure provides a method to preserve the efficacy of allogeneic cell therapy products and to overcome HvG in patients treated with a cell therapy, e.g., by overexpressing a CD31 polypeptide in the cell. In one aspect, the disclosure provides an allogeneicAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P cell therapy product that overexpresses a CD31 polypeptide. The cell may not express an endogenous CD31. The cell may express an endogenous CD31.

[0158] The present disclosure relates to a CD31 polypeptide comprising an extracellular domain of CD31. The polypeptide may further comprise a hinge region, a transmembrane domain and / or an intracellular domain. The hinge region, transmembrane domain and / or intracellular domain may be derived from CD31 or a different molecule (e.g., CD8 or CD28). The CD31 polypeptide may include a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0159] The present disclosure is related to engineered cells (e.g., CAR-T cells, CAR-NK cells, TCR-T cells) overexpressing a CD31 polypeptide. The engineered cell may be an allogeneic cell isolated from a donor. The engineered cell may express an exogenous CD31 polypeptide. The exogenous CD31 polypeptide may protect the modified cells from NK cell- or T cell-mediated killing. The exogenous CD31 polypeptide may be the only exogenous peptide that the modified cells express.

[0160] The present disclosure is also related to engineered cells that express a CD31 polypeptide comprising an extracellular domain, wherein the extracellular domain comprises at least a portion of C2 Ig domains of CD31.

[0161] The CD31 polypeptide may comprise an extracellular domain of CD31. The full-length sequence of CD31 is shown in SEQ ID NO: 8. The full-length nucleotide sequence of CD31 is shown in SEQ ID NO: 23. The extracellular domain of CD31 is amino acids 28-601 of SEQ ID NO: 8. The intracellular domain of CD31 is amino acids 621-738 of SEQ ID NO: 8. The CD31 polypeptide may comprise the entire sequence or any portion of the extracellular domain of CD31. The CD31 polypeptide may comprise the entire sequence or any portion of the extracellular domain of SEQ ID NO: 1. The extracellular domain of the CD31 polypeptide may have an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. The extracellular domain of the CD31 polypeptide can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 mutations. The mutation can be a deletion, an insertion, a substitution, or a combination thereof. The extracellular domain of CD31 may include a K89A mutations and a N25Q mutation, wherein the position is based on alignment with SEQ ID NO: 1. N25Q and K89A are located in C2 Ig domain 1 of CD31 extracellular domain. The two sites are involved in homophilic binding.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0162] The extracellular domain of CD31 may comprise one or more C2 Ig domain(s) of the full-length extracellular domain of CD31. The full-length extracellular domain of CD31 includes 6 C2 Ig domains. C2 Ig domain 1 is amino acids 8-94 of SEQ ID NO: 1 and has an amino acid sequence of SEQ ID NO: 16. C2 Ig domain 2 is amino acids 118-206 of SEQ ID NO: 1 and has an amino acid sequence of SEQ ID NO: 17. C2 Ig domain 3 is amino acids 209-288 of SEQ ID NO: 1 and has an amino acid sequence of SEQ ID NO: 18. C2 Ig domain 4 is amino acids 301- 374 of SEQ ID NO: 1 and has an amino acid sequence of SEQ ID NO: 19. C2 Ig domain 5 is amino acids 397-466 of SEQ ID NO: 1 and has an amino acid sequence of SEQ ID NO: 20. C2 Ig domain 6 is amino acids 472-564 of SEQ ID NO: 1 and has an amino acid sequence of SEQ ID NO: 21. An extracellular domain of CD31 may include one or more of the C2 Ig domains (e.g., 1, 2, 3, 4, 5 or 6 C2 Ig domains) or any fragment(s) of the C2 Ig domains (e.g., a fragment of C2 Ig domain 6 having an amino acid sequence of SEQ ID NO: 21) of the extracellular domain of CD31. The C2 Ig domain 1, 2, 3, 4, 5, or 6 may have some mutations, including e.g., substitutions, deletions or insertions.

[0163] An extracellular domain of CD31 may include an amino acid sequence that is about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the C2 Ig domains (e.g., 1, 2, 3, 4, 5 or 6 C2 Ig domains) or any fragment(s) of the C2 Ig domains (e.g., a fragment of C2 Ig domain 6 having an amino acid sequence of SEQ ID NO: 21) of the extracellular domain of CD31.

[0164] The extracellular domain may comprise or consist of 1, 2, 3, 4 or 5 C2 Ig domains selected from C2 Ig domains 1, 2, 3, 4, 5 and 6 of CD31, optionally with a leader sequence before the first C2 Ig domain and / or a sequence connecting any of the two adjoining C2 Ig domains, and optionally a sequence after the last C2 Ig domain. The extracellular domain may also include a membrane proximal region.

[0165] The present disclosure relates to a CD31 polypeptide comprising or consisting of a membrane proximal region. As used herein, a membrane proximal region of CD31 is a region of the extracellular domain of CD31 that is proximal to the cell membrane. The membrane proximal region of CD31 may be N-terminal to the transmembrane domain of CD31. The membrane proximal region of CD31 may have a length of about 1-50, 1-40, 1-30, 1-20, 1-10, 10-50, 10-40, 10-30, 10-20 or 20-30 amino acids. The membrane proximal region of CD31 may have a length of about 23 amino acids. The membrane proximal region of CD31 may include an amino acidAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P sequence of that is about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12.

[0166] The CD31 polypeptide may comprise an extracellular domain of CD31, a transmembrane domain of CD31 and an intracellular domain of CD31. The intracellular domain of CD31 may be a truncated intracellular domain of CD31. The intracellular domain of CD31 is shown in SEQ ID NO: 9. The truncated intracellular domain of CD31 can have any suitable length and sequence. The truncated intracellular domain of CD31 may have no more than 118, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of the intracellular domain of CD31. The truncated intracellular domain of CD31 may have no more than 5 amino acids of the intracellular domain of CD31. The truncated intracellular domain of CD31 may have 2 amino acids of the intracellular domain of CD31. The truncated intracellular domain of CD31 may have amino acids Lysine (K) and Cystine (C).

[0167] The transmembrane domain may be a CD31 transmembrane domain. The transmembrane domain of CD31 can have any suitable length and sequence. The transmembrane domain of the CD31 polypeptide may comprise an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.

[0168] The transmembrane domain may be a CD8 transmembrane domain. The transmembrane domain of CD8 can have any suitable length and sequence. The transmembrane domain of CD8 may comprise a nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25. The transmembrane domain of CD8 may comprise an amino acid sequence of SEQ ID NO: 30 or an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30.

[0169] The transmembrane domain may be a CD28 transmembrane domain. The transmembrane domain of CD28 can have any suitable length and sequence. The transmembrane domain of CD28 may comprise a nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27. The transmembrane domain of CD28 may comprise an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32.

[0170] The transmembrane domain of CD28 with an YT / LL mutation (see, e.g., Scott A Leddon et al., The CD28 Transmembrane Domain Contains an Essential Dimerization Motif. Front Immunol.2020 Jul 16; 11: 1519) may comprise a nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28. The transmembrane domain of CD28 with an YT / LL mutation may comprise an amino acid sequence of SEQ ID NO: 33 or an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33.

[0171] The CD31 polypeptide may include a hinge region. The hinge region may be a CD8 hinge domain. The hinge region of CD8 can have any suitable length and sequence. The hinge region of CD8 may comprise a nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24. The hinge region of CD8 may comprise an amino acid sequence of SEQ ID NO: 29 or an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29.

[0172] The CD31 polypeptide may include a hinge region. The hinge region may be a CD28 hinge domain. The hinge region of CD28 can have any suitable length and sequence. The hinge region of CD28 may comprise a nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26. The hinge region of CD28 can have any suitable length and sequence. The hinge region of CD28 may comprise an amino acid sequence of SEQ ID NO: 31 or an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0173] The CD31 polypeptide may comprise an extracellular domain of CD31, a transmembrane domain of CD31 and an intracellular domain of CD31, and the extracellular domain of CD31 comprises no more than 5 C2 Ig domains of the full-length extracellular domain of CD31 (e.g., 1, 2, 3, 4 or 5 C2 Ig domain(s)). The extracellular domain of CD31 may comprise any 1, 2, 3, 4 or 5 of the C2 Ig domain(s).

[0174] The CD31 polypeptide may comprise an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31. The extracellular domain of CD31 may comprise any 1, 2, 3, 4, 5 or 6 of the C2 Ig domain(s).

[0175] In one aspect, the present disclosure provides a CD31 polypeptide that comprises a full- length extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31. The CD31 polypeptide may comprise an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

[0176] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 3, 4, 5 and 6 of the extracellular domain of CD31, a transmembrane domain of CD31 and a full-length intracellular domain of CD31.

[0177] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1, 2 and 6 of the extracellular domain of CD31, a transmembrane domain of CD31 and a full-length intracellular domain of CD31.

[0178] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1, 2, 3, 4 and 5 of the extracellular domain of CD31, a transmembrane domain of CD31 and a full-length intracellular domain of CD31.

[0179] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1 and 2 of the extracellular domain of CD31, a transmembrane domain of CD31 and a full-length intracellular domain of CD31.

