Cells overexpressing b3GNT2 and methods of use thereof

Abstract

Provided herein are engineered cells overexpressing a UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) polypeptide and methods of use thereof. In some embodiments, overexpression of the B3GNT2 polypeptide confers prevention or mitigation of T cell- or NK cell-mediated killing. Also provided herein viral particles comprising B3GNT2 polypeptide and methods of making and using the viral particles.

Description

Atorney Docket No.: 51624-0098WO1 / LG-U2024143WOCELLS OVEREXPRESSING B3GNT2 AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of priority of U.S. Patent Application No. US 63 / 690,199 filed on September 03, 2024, the content of which is incorporated herein by reference in its entirety.SEQUENCE STATEMENT

[0002] This application contains a Sequence Listing that has been submitted electronically as an XML file named “51624-0098WO1.” The XML file, created on September 2, 2025, is 10,582 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 UDP-GlcNAc:betaGal beta-1, 3-N- acetylglucosaminyltransf erase 2 (B3GNT2) 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, theAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO disclosure relates to engineered cells that overexpress a B3GNT2 polypeptide for the prevention or mitigation of T cell- or NK cell-mediated killing.

[0006] In one aspect, the disclosure is related to engineered cells that overexpress a B3GNT2 polypeptide. In some embodiments, the engineered cell is an allogeneic cell isolated from a donor (e g., for being administered to a subject). In some embodiments, overexpression of the B3GNT2 polypeptide confers prevention or mitigation of T cell- or NK cell-mediated killing.

[0007] In some embodiments, the engineered cell expresses an exogenous B3GNT2 polypeptide.

[0008] In one aspect, the disclosure is related to an engineered cell that overexpresses a Beta- 1,3-N-Acetylglucosaminyltransferase 2 (B3GNT2) polypeptide.

[0009] In some embodiments, the engineered cell expresses an exogenous B3GNT2 polypeptide.

[0010] In some embodiments, the engineered cell has an enhanced transcriptional activity of an endogenous B3GNT2 polypeptide. In some embodiments, the engineered cells described herein may express one or more regulators that can up-regulate the expression of B3GNT2.

[0011] In one aspect, the disclosure is related to an engineered cell, comprising an exogenous nucleic acid sequence that encodes a B3GNT2 polypeptide.

[0012] In some embodiments, the B3GNT2 polypeptide comprises a mutation (e.g., a deletion, an insertion, a substitution, or a combination thereof), wherein the B3GNT2 retains or enhances the poly-N-acetyl-lactosamine synthase activity of wildtype B3GNT2.

[0013] In some embodiments, the B3GNT2 polypeptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.

[0014] In some embodiments, the B3GNT2 peptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 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: 2.

[0015] In some embodiments, the engineered cell is an allogeneic cell isolated from a donor for being administered to a subject or an autologous cell.

[0016] In some embodiments, the engineered cell is a T cell, a NK cell, a Treg cell, a B cell, a monocyte, a macrophage cell, a dendritic cell, a peripheral blood mononuclear cell (PBMC), aAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO hematopoietic stem cell, a pluripotent stem cell, a mesenchymal stem cell or an embryonic stem cell.

[0017] In some embodiments, the T cell is a natural killer T (NK-T) cell, a y5 T cell, or an a.p T cell.

[0018] In some embodiments, the expression level of the B3GNT2 in the engineered cell is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a wild-type cell or the control cell.

[0019] In some embodiments, the engineered cell expresses an engineered receptor (e.g., chimeric antigen receptor (CAR), TCR, or TAC receptor).

[0020] In some embodiments, the engineered receptor is a CAR, and the CAR specifically targets an antigen.

[0021] In some embodiments, the antigen is a tumor antigen, preferably, the tumor antigen is BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD 19, 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-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, LI cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MARTI, 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, or PD-L2.