[0180] In one aspect, the present disclosure provides a CD31 polypeptide that comprises an extracellular domain of CD31 having the K89A and the N25Q mutations, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0181] In one aspect, the present disclosure provides a CD31 polypeptide that comprises a full- length extracellular domain of CD31, a transmembrane domain of CD28 and a truncated intracellular domain of CD31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0182] In one aspect, the present disclosure provides a CD31 polypeptide that comprises a full- length extracellular domain of CD31, a transmembrane domain of CD28 with an YT / LL mutation (e.g., with a nucleotide sequence as shown in SEQ ID NO: 28) and a truncated intracellular domain of CD31.

[0183] In one aspect, the present disclosure provides a CD31 polypeptide that comprises a membrane proximal region of CD31, a hinge region of CD8 and a truncated intracellular domain of CD31.

[0184] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1 and 2 of the extracellular domain of CD31, a hinge region of CD8, a transmembrane domain of CD8 and a truncated intracellular domain of CD31.

[0185] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1 and 2 of the extracellular domain of CD31, a hinge region of CD28, a transmembrane domain of CD28 and a truncated intracellular domain of CD31.

[0186] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1, 2, 3, 4 and 5 of the extracellular domain of CD31, a hinge region of CD8, a transmembrane domain of CD8 and a truncated intracellular domain of CD31.

[0187] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1, 2, 3, 4 and 5 of the extracellular domain of CD31, a hinge region of CD28, a transmembrane domain of CD28 and a truncated intracellular domain of CD31.

[0188] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 3, 4, 5 and 6 of the extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0189] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1, 2 and 6 of the extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0190] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1, 2, 3, 4 and 5 of the extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

[0191] In one aspect, the present disclosure provides a CD31 polypeptide that comprises C2 Ig domains 1 and 2 of the extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0192] In one aspect, the present disclosure provides a CD31 polypeptide that comprises a membrane proximal region of CD31, a transmembrane domain of CD31 and a full-length intracellular domain of CD31. The CD31 polypeptide may comprise an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10.

[0193] In one aspect, the present disclosure provides a CD31 polypeptide that comprises a membrane proximal region of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31. The CD31 polypeptide may comprise an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11.

[0194] In one aspect, the present disclosure provides a CD31 polypeptide that comprises a membrane proximal region of CD31. The CD31 polypeptide may comprise an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12.

[0195] The CD31 polypeptide may comprise a signal peptide. The signal peptide may comprise an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5.

[0196] The CD31 polypeptide expressed in the engineered cell may comprise an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is about or at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. The CD31 polypeptide may have an amino acid sequence that is identical to SEQ ID NO: 2. The CD31 polypeptide can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 mutations. The CD31 polypeptide can have only one mutation. The mutation can be a deletion, an insertion, a substitution, or a combination thereof. Engineered Receptor (e.g., CAR and TCR)

[0197] In one aspect, the present disclosure provides cells (e.g., immune cells) that express engineered receptor and the CD31 polypeptide. The engineered receptor may comprise an extracellular ligand binding domain or an extracellular antigen binding domain, and optionally an intracellular signaling domain. Exemplary engineered receptor include, but are not limited to,Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P CAR, engineered TCR, and TAC receptors. The engineered receptor may comprise an extracellular domain comprising an antigen binding domain that specifically binds to an antigen (e.g., a tumor antigen), a transmembrane region, and an intracellular signaling domain. The intracellular signaling domain may comprise a primary intracellular signaling domain and / or a co- stimulatory signaling domain. The intracellular signaling domain may comprise an intracellular signaling domain of a TCR co-receptor. The engineered receptor may be encoded by a heterologous polynucleotide operably linked to a promoter (such as a constitutive promoter or an inducible promoter).

[0198] The engineered receptor may comprise one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or co-stimulatory signaling domains.

[0199] The engineered receptor may be a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and can be suitable for the modified cells comprising the CD31 polypeptide described herein. CARs can also be constructed with a specificity for any cell surface marker by utilizing antigen binding fragments or antibody variable domains of, for example, antibody molecules.

[0200] CARs of the present disclosure comprise an extracellular domain comprising at least one antigen binding domain that specifically binds at least one tumor antigen, a transmembrane region, and an intracellular signaling domain. The intracellular signaling domain may generate a signal that promotes an immune effector function of the CAR-containing cell, e.g., a CAR-T cell. “Immune effector function or immune effector response” refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response can refer to a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. Examples of immune effector function, e.g., in a CAR-T cell, include cytolytic activity and helper activity (such as the secretion of cytokines). The CAR may have an intracellular signaling domain with an attenuated immune effector function. The intracellular signaling domain may generate a signal that promotes proliferation and / or survival of the CAR containing cell. The CAR may comprise one or more intracellular signaling domains selected from the signaling domains of CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. The signaling domain of a naturally occurring molecule canAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P comprise the entire intracellular or cytoplasmic portion, or the entire native intracellular signaling domain, of the molecule, or a fragment or derivative thereof.

[0201] The intracellular signaling domain of a CAR may comprise a primary intracellular signaling domain. “Primary intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. The primary intracellular signaling domain may contain a signaling motif known as Immunoreceptor Tyrosine- based Activation Motif, or ITAM. The primary intracellular signaling domain may comprise a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, FcgammaRIIa, DAP10, and DAP12. The primary intracellular signaling domain may comprise a nonfunctional or attenuated signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, FcgammaRIIa, DAP10, and DAP12. The nonfunctional or attenuated signaling domain can be a mutant signaling domain having a point mutation, insertion or deletion that attenuates or abolishes one or more immune effector functions, such as cytolytic activity or helper activity, including antibody-dependent cellular toxicity (ADCC). The CAR may comprise a nonfunctional or attenuated CD3 zeta (i.e. CD3ζ or CD3z) signaling domain. The intracellular signaling domain may not comprise a primary intracellular signaling domain. An attenuated primary intracellular signaling domain may induce no more than about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of an immune effector function (such as cytolytic function against target cells) compared to CARs having the same construct, but with the wild-type primary intracellular signaling domain.

[0202] The intracellular signaling domain of a CAR may comprise one or more (such as any of 1, 2, 3, or more) co-stimulatory signaling domains. “Co-stimulatory signaling domain” can be the intracellular portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co- stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. A co- stimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activationAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P molecules (SLAM proteins), and activating NK cell receptors. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA- 1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and a ligand that specifically binds with CD83.

[0203] The CAR may comprise a single co-stimulatory signaling domain. The CAR may comprise two or more co-stimulatory signaling domains. The intracellular signaling domain may comprise a functional primary intracellular signaling domain and one or more co-stimulatory signaling domains. The CAR may not comprise a functional primary intracellular signaling domain (such as CD3ζ). The CAR may comprise an intracellular signaling domain consisting of or consisting essentially of one or more co-stimulatory signaling domains. The CAR may comprise an intracellular signaling domain consisting of or consisting essentially of a nonfunctional or attenuated primary intracellular signaling domain (such as a mutant CD3ζ) and one or more co- stimulatory signaling domains. Upon binding of the antigen binding domain to tumor antigen, the co-stimulatory signaling domains of the CAR can transduce signals for enhanced proliferation, survival and differentiation of the modified immune cells having the CAR (such as T cells), and inhibit activation induced cell death. The one or more co-stimulatory signaling domains may be derived from one or more molecules selected from the group consisting of CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specially bind to CD83.

[0204] The intracellular signaling domain of a CAR may comprise a co-stimulatory signaling domain derived from CD28. The intracellular signaling domain may comprise a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28. The intracellularAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P signaling domain in the chimeric receptor of the present application may comprise a co-stimulatory signaling domain derived from 4-1BB (i.e., CD137). The intracellular signaling domain may comprise a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of 4- 1BB.

[0205] The intracellular signaling domain of the CAR may comprise a co-stimulatory signaling domain of CD28 and a co-stimulatory signaling domain of 4-1BB. The intracellular signaling domain may comprise a cytoplasmic signaling domain of CD3ζ, a co-stimulatory signaling domain of CD28, and a co-stimulatory signaling domain of 4-1BB. The intracellular signaling domain may comprise a polypeptide comprising from the N-terminus to the C-terminus: a co-stimulatory signaling domain of CD28, a co-stimulatory signaling domain of 4-1BB, and a cytoplasmic signaling domain of CD3ζ.

[0206] The antigen binding domain of a CAR may be an antibody or an antibody fragment, such as an scFv, a Fv, a Fab, a (Fab′)2, a single domain antibody (sdAb), or a VHH domain. The antigen binding domain of a CAR may comprise a ligand or an extracellular portion of a receptor that specifically binds to a tumor antigen. The CAR may be a monospecific, bispecific or multispecific CAR. The antigen binding domain of a CAR may specifically bind a single tumor antigen. The antigen binding domain of a CAR may bind two or more tumor antigens.

[0207] The tumor antigen may be selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta- like 3 (DLL3), CD70, CS-1, c-Met, Glycolipid F77, PD-L1, and PD-L2, and other tumor antigens with clinical significance, and combinations thereof. The tumor antigen may be derived from an intracellular protein of tumor cells. The tumor antigen may be expressed on the surface of tumor cells.

[0208] Many CARs targeting different tumor antigens have been widely disclosed in the field, such as CD19 CARs or BCMA CARs. The extracellular antigen-binding domain of CD19 CARsAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P can be or include the CD19 binding fragment (e.g., FMC63, SJ25C1, or those disclosed in different patents such as WO 2022 / 012683, etc). BCMA CARs also have been well described, related patents include but not limited to WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760, and WO 2018 / 028647, etc.

[0209] The transmembrane region of a CAR may comprise or be chosen from the transmembrane region of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. The transmembrane region of the CAR may be a CD4, CD3, CD8α, or CD28 transmembrane region. The transmembrane region of the CAR may comprise a transmembrane region of CD8α.

[0210] The extracellular domain may be connected to the transmembrane region by a hinge region. The hinge region may comprise a hinge region of CD8α.

[0211] The CAR may comprise a signal peptide (SP), such as a CD8α signal peptide.

[0212] The CAR may be a BCMA CAR. A wide variety of antigen binding domain sequences can be used as the antigen binding domain of the CAR.

[0213] The CAR may be a single CAR, dual CAR (or dual-targeting CAR), tandem CAR or split CAR.

[0214] The BCMA CAR may comprise an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. The BCMA CAR may comprise an amino acid sequence of SEQ ID NO: 3. The BCMA CAR may comprise a nucleotide sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. The CAR may specifically bind to BCMA-positive tumor cells.

[0215] The engineered receptor may be a modified T-cell receptor. The engineered TCR may be specific for a tumor antigen. The tumor antigen may be selected from the group consisting of CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD30, CD33, CD38, CEA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, Glypican 3 (GPC3), Guanylate cyclase 2C (GCC), DLL3, Claudin18.2, Claudin6, Glycolipid F77, PD-L1, PD-L2, and other tumor antigens with clinical significance, and combinations thereof. The tumor antigen may be derived from an intracellular protein of tumor cells. The tumor antigen may be expressed on the surface of tumor cells. Many TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigens, TCRs for tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigens), and breast cancer (e.g., HER2, NY-BR1). Any of the TCRs known in the art can be used. The TCR may have an enhanced affinity to the tumor antigen. Exemplary TCRs and methods for introducing the TCRs to immune cells have been described, for example, in U.S. Pat. No.5,830,755, and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol.2, 957-961 (2001), which are incorporated herein by reference in the entirety.

[0216] The TCR receptor complex is an octomeric complex formed by variable TCR receptor α and β chains (or γ and δ chains on case of γδ T cells) with three dimeric signaling modules CD3δ / ε, CD3γ / ε and CD247 (T-cell surface glycoprotein CD3 zeta chain) ζ / ζ or ζ / η. Ionizable residues in the transmembrane region of each subunit form a polar network of interactions that hold the complex together. TCR complex has the function of activating signaling cascades in T cells.

[0217] The engineered receptor may be an engineered TCR comprising one or more T-cell receptor (TCR) fusion proteins (TFPs). Exemplary TFPs have been described, for example, in US20170166622A1, which is incorporated herein by reference in its entirety. The TFP may comprise an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit,Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The TFP may comprise a transmembrane region that comprises a transmembrane region of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The TFP may comprise a transmembrane region that comprises a transmembrane region of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0218] The TFP comprising a TCR subunit may comprise at least a portion of a TCR extracellular domain, and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T cell.

[0219] The engineered receptor may be a T-cell antigen coupler (TAC) receptor. Exemplary TAC receptors have been described, for example, in US20160368964A1, which is incorporated herein by reference. The TAC may comprise an antigen binding domain, a TCR-binding domain that specifically binds a protein associated with the TCR complex, and a T-cell receptor signaling domain. The antigen binding domain may be an antibody fragment, such as scFv or VHH, which specifically binds to a tumor antigen. The antigen binding domain may be a designed Ankyrin repeat (DARPin) polypeptide. The tumor antigen may be selected from the group consisting of CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD30, CD33, CD38, CEA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, Glypican 3 (GPC3), Guanylate cyclase 2C (GCC), DLL3, Claudin18.2, Claudin6, Glycolipid F77, PD-L1, PD-L2, and other tumor antigens with clinical significance, and combinations thereof. The tumor antigen may be derived from an intracellular protein of tumor cells. The tumor antigen may be expressed on the surface of tumor cells. The protein associated with the TCR complex may be CD3, such as CD3E. The TCR-binding domain may be a single chain antibody, such as scFv, or aAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P VHH. The TCR-binding domain may be derived from UCHT1. The TAC receptor may comprise a cytosolic domain and a transmembrane region. The T-cell receptor signaling domain may comprise a cytosolic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. The TAC receptor may comprise a transmembrane region and a cytosolic domain derived from CD4. The TAC receptor may comprise a transmembrane region and a cytosolic domain derived from CD8 (such as CD8α).

[0220] T cell co-receptors are expressed as membrane protein on T cells. They can provide stabilization of the TCR: peptide: MHC complex and facilitate signal transduction. The two subtypes of T cell co-receptor, CD4 and CD8, display strong specificity for particular MHC classes. The CD4 co-receptor can only stabilize TCR: MHC II complexes while the CD8 co- receptor can only stabilize the TCR: MHC I complex. The differential expression of CD4 and CD8 on different T cell types results in distinct T cell functional subpopulations. CD8+ T cells are cytotoxic T cells.

[0221] The engineered receptor (such as CAR, TCR, or TAC) may target one or more tumor antigens. Tumor antigens are proteins that are produced by tumor cells that can elicit an immune response, particularly T-cell mediated immune responses. The selection of the targeted antigen will depend on the particular type of cancer to be treated. Exemplary tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70- 2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF- I receptor and mesothelin.

[0222] The tumor antigen may comprise one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2 / Neu / ErbB-2. Yet another group ofAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.

[0223] The tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell, and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development, when the immune system is immature, and unable to respond or they can be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.

[0224] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2; tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMA, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS. Nucleic Acids (Polynucleotides)

[0225] The present disclosure provides (i) nucleic acids (e.g., expression vectors) encoding the CD31 polypeptide, and (ii) nucleic acids (e.g., expression vectors) encoding an engineeredAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P receptor (e.g., CAR or TCR). The nucleic acids of the present disclosure can comprise a nucleic acid sequence encoding any one of the CD31 polypeptide, CARs, and / or TCRs disclosed herein. The nucleic acid may encode both an engineered receptor and an CD31 polypeptide.

[0226] A polynucleotide of the present disclosure may comprise a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequence can be separated by a linker. A linker for use in the present disclosure allows for multiple proteins to be encoded by the same nucleic acid sequence (e.g., a multicistronic or bicistronic sequence), which are translated as a polyprotein that is dissociated into separate protein components. The polynucleotide may comprise from 5’ to 3’ the first polynucleotide sequence, the linker, and the second polynucleotide sequence. The polynucleotide may comprise from 5' to 3' the second polynucleotide sequence, the linker, and the first polynucleotide sequence. The first polynucleotide sequence may encode an engineered receptor (e.g., CAR) described herein and the second polynucleotide sequence may encode a CD31 polypeptide described herein.

[0227] The linker may comprise a nucleic acid sequence that encodes for an internal ribosome entry site (IRES). As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e.g., immunogloublin heavy-chain-binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those of skill in the art would be able to select the appropriate IRES.

[0228] The linker may comprise a nucleic acid sequence that encodes for a self-cleaving peptide. As used herein, a “self-cleaving peptide” or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and- mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), andAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P porcine teschovirus-1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively. Those of skill in the art would be able to select the appropriate self-cleaving peptide.

[0229] The linker can comprise a spacer sequence. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, (GSGGS)n (SEQ ID NO: 6) and (GGGS)n (SEQ ID NO: 7), where n represents an integer of at least 1. The linker may be an SSGGGGS linker. Those of skill in the art would be able to select the appropriate spacer sequence.

[0230] A polynucleotide of the present disclosure can be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

[0231] The promoter may be a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used; see, e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by use of an Ncr1 (p46) promoter; see, e.g., Eckelhart et al. Blood (2011) 117:1565.

[0232] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences can also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the elongation-factor-1-alpha promoter (EF-1 alpha promoter, EF-1α promoter), as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expressionAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0233] A polynucleotide of the present disclosure may enable the production of (i) a CD31 polypeptide described herein, and / or (ii) an engineered receptor described herein (e.g., in a mammalian cell). A polynucleotide of the present disclosure may enable replication of the polynucleotide.

[0234] An expression vector (e.g., a lentiviral vector) can be used to introduce the CAR or TCR into an immune cell or precursor thereof (e.g., a T cell). Accordingly, an expression vector (e.g., a lentiviral vector) of the present disclosure can comprise a polynucleotide encoding for a CAR or a TCR. The expression vector (e.g., lentiviral vector) can comprise additional elements that will aid in the functional expression of the CAR or TCR encoded therein. An expression vector comprising a polynucleotide encoding for a CAR or TCR may further comprise a mammalian promoter. The vector may comprise an EF-1α promoter. The use of an EF-1α promoter can increase the efficiency in expression of downstream transgenes (e.g., a CAR- or TCR-encoding polynucleotide). Physiologic promoters (e.g., an EF-1α promoter) can be less likely to induce integration mediated genotoxicity, and can abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector (e.g., lentiviral vector) are known to those of skill in the art and can be incorporated into a vector of the present disclosure. The vector (e.g., lentiviral vector) may further comprise a non-requisite cis acting sequence that can improve titers and gene expression.

[0235] The polynucleotide may encode a naked CAR. The polynucleotide may comprise from the 5' end to the 3' end, a CD8α signal peptide, an antigen binding domain, a CD8α hinge region, a CD8α transmembrane region, a CD137 co-stimulatory signaling domain, a CD3ζ cytoplasmic domain.

[0236] The polynucleotide may encode a CAR and a CD31 polypeptide. The polynucleotide may comprise from the 5' end to the 3' end, the coding sequences of a CD31 polypeptide, a 2A cleavable linker, and a CAR. The polynucleotide may encode an amino acid sequence of CD31 that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2. The polynucleotide may encode an amino acid sequence of CAR that is at leastAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0237] The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein. In some cases, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. The nucleic acid sequence may be less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, or 5000 nucleotides. The amino acid sequence may be less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 amino acid residues.

[0238] The amino acid sequence may (i) comprise an amino acid sequence; or (ii) consist of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

[0239] The nucleic acid sequence may (i) comprise a nucleic acid sequence; or (ii) consist of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

[0240] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes may be at least 80% of the length of the reference sequence, and may be at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Introduction of Polynucleotides into Host Cells

[0241] The polynucleotides (e.g., vectors) described herein can be introduced as one or more polynucleotides or constructs, optionally comprising a marker that will allow for selection of host cells that contain the construct(s). The genes and regulatory regions can be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing. Particularly, using PCR, individual fragments including all or portions of a functional unit can be isolated, where one or more mutations can be introduced using "primer repair", ligation, in vitro mutagensis, etc. as appropriate. The polynucleotides obtained and demonstrated to have the appropriate sequences can then be introduced into the host cell by any convenient means. The polynucleotides can be integrated and packaged into non-replicating, defective viral genomes like lentivirus, Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors, for infection or transduction into cells. The polynucleotides can include viral sequences for transfection, if desired. Alternatively, the polynucleotides can be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cells can be grown and expanded in culture before introduction of the construct(s), followed by the appropriate treatment for introduction of the construct(s) and integration of the construct(s). The cells are then expanded and screened by virtue of a marker present in the construct. Various markers that can be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0242] The CD31 polypeptide and engineered receptor can be introduced into the modified cells as an RNA for transient expression. RNA can be delivered to the immune cells of the disclosure by various means including microinjection, electroporation, and lipid-mediated transfection, for example. Introduction of constructs into the cell's genome can occur via transposons. An example of a synthetic transposon for use is the Sleeping Beauty transposon that comprises an expression cassette including the appropriate gene of active fragment thereof. TheAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P construct can be integrated at a particular locus in the genome of the host cell. An endogenous gene can be replaced with the gene encoded for by the construct using homologous recombination.

[0243] A construct encoding both a CD31 polypeptide and a CAR can be introduced into the host cell using a lentiviral delivery system. A construct encoding both a CD31 polypeptide and a CAR can be introduced into the host cell using a retroviral delivery system. The host cells can be human cells. The host cells can be human T cells. The human T cells can be purified from commercialized PBMCs. The host cells can be αβT cells. The host cells can be γδT cells. The host cells can be Vδ1 γδT cells. The host cells can be Vδ2 γδT cells. The host cells can be Vδ3 γδT cells. The host cells can be tumor-infiltrating lymphocytes (TIL). The host cells can be NK cells. Engineered Cells

[0244] In one aspect, the present disclosure provides engineered cells comprising the CD31 polypeptide described herein. The engineered cells comprising the CD31 polypeptide described herein may further comprise an engineered receptor (e.g., CAR). The engineered cells comprising the CD31 polypeptide described herein may further comprise a CAR (CAR armored with CD31 polypeptide). The cell can be an immune cell. The cell can be a T cell, a NK cell, a NK-T cell, a Treg cell, a B cell, a monocyte, a macrophage cell, a dendritic cell, or a combination thereof. The cell can be selected from a group consisting of T cell, αβT cell, γδT cell, NK cell, NKT, tumor- infiltrating lymphocytes (TIL), peripheral blood mononuclear cell (PBMC), hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, and a combination thereof.

[0245] In one aspect, the present disclosure provides engineered cells comprising a CD31 polypeptide (e.g., an exogenous CD31 polypeptide).

[0246] The cell can be an allogeneic T cell, e.g., an allogeneic T cell lacking expression of endogenous T cell receptor (TCR) and / or human leukocyte antigen (HLA), e.g., HLA class I and / or HLA class II. The engineered immune cell can be a T cell lacking a functional endogenous TCR. A T cell lacking a functional endogenous TCR can be, e.g., engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that comprise a functional TCR (e.g., engineered such that it does not express (or exhibits reduced expression of) TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε and ζ-chain or engineered such that it produces very little functional TCR on its surface. Alternatively, the T cell can express a substantially impaired TCR, e.g., by expression of mutated or truncated forms of one or more ofAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P the subunits of the TCR. The term "substantially impaired TCR" means that this TCR will not elicit an adverse immune reaction in a host. The engineered receptor (e.g., CAR) may redirect the specificity of the engineered cells through the expression of a chimeric antigen receptor (CAR) or TCR on these cells. CAR expression can be induced through electroporation of engineered cells for the insertion of genetic material, or by infecting these cells with viral vectors, such as lentiviruses or retroviruses containing the desired genetic material. Such genetic editing can improve the potency of the engineered cells by improving homing, cytokine production, recycle killing, and / or improved engraftment.

[0247] The engineered cells expressing the CD31 polypeptide described herein may express more than one engineered receptors, such as any combination of CAR, TCR, or TAC receptor.

[0248] The engineered cell expressing the CD31 polypeptide described herein may be used to treat cancer.

[0249] Comparing to a cell without the expression of the CD31 polypeptide described herein, the modified cell comprising the CD31 polypeptide described herein may have a higher cytotoxicity against tumor cells. Comparing to a cell without the expression of the CD31 polypeptide described herein, the modified cell comprising the CD31 polypeptide described herein may have a higher persistence and / or proliferation. Persistence, as referred to herein, may indicate anti HvG activity and / or survival of cells.

[0250] In one aspect, the present disclosure provides engineered cells comprising (i) a CD31 polypeptide described herein and (ii) an engineered receptor (e.g., CAR) described herein. The modified cell may be an immune cell. The modified cell may comprise one or more polynucleotides encoding (i) a CD31 polypeptide described herein and (ii) an engineered receptor (e.g., CAR) described herein. Accordingly, such engineered cells possess the specificity directed by the engineered receptor (e.g., CAR) that is expressed therein. For example, a modified cell of the present disclosure comprising a CAR possesses specificity for one or more antigen(s) on a target cell (e.g., one or more tumor antigen(s) on a cancer cell).

[0251] The engineered cells may be modified immune cells. The engineered cells may be T cells. The engineered cells may be NK cells. The engineered cells may be αβ T cells. The engineered cells may be γδ T cells. The engineered cells may be Vδ1 T cells. The engineered cells may be Vδ2 T cells.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0252] The engineered cells may be autologous cells, syngeneic cells, allogeneic cells, or xenogeneic cells with respect to the individual receiving them. The engineered cells may be modified by changing the major histocompatibility complex (MHC) profile, by inactivating β2- microglobulin to prevent the formation of functional Class I MHC molecules, or by inactivating Class II MHC molecules. The engineered cell expressing the CD31 polypeptide described herein may have a disruption at the endogenous B2M gene, or has reduced expression or activity of B2M.

[0253] The engineered cells described herein may be eukaryotic cells, e.g., mammalian cells. The engineered cells may be human cells. The engineered cells may be equine, bovine, murine, ovine, canine, or feline cells.

[0254] The engineered cells may be autologous cells obtained from the human subject receiving them. The engineered cells may be autologous T cells obtained from the human subject receiving them.

[0255] The engineered cells may be from a cell line, e.g., a T2 human hybrid cell line (T and B lymphoblast).

[0256] Comparing to unmodified cells that do not comprise the CD31 polypeptide, the engineered cells may have an enhanced proliferation. Comparing to unmodified cells that do not comprise the CD31 polypeptide, the engineered cells may have an enhanced cytotoxicity toward tumor cells.

[0257] The engineered receptor may be a CAR. The CAR may comprise from N-terminus to C-terminus: a CD8α signal peptide, an antigen binding domain, a CD8α hinge region, a CD8α transmembrane region, a CD137 co-stimulatory signaling domain, and a CD3ζ cytoplasmic domain. The antigen binding domain of the CAR may be an antibody or an antibody fragment, such as an scFv, a sdAb, or a VHH domain. The antigen binding domain of the CAR may bind to BCMA. The CAR may comprise an amino acid sequence having at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to in SEQ ID NO: 3.

[0258] The extracellular domain of the CD31 polypeptide may comprise an amino acid sequence having at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to in SEQ ID NO: 1.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0259] The CD31 polypeptide may comprise an amino acid sequence having at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to in SEQ ID NO: 2.

[0260] Comparing to a cell without the overexpression of the CD31 polypeptides, the engineered cell described herein may have an increased expression of CD31 by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90%. The engineered cell described herein may have an increased expression of CD31 by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more folds compared to cells without the overexpression of the CD31 polypeptide.

[0261] The engineered cells may comprise both a CD31 polypeptide and a CAR (CAR armored with CD31 polypeptide). The engineered cells may be modified CAR-T cells (CD31 polypeptide armored CAR-T cells). The expression of CAR and CD31 polypeptide by the engineered cells can be determined by flow cytometry (FACS). The engineered cells can have a CAR positive rate of more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90%. The engineered cells may have a CAR positive rate of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. The engineered cells may have a CAR positive rate of 70%-80%, 70%-90%, 75%-85%, or 75%-90%.

[0262] The purity of the engineered cells can be determined by flow cytometry (FACS). The engineered cells have a purity of more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90%. The engineered cells may have a purity of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%,Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P less than 80%, or less than 90%. The engineered cells may have a purity of 90%-100%, 95%-100%, 98%-100%, or 98%-99%.

[0263] The overexpression of CD31 can promote the expansion of the engineered cells (e.g., γδT cells). The expansion of the engineered cells may be increased by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90% compared to cells without the overexpression of the CD31 polypeptide. The expansion of the engineered cells may be increased by about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more folds compared to cells without the overexpression of the CD31 polypeptide. Cell expansion or proliferation can be measured by an in vitro cell proliferation assay. Comparing to cells without the CD31 polypeptide described herein, the modified cells comprising the CD31 polypeptide described herein has a cell number that is higher by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90%. Comparing to a cell without the CD31 polypeptide described herein, the modified cells comprising the CD31 polypeptide described herein may have a cell number that is higher by less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%.

[0264] The engineered cells can kill tumor cells. The cytotoxicity of the engineered cells against tumor cells can be determined by an in vitro long-term cytotoxicity assay. The effector cell: target cell (E:T) ratio can be about 0.5:1, 1:1, 2:1, 2.5:1, 5:1, or 10:1. The in vitro cytotoxicity of the engineered cells against tumor cells can be evaluated in a long-term cytotoxicity assay, where the engineered cells are co-cultured with tumor cells. The effector cell: target cell (E:T) ratio can be 1:40, 1:30, 1:10, 0.5:1, 1:1, 2:1, 2.5:1, 5:1, or 10:1. The E:T ratio can be 1:1. The engineered cells may have a cytotoxicity of more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%, after 1 round, 2 rounds, 3 rounds, 4 rounds, or 5 rounds of stimulation in a re- challenge assay. The engineered cells may have a cytotoxicity of less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less thanAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 70%, less than 80%, or less than 90%, after 1 round, 2 rounds, 3 rounds, 4 rounds, or 5 rounds of stimulation in a re-challenge assay. The engineered cells may have a cytotoxicity of 10-100%, 10%-50%, 20-100%, 20-60%, 20-40%, 30-70%, 40-80%, 50-90%, 70-100%, 80-100%, or 90- 100%, after 1 round, 2 rounds, 3 rounds, 4 rounds, or 5 rounds of stimulation in a re-challenge assay. Comparing to unmodified cells that do not comprise the CD31 polypeptide, the cytotoxicity of the engineered cells comprising the CD31 polypeptide may increase by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 10,00%, after 1 round, 2 rounds, 3 rounds, 4 rounds, or 5 rounds of stimulation in a re-challenge assay.

[0265] The overexpression of CD31 can protect the engineered cells described herein from NK cell-mediated killing (e.g., immune clearance). The NK cell-mediated killing of the engineered cells may be decreased by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90% compared to cells without the overexpression of the CD31 polypeptide. The NK cell-mediated killing of the engineered cells may be decreased by about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more folds compared to cells without the overexpression of the CD31 polypeptide.

[0266] The overexpression of CD31 can protect the engineered cells described herein from T cell-mediated killing (e.g., immune clearance). The T cell-mediated killing of the engineered cells may be decreased by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90% compared to cells without the overexpression of the CD31 polypeptide. The T cell- mediated killing of the engineered cells may be decreased by about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more folds compared to cells without the overexpression of the CD31 polypeptide. Allogeneic Cell or Allogeneic Cells

[0267] In one aspect, the present disclosure provides allogeneic cells (or allogenic cells), the terms “allogeneic cells”, “allogeneic immune cells” or “allogeneic engineered immune cells” areAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P used interchangeably herein to refer to the cells are obtained from allogeneic donor. The allogeneic cells may T cells or NK cells. The T cell may a γδ T cell.

[0268] In some aspects, the allogeneic cell can be an allogeneic T cell, e.g., an allogeneic T cell lacking expression of endogenous T cell receptor (TCR) and / or human leukocyte antigen (HLA), e.g., HLA class I and / or HLA class II.

[0269] In some aspects, the allogeneic cell can be a T cell lacking a functional endogenous TCR. A T cell lacking a functional endogenous TCR can be, e.g., engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that comprise a functional TCR (e.g., engineered such that it does not express (or exhibits reduced expression of) TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε and ζ-chain or engineered such that it produces very little functional TCR on its surface. Alternatively, the T cell can express a substantially impaired TCR, e.g., by expression of mutated or truncated forms of one or more of the subunits of the TCR. The term "substantially impaired TCR" means that this TCR will not elicit an adverse immune reaction in a host.

[0270] In some aspects, the T cell or NK cell described herein can be, e.g., engineered such that it does not express a functional HLA on its surface. For example, a cell described herein can be engineered such that cell surface HLA, e.g., HLA class I and / or HLA class II, is downregulated. In some aspects, downregulation of HLA may be accomplished by reducing or eliminating expression of beta-2 microglobulin (B2M).

[0271] In some aspects, the cell can lack a functional TCR and a functional HLA, e.g., HLA class I and / or HLA class II. Modified cells that lack expression of a functional TCR and / or HLA can be obtained by any suitable means, including a knockout or knock down of one or more subunit of TCR or HLA. For example, the T cell or NK cell can include a knock down of TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription-activator like effector nuclease (TALEN), or zinc finger endonuclease (ZFN). Methods of Treatment

[0272] The CD31 polypeptide described herein, the polynucleotides described herein and the modified cells described herein can be used in a variety of experimental, therapeutic and commercial applications.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0273] In one aspect, the disclosure provides a method of modulating an immune response comprising administering an effective amount of modified cells described herein to a subject in need thereof.

[0274] In one aspect, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject, an effective amount of the engineered cell described herein.

[0275] In one aspect, the disclosure provides a method of inhibiting immune clearance of an engineered cell in a subject, comprising: (a) introducing a vector expressing an exogenous CD31 polypeptide into the engineered cell, wherein the exogenous CD31 polypeptide comprises an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31, thereby overexpressing the CD31 or a portion thereof; (b) administering the engineered cell from step (a) to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell.

[0276] In one aspect, the disclosure provides a method of increasing the in vivo expansion of an engineered cell in a subject, comprising: (a) introducing a vector expressing an exogenous CD31 polypeptide into the engineered cell, wherein the exogenous CD31 polypeptide comprises an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31, thereby overexpressing the CD31 or a portion thereof; (b) administering the engineered cell from step (a) to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell.

[0277] The term “effective amount” as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired results.

[0278] In another aspect, the present disclosure provides a method for treating cancer comprising administering an effective amount of modified cells described herein to a subject in need thereof. Examples of cancer that can be treated include, but are not limited to, leukemias including chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, and T cell and B cell leukemias, lymphomas (Hodgkin's and non-Hodgkins), lymphoproliferative disorders, plasmacytomas, histiocytomas, melanomas, adenomas, sarcomas, carcinomas of solid tissues, hypoxic tumors, squamous cell carcinomas, genitourinary cancers such as cervical and bladder cancer, hematopoietic cancers, head and neck cancers, and nervous system cancers.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0279] The disclosure further includes the use of the modified cells described herein in the manufacture of a medicament or pharmaceutical composition to modulate an immune response, to treat an infection or to treat cancer as described hereinabove.

[0280] The modified cells can also be used in experimental models, for example, to further study and elucidate the function of the cells.

[0281] One or more of the modified cells described herein can be administered to a subject in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions or injections, or subcutaneous injections. In some cases, the modified cells can expand within a subject's body, in vivo, after administration to a subject. The modified cells can be frozen to provide cells for multiple treatments with the same cell preparation. The modified cells of the disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. A kit can include instructions (e.g., written instructions) on the use of the modified cells and compositions comprising the same.

[0282] In one aspect, the present disclosure provides a method of treatment that comprises administering to a subject a therapeutically-effective amount of the modified cells. The therapeutically-effective amount of the modified cells may be administered for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. The therapeutically-effective amount of the modified cells may be administered for at least one week. The therapeutically-effective amount of the modified cells may be administered for at least two weeks.

[0283] The modified cells described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the modified cells can vary. For example, the modified cells can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The modified cells can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the modified cells can be initiated immediately within the onset of symptoms, within the first 3 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within 48 hours of the onset of the symptoms, or within any period of time from the onset of symptoms. The initial administration can be via any route practical (e.g., intravenous infusions or injections), such as by any route described herein using anyAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P formulation described herein. The administration of the modified cells of the disclosure can be an intravenous administration. One or multiple dosages of the modified cells can be administered as soon as is practicable after the onset of a cancer or an infectious disease, and for a length of time necessary for the treatment of the disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. For the treatment of cancer, one or multiple dosages of the modified cells can be administered years after onset of the cancer and before or after other treatments. The modified cells can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years at least 3 years, at least 4 years, or at least 5 years. The length of treatment can vary for each subject.

[0284] Methods for administration of modified cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No.2003 / 0170238 to Gruenberg et al; US Patent No.4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol.31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. The cell therapy, e.g., adoptive T cell therapy may be carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0285] The cell therapy (e.g., adoptive T cell therapy) may be carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. The cells may then be administered to a different subject, e.g., a second subject, of the same species. The first and second subjects may be genetically identical. The first and second subjects may beAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P genetically similar. The second subject may express the same HLA class or supertype as the first subject.

[0286] The subject may have been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. The subject may have persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. The administration may effectively treat the subject despite the subject having become resistant to another therapy.

[0287] The subject may be responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. The subject may not have relapsed. The subject may be determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0288] The subject may have persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. The administration may effectively treat the subject despite the subject having become resistant to another therapy.

[0289] The modified cells described herein can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the modified cells can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the modified cells or pharmaceutical compositions include, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplary cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancers can be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors / cancers and pediatric tumors / cancers are alsoAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P included. The cancer can be a solid tumor or a hematological tumor. The cancer can be a carcinoma. The cancer can be a sarcoma. The cancer can be a leukemia. The cancer can be a solid tumor.

[0290] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

[0291] Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0292] Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0293] The modified cells (e.g., immune cells, T cells, or NK cells) described herein can be included in a composition for immunotherapy. The composition can include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified cells can be administered.

[0294] The modified cells can be immediately used in the above therapeutic, experimental or commercial applications or the cells can be cryopreserved for use at a later date. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0295] The modified cells disclosed herein can be formulated in unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage forms comprise additional lymphocytes. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative or without a preservative. In some examples, the pharmaceutical composition does not comprise a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative. EXAMPLES The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P Example 1. Materials and Methods Plasmid

[0296] CD24 (NM_001291737), CD31 (SEQ ID NO: 1 or SEQ ID NO: 2), CD47 (NM_001777.4), CD52 (NM_001803.3), CD106 (NM_001078.4), BCMA-CAR (SEQ ID NO: 3) were inserted into the GFP section of Sin-CMV plasmid (Biovec pharma).

[0297] BCMA CAR+CD31 full length (BCMA CAR+CD31fl), BCMA CAR+CD31 intracellular domain truncated (BCMA CAR+CD31tICD), BCMA CAR+CD31v3-1, BCMA CAR+CD31v3-2, BCMA CAR+CD31v3-3 were cloned into Sin-CMV plasmids (Biovec Pharma). Cell lines

[0298] 293Vec-BaEV stable cell line was obtained from Biovec pharma.293Vec-BaEV cells were cultured with DMEM (Gibco) medium completed with 10% Fetal Bovine Serum (Gibco) at 37°C in a humidified atmosphere with 5% CO2.

[0299] T2 (174 × CEM.T2) cells were derived from a variant of the T1 cell line (CRL-1991 from ATCC) produced by selection the SFR1-MI.3 monoclonal antibody (against a monomorphic determinant on HLA DR). T2 cells were cultured using RPMI1640 (Gibco) medium completed with 10% Fetal Bovine Serum (Gibco) at 37°C in a humidified atmosphere with 5% CO2.

[0300] NCI-H929 cells were purchased from ATCC and cultured in RPMI1640 media (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C incubator with 5% CO2. Healthy blood donors

[0301] PBMCs were obtained through Hemacare leukopak. Fresh PBMCs were isolated using Ficoll-Paque Plus (GE HealthCare Life Sciences) and subjected to density centrifugation. The isolated PBMCs were frozen using CryoStor® CS5 Cell Freezing Medium (Stemcell Technologies) and stored in liquid nitrogen tank. γδ T cell expansion

[0302] PBMCs were expanded under using zoledronic acid (Sigma) and IL-2 (Life Technology) in CTS™ OpTmizer™ T Cell Expansion medium (Fisher Scientific). On days 0, 2, and 9, PBMCs cell number were determined using Vi-CELL Cell Counters (Beckman Coulter). On days 0 and 9, PBMCs were immunophenotyped for γδ T cell (CD3+Vγ9+Vδ2+) and naturalAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P killer (NK) cell content (CD3−CD56+) using a Cytek Aurora 5L (UV, V, B, YG, R) cytometer. Unmixed data were exported and analyzed in FlowJo (v10.8). All information regarding antibodies for immunophenotyping is provided in Table 2. Table 2. Antibodies for Immunophenotyping NameCompanyCat#PE anti-human CD106 AntibodyBiolegend3058062PBNK cell expansion

[0303] PBNK cells were expanded using feeder free protocol. Retroviral packaging and transduction

[0304] The 293Vec-BaEV cells were seeded in T225 culture flask (Falcon) at a density of 20×106cells the day before transfection. The retroviral plasmid Sin-CMV containing the immune tolerance gene or BCMA-CAR was transfected into 293Vec-BaEV cells using Lipofectamine 3000 (Life Technology) for 6 h at 37°C in 5% CO2according to the manufacturer's protocol. Following 48–72 h, supernatant containing retroviral particles was harvested and filtered through a 0.45-µm filter (EMD Millipore) to remove cell debris. The supernatants were concentrated by adding Retro-Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P X concentrator (Takara) according to the manufacturer's protocol. The retroviral particle pellet was re-suspended in cold PBS and stored at −80°C. The viral titers of concentrated retroviral particles were measured by infecting primary T cells seeded at a density of 1×106cells / well in a 24-well plate (VWR) with viral serial dilutions. Three days later, gene expression was detected using flow cytometry and the viral titer was calculated using the following equation: viral titer (Tu / µl) = (% gene+ cells × number of cells transduced) / virus volume.

[0305] For retroviral transduction, PBMCs stimulated with zoledronic acid and IL-2 were seeded at 5×106cells in 2 ml CTS medium in 6-well plates (VWR), and the concentrated retrovirus was added at MOI=1. The cell and virus mixture were spin inoculated for 2hr at 2,500 rpm at 32°C. After centrifugation, the plate was put back to incubator overnight. The medium was replaced on second day. After 3 days of culture, the cells were collected, and the expression of gene or BCMA- CAR was detected using flow cytometry, as described above. Immunophenotyping

[0306] γδ T cells and NK cells were characterized with different markers using flow cytometry. The detailed information of antibodies is provided in Table 2. γδ T cells or NK cells were washed with FACs staining buffer (Biolegend). Cells were resuspended in FACs staining buffer containing FC receptor block (BD Biosciences) and incubated for 10 min at room temperature. Immediately following the incubation, cells were stained with the antibody panel (Table 2) for 45 min at 4°C. After staining, cells were washed twice with FACs staining buffer and immediately analyzed for cell surface marker expression on a Cytek Aurora 5 L (UV, V, B, YG, R) instrument. Data analysis was performed using FlowJo (v10.8). NK cell mediated cytotoxicity assay

[0307] Target cells (T2 tumor cells or primary γδT cells with or without modification) were labeled with cell trace violet (Life Technology) for 20 minutes at 37°C following the manufacturer’s protocol. Labeled cells were then washed and re-suspended in RPMI media containing 10% FBS. Effector cells were harvested and re-suspended in RPMI media (Gibco) containing 10% FBS (Gibco) and titrated at designated effector: target ratios (10:1, 5:1, 2.5:1, 1.25:1, 0.6:1, 0.3:1). After target cell labeling, the titrated effector cells were added to target cells for 4hr or overnight. Immediately prior to analysis, the cells were added with 7-AAD (Biolegend)Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P to discriminate live vs. dead cells. After 5 mins incubation, samples were analyzed for viable target cells on a Cytek Aurora 5 L (UV, V, B, YG, R) instrument. Cytotoxicity was calculated by subtracting background cell death of each target cell from the experimental sample. E7 TCR-T cell mediated killing assay

[0308] Target cells (T2 cells or γδT cells with or without modification) were labeled with cell trace violet (Life Technology) for 20 minutes at 37°C following the manufacturer’s protocol. Labeled cells were then washed and re-suspended in RPMI media (Gibco) containing 10% FBS (Gibco). After labeling, the target cells were stimulated by E7 peptide for 2hr in serum free AIM V medium (Gibco). After 2hr stimulation, the target cells were counted and seeded in 96 well round bottom plate. Effector cells were titrated at designated effector: target ratios (10:1, 5:1, 2.5:1, 1.25:1, 0.6:1, 0.3:1). The titrated effector cells were then added to labeled target cells for overnight incubation. Immediately prior to analysis, the cells were added with 7-AAD (Biolegend) to discriminate live vs. dead cells. After 5 mins of incubation, samples were analyzed for viable target cells on a Cytek Aurora 5 L (UV, V, B, YG, R) instrument. Cytotoxicity was calculated by subtracting background cell death of each target cell from the experimental sample. 3-way mixed lymphocyte reaction assay (3-w MLR)

[0309] Allogeneic HLA-A2-positive or autologous HLA-A2-negative PBMC cells were labelled with cell trace violet (CVT) and then mixed with the g9d2 T cells and NCI-H929 tumor cells in complete AIM-V media supplemented with 10% FBS. The ratio of PBMC-CVT: g9d2 T cells: NCI-H929 cells is 200:1:1 or 400:1:1 at the 3-w MLR assay set up. Half media were replenished every other day. The cells in each sample were counted and evaluated by flow cytometry on day 4 and 8 or 9. Serial NCI-H929 tumor cell challenges

[0310] NCI-H929 tumor cells and BCMA CAR g9d2 T cells were cocultured together in complete AIM-V media supplemented with 10% FBS at ET ratio 1:1. The same number of NCI- H929 cells used at round 1 were continuously added into the coculture to rechallenge the g9d2 T cells every 24h after round 1 till round 5. Half media was replenished every other day. Cell count and flow cytometry analysis were performed at round 1, 3 and 5 tumor cell challenge.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P Example 2: CD31tICD-expressing T2 cells are Resistant to NK cell and E7 TCR-T Mediated Killing

[0311] T2 cells are human hybrid cell line (T and B lymphoblast). These cells are HLA-A2 positive which makes them sensitive to E7 TCR-T mediated killing. T2 cells are TAP2 deficient which make the MHC class I molecule not stable and make these cells sensitive to NK cell mediated killing. T2 cells were used as target cells to determine whether the overexpression of CD31tICD can prevent both NK cell and TCR-T mediated killing. RV-CD31tICD was transduced in T2 cells. The expression of CD31tICD in T2 cells is about 82.8% (FIG. 1A). CD31tICD expressing T2 cells or other immune tolerance inducing gene expressing T2 cells were co-cultured with NK92 cells for 4hr. After 4hr, NK92 had a strong killing capability on T2 cells. There was no killing capability on CD31tICD expressing T2 cells or other immune tolerance gene expressing T2 cells. CD31tICD overexpression protected T2 cells from NK92 mediated killing (FIG.1B).

[0312] For TCR-T cell mediated killing on T2 cells or CD31tICD expressing T2 cells, TCR- T cells had strong killing capability on T2 cells after overnight incubation. Compared with the killing capability of TCR-T cells on other immune tolerance gene expressing T2 cells, CD31tICD expressing T2 cells were completely resistant to the killing of TCR-T cells. CD31tICD was better to prevent the killing mediated by NK92 cells or TCR-T cells compared with other immune tolerance gene such as CD24, CD52 or CD106 (FIG.1C).

[0313] One possible mechanism is that CD31 is a member of “Don’t eat me signal” family like CD24 or CD47, which can prevent the killing from NK92 cells or TCR-T cells. Another possible mechanism is that CD31 weakens the immune synapses between effector cells and CD31 expressing T2 cells. Therefore, the killing efficiency of NK92 cells or TCR-T cells on CD31 expressing T2 cells is reduced. Example 3: Overexpression of CD31tICD in Type II gdT Cells

[0314] Experiments were performed to determine whether the overexpression of CD31tICD in primary γδT cells can prevent NK cell or TCR-T cell mediated killing. PBMC samples from two different donors (D4 and D22) were used to expand the typeII γδT cells using 5 µM Zoledronic acid and IL2 in CTS OpTmizer T cell expansion medium. On day 9, the purity of γδT cells is around 80% in these two samples (FIG. 2A). RV-CD31tICD, RV-CD47 and RV-BCMA-CARAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P were transduced in these γδT cells on day 2. The transduction efficiency in these samples from the two donors ranged from 50% to 90% (FIG. 2B). Interestingly, the overexpression of CD31tICD promoted the expansion of γδT cells. While the overexpression of other candidate genes, the expansion of γδT cells was negatively affected especially by CD47 (FIGs.3A-3D). Example 4: Overexpression of CD31tICD Does Not Impact the Killing Function of BCMA- CAR

[0315] To examine whether the overexpression of CD31tICD will affect the killing capability of BCMA-CAR on tumor cells, two donor (D4&D22) BCMA-CAR γδT cells with or without the expression of CD31tICD were co-cultured with H929 tumor cells for 4hr. The expression of CD31tICD in D4 BCMA-CAR γδT cells was around 57% (FIG.2B). The expression of CD31tICD in D22 BCMA-CAR γδT cells is around 54% (FIG.2B). After 4hr incubation, the killing capability of BCMA-CAR γδT cells was analyzed by flow cytometry. There was no difference on the killing capability of BCMA-CAR γδT cells with or without the expression of CD31tICD (FIGs.4A-4B). Even without the expression of BCMA-CAR, γδT cells as innate killer cells have killing capability on H929 cells (FIGs.4A-4B). Example 5: The Expression of CD31tICD Protected γδT cells from NK Cell Mediated Killing

[0316] To determine whether γδT cells are sensitive to NK cell mediated killing, 5 different donor derived NK cells (D15, D17, D19, D32, D0081) were co-cultured with two different donor expanded γδT cells (D4 and D22) for overnight at different E:T ratio. The killing capability of NK cells on γδT cells is weak even at high E:T ratio (50:1) and with longer incubation time (FIG.5A- 5B). To make the γδT cells sensitive to NK cell mediated killing, B2M was knocked out in γδT cells using CRISPR / Cas9 system. The knockout efficiency of B2M gene in γδT cells was very high (95% or higher) (FIG. 5C). The killing capability of NK cells on B2M KO γδT cells was dramatically improved compared with γδT cells. The overexpression of CD31tICD in B2M KO γδT cells make the cells insensitive to NK cell mediated killing at different incubation time (4hr or overnight) (FIGs.5D-5E).Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P Example 6: The Expression of CD31tICD Protected γδT Cells From TCR-T Cell-Mediated Killing

[0317] Both T2 cells and D22 γδT cells are HLA-A2+ which make these cells sensitive to TCR-T mediated killing (FIG.6). After E7 NCI peptide stimulation, E7 TCR-T cells had strong killing capability on T2 cells and D22 γδT cells. The expression of CD31tICD and CD47 in D22 γδT cells protected D22 γδT cells from TCR-T mediated killing to some extent. D4 B2M KO γδT cells were used as negative control because D4 γδT cells are HLA-A2 negative which make the D4 γδT cells insensitive to E7 TCR-T cell mediated killing. Furthermore, the KO of B2M gene made D4 γδT cells lose the MHC class-I peptide presentation capability. Therefore, the killing capability on D4 B2M KO γδT cells is dramatically reduced (FIG.6). Example 7: Engineered CD31 Variants Protected γδT Cells From TCR-T Cell-Mediated Killing

[0318] CD31 variants (CD31v3-1, CD31v3-2 and CD31v3-3) were designed to determine anti-allogeneic response of these series (FIG.7). The amino acid sequence of CD31v3-1 is shown in SEQ ID NO: 10 and the nucleotide sequence of CD31v3-1 is shown in SEQ ID NO: 13. The amino acid sequence of CD31v3-2 is shown in SEQ ID NO: 11 and the nucleotide sequence of CD31v3-2 is shown in SEQ ID NO: 14. The amino acid sequence of CD31v3-3 is shown in SEQ ID NO: 12 and the nucleotide sequence of CD31v3-3 is shown in SEQ ID NO: 15. These series are much smaller in size compared to full length CD31 as well as CD31 with a truncated intracellular domain (CD31tICD).

[0319] BCMA CAR+CD31variants were prepared with similar BCMA CAR percentage and MFI values with BCMA CAR control (FIGs.8A-8B). Compared to control BCMA CAR ^9 ^2 T cells, more BCMA CAR+CD31 variant ^9 ^2 T cells were consistently present in all three allogeneic PBMC ^9 ^2 T cell pairs (FIG.9), indicating that these cells have better persistence in allogeneic PBMCs.

[0320] In both ET ratios of 200:1:1 and 400:1:1, BCMA CAR+CD31 variant ^9 ^2 T cells persist better in 3-w MLR (FIGs.10A and 10B), compared to control BCMA CAR and full length CD31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P

[0321] According to 3-way MLR assay results, BCMA CAR+CD31v3-2 and 3 are further investigated by their tumor killing capabilities and corresponding persistence. In series tumor killing assays, BCMA CAR+CD31v3-2 and 3 ^9 ^2 T cells have comparable tumor killing capabilities and persist better (FIGs.11A-11B), compared to control BCMA CAR ^9 ^2 T cells.

[0322] Table 3 further lists examples of engineered CD31 constructs. Table 3. Examples of engineered CD31 constructs Construct name Anti-HvG activity BCMA CAR CD31fl onp6: membrane proximal region sequence of CD31 extracellular region 8H: CD8 hinge region 8HTM: CD8 hinge + TM 28TM: CD28 TM 28TMLL: CD28 TM with YT / LL mutationAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 28HTM: CD28 hinge + TM “-”, “+”, “++”, “+++”: means the persistence of CD31 polypeptides of CAR g9d2T cells in 3-w MLR compared to that of control CAR g9d2T cells “-”: no of persistence “+”: persistence better persistence “+++: much better persistence OTHER EMBODIMENTS

[0323] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P WHAT IS CLAIMED IS:

1. An engineered cell that overexpresses a CD31 polypeptide, wherein the engineered cell is an allogeneic cell isolated from a donor for being administered to a subject.

2. The engineered cell of claim 1, wherein the engineered cell expresses an exogenous CD31 polypeptide.

3. The engineered cell of claim 1 or 2, wherein the CD31 polypeptide comprises an extracellular domain of CD31.

4. The engineered cell of any one of claims 1-3, wherein the CD31 polypeptide comprises an extracellular domain of CD31, a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

5. The engineered cell of claim 4, wherein the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

6. The engineered cell of claim 5, wherein the truncated intracellular domain of CD31 comprises 2 amino acids of the intracellular domain of CD31.

7. The engineered cell of any one of claims 3-6, wherein the extracellular domain of CD31 comprises an amino acid sequence set forth in SEQ ID NO:1 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO:

1.

8. The engineered cell of any one of claims 1-7, wherein the CD31 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 9. An engineered cell that expresses a CD31 polypeptide comprising an extracellular domain, wherein the extracellular domain comprises at least a portion of C2 Ig domains of CD31.

10. The engineered cell of claim 9, wherein the extracellular domain comprises only 1, 2, 3, 4 or 5 of C2 Ig domains of CD31.

11. The engineered cell of claim 9 or 10, wherein the extracellular domain comprises no more than 5 of C2 Ig domains of CD31.

12. The engineered cell of any one of claims 9-11, wherein the C2 Ig domains are selected from C2 Ig domains 1, 2, 3, 4, 5 and 6 of CD31.

13. The engineered cell of any one of claims 9-12, wherein the extracellular domain of CD31 comprises an amino acid sequence set forth in one or more of SEQ ID NOs:16-21 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in one or more of SEQ ID NOs: 16-21.

14. The engineered cell of claim 9, wherein the extracellular domain comprising a membrane proximal region of CD31.

15. The engineered cell of claim 14, wherein the membrane proximal region comprises a portion of C2 Ig domain 6 of CD31.

16. The engineered cell of claim 14 or 15, wherein the membrane proximal region comprises or consists of an amino acid sequence set forth in SEQ ID NO:12 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

12.

17. The engineered cell of any one of claims 9-16, wherein the CD31 polypeptide comprises a transmembrane domain of CD31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 18. The engineered cell of claim 17, wherein the transmembrane domain of CD31 comprises an amino acid sequence set forth in SEQ ID NO: 4 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

4.

19. The engineered cell of any one of claims 9-18, wherein the CD31 polypeptide comprises an intracellular domain of CD31.

20. The engineered cell of claim 19, wherein the intracellular domain of CD31 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 9 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

9.

21. The engineered cell of claim 19, wherein the intracellular domain is a truncated intracellular domain, and wherein the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

22. The engineered cell of claim 21, wherein the truncated intracellular domain of CD31 comprises 2 amino acids of the intracellular domain of CD31.

23. The engineered cell of any one of claims 14-22, wherein the CD31 polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 10-12 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.

24. The engineered cell of any one of claims 1-23, wherein the engineered cell is selected from a group consisting of a T cell, a NK cell, a Treg cell, a B cell, a monocyte, a macrophage cell, a dendritic cell or a combination thereof.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 25. The engineered cell of claim 24, wherein the T cell is selected from a group consisting of a natural killer T (NK-T) cell, a γδ T cell, and an αβ T cell.

26. The engineered cell of claim 25, wherein the αβ T cell is modified to be suitable for allogeneic cell therapy.

27. The engineered cell of any one of claims 1-26, wherein the expression of the CD31 on the engineered cell is increased about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a wild-type cell or the control cell.

28. The engineered cell of any one of claims 1-27, further expressing a chimeric antigen receptor (CAR), TCR, or TAC receptor.

29. The engineered cell of claim 28, wherein the CAR specifically targets an antigen.

30. The engineered cell of claim 29, wherein the antigen is a tumor antigen, preferably, the tumor antigen selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER- 2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta-like 3 (DLL3), CD70, CS-1, c-Met, Glycolipid F77, PD-L1, and PD-L2.

31. The engineered cell of any one of claims 28-30, wherein the CAR comprises an amino acid sequence set forth in SEQ ID NO: 3 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 3.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 32. A composition comprising the engineered cell of any one of claims 1-31, and a pharmaceutically acceptable carrier.

33. A method of making the engineered cell of any one of claims 1-31, comprising introducing a vector expressing the CD31 polypeptide into a cell, thereby making the engineered cell.

34. The method of claim 33, wherein the vector is a retroviral vector.

35. A method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of the engineered cell of any one of claims 1-31, or the composition of claim 32.

36. The method of claim 35, wherein the disease or disorder is cancer, autoimmune disease, or infection.

37. A method of inhibiting immune clearance of an engineered cell in a subject, comprising administering the engineered cell of any one of claims 1-31 or the composition of claim 32 to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell.

38. The method of claim 37, wherein the immune clearance of the engineered cell is through T cell-mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

39. A method of increasing the in vivo expansion of an engineered cell in a subject, comprising administering the engineered cell of any one of claims 1-31 or the composition of claim 32 to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell.

40. A method of reducing Host-versus-Graft (HvG) reactions in a subject, comprising administering the engineered cell of any one of claims 1-31 or the composition of claim 32 to a subject in need thereof, thereby reducing HvG reactions in the subject.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 41. The method of claim 40, wherein the subject has undergone or will receive a cell or organ transplantation.

42. The method of claim 41, wherein the cell transplantation is a stem cell transplantation.

43. The method of any one of claims 33-42, wherein the engineered cell is a CAR-T cell.

44. The method of any one of claims 35-43, wherein the subject is a human subject.

45. A CD31 polypeptide comprising or consisting of an extracellular domain, wherein the extracellular domain comprises at least a portion of C2 Ig domains of CD31.

46. The CD31 polypeptide of claim 45, wherein the extracellular domain comprises only 1, 2, 3, 4 or 5 C2 Ig domains of CD31.

47. The CD31 polypeptide of claim 45 or 46, wherein the extracellular domain comprises no more than 5 C2 Ig domains of CD31.

48. The CD31 polypeptide of any one of claims 45-47, wherein the C2 Ig domains are selected from C2 Ig domains 1, 2, 3, 4, 5 and 6 of CD31.

49. The CD31 polypeptide of any one of claims 45-48, wherein the extracellular domain of CD31 comprises an amino acid sequence set forth in one or more of SEQ ID NOs:16-21 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in one or more of SEQ ID NOs: 16-21.

50. The CD31 polypeptide of claim 45, wherein the extracellular domain comprising a membrane proximal region of CD31.

51. The CD31 polypeptide of claim 50, wherein the membrane proximal region comprises a portion of C2 Ig domain 6 of CD31.Attorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P 52. The CD31 polypeptide of claim 50 or 51, wherein the membrane proximal region comprises or consists of an amino acid sequence set forth in SEQ ID NO:12 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

12.

53. The CD31 polypeptide of any one of claims 45-52, wherein the CD31 polypeptide comprises a transmembrane domain of CD31.

54. The CD31 polypeptide of claim 53, wherein the transmembrane domain of CD31 comprises an amino acid sequence set forth in SEQ ID NO: 4 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

4.

55. The CD31 polypeptide of any one of claims 45-54, wherein the CD31 polypeptide comprises an intracellular domain of CD31.

56. The CD31 polypeptide of claim 55, wherein the intracellular domain of CD31 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 9 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

9.

57. The CD31 polypeptide of claim 55, wherein the intracellular domain is a truncated intracellular domain, and wherein the truncated intracellular domain of CD31 comprises no more than 5 amino acids of the intracellular domain of CD31.

58. The CD31 polypeptide of claim 57, wherein the truncated intracellular domain of CD31 comprises 2 amino acids of the intracellular domain of CD31.

59. The CD31 polypeptide of any one of claims 45-58, wherein the CD31 polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 10-12 or aAttorney Docket No.: 51624-0080WO1 / Client Ref# LG-U2023114P sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.

60. The CD31 polypeptide of any one of claims 45-59, comprising a truncated intracellular domain of CD31 and one of the following: (1) a CD31 extracellular domain comprising amino acids of SEQ ID NO: 1 and a K89A mutation and a N25Q mutation; (2) a CD31 extracellular domain comprising an amino acid sequence of SEQ ID NO: 12 and a CD8 hinge region; (3) a CD31 extracellular domain comprising amino acids of SEQ ID NOs: 16-17 and a CD28 hinge and transmembrane domain; (4) a CD31 extracellular domain comprising the amino acid of SEQ ID NO: 12 and a transmembrane domain of CD31 and an intracellular domain of CD31; and (5) a CD31 extracellular domain comprising the amino acid of SEQ ID NO: 12 and a transmembrane domain of CD31 and a truncated intracellular domain of CD31.

61. A nucleic acid, comprising one or more nucleic acid sequences encoding: (a). a CD31 polypeptide of any one of claims 45-60; and optionally (b). an engineered receptor.

62. A vector comprising the nucleic acid of claim 61.

63. An engineered cell comprising the CD31 polypeptide of any one of 45-60, the nucleic acid of claim 61, or the vector of claim 62.

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