[0022] In some embodiments, (1) the HLA (MHC Class I and MHC Class II molecules) gene of the engineered cell is not genetically modified; (2) only the expression of endogenous MHC Class I molecules is eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the engineered cells; (3) only the expression of endogenous MHC Class II molecules is eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class II molecules is achieved by disrupting endogenous Class II major histocompatibility complex transactivator (CIITA) gene of the engineered cells; or (4) both the expression of endogenous MHC Class I molecules and MHC Class II molecules are eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the engineered cells, and whereinAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO the eliminated or reduced expression of endogenous MHC Class TI molecules is achieved by disrupting endogenous Class II major histocompatibility complex transactivator (CIITA) gene of the engineered cells.

[0023] In some embodiments, the expression of the B3GNT2 polypeptide confers resistance to T cell-mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

[0024] In one aspect, the disclosure is related to a method of reducing immunogenicity of an engineered cell, comprising one of the following: (1) inhibiting immune clearance of an engineered cell; (2) inhibiting resistance of an engineered cell to T cell-mediated cytotoxicity and / or NK cell- mediated cytotoxicity; (3) increasing the in vivo expansion of an engineered cell in a subject; or (4) reducing Host-versus-Graft (HvG) reaction of an engineered cell in a subject; wherein the method comprising overexpressing a B3GNT2 polypeptide or expressing an exogenous B3GNT2 polypeptide in the engineered cell.

[0025] In some embodiments, the method comprises introducing a nucleic acid encoding a B3GNT2 polypeptide into a cell.

[0026] In some embodiments, the nucleic acid comprises a non-native regulatory element (e.g., a promoter).

[0027] In some embodiments, the method comprises introducing a nucleic acid encoding a transcription activator that enhances the transcriptional activity of an endogenous B3GNT2 polypeptide.

[0028] In some embodiments, the B3GNT2 polypeptide comprises a mutation, wherein the B3GNT2 retains or enhances the poly-N-acetyl-lactosamine synthase activity of wildtype B3GNT2.

[0029] In some embodiments, the B3GNT2 polypeptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.

[0030] In some embodiments, the B3GNT2 peptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 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: 2.

[0031] In some embodiments, the engineered cell is an allogeneic cell isolated from a donor for being administered to a subject or an autologous cell.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0032] In some embodiments, the engineered cell is a T cell, a NK cell, a Treg cell, a B cell, a monocyte, a macrophage cell, a dendritic cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, a mesenchymal stem cell or an embryonic stem cell.

[0033] In some embodiments, the T cell is a natural killer T (NK-T) cell, a yd T cell, or an u.p T cell.

[0034] In some embodiments, the expression of the B3GNT2 on the engineered cell is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a wild-type cell or the control cell.

[0035] In some embodiments, the engineered cell expresses an engineered receptor (e.g., chimeric antigen receptor (CAR), TCR, or TAC receptor).

[0036] In some embodiments, (1) the HLA (MHC Class I and MHC Class II molecules) gene of the engineered cell is not genetically modified; (2) only the expression of endogenous MHC Class I molecules is eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the engineered cells; (3) only the expression of endogenous MHC Class II molecules is eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class II molecules is achieved by disrupting endogenous Class II major histocompatibility complex transactivator (CIITA) gene of the engineered cells; or (4) both the expression of endogenous MHC Class I molecules and MHC Class II molecules are eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the engineered cells, and wherein the eliminated or reduced expression of endogenous MHC Class II molecules is achieved by disrupting endogenous Class II major histocompatibility complex transactivator (CIITA) gene of the engineered cells.

[0037] In some embodiments, the expression of the B3GNT2 polypeptide confers resistance to T cell-mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

[0038] In one aspect, the disclosure is related to an engineered cell prepared by the method described herein.

[0039] In one aspect, the disclosure is related to a viral particle comprising B3GNT2 polypeptide that displays B3GNT2 polypeptide on the surface of the particle.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0040] In one aspect, the disclosure is related to a nucleic acid, comprising one or more nucleic acid sequences encoding: an engineered receptor, and a B3GNT2 polypeptide.

[0041] In some embodiments, the viral particle is a virus particle or a virus-like particle (VLP), optionally a retroviral particle or a retroviral -like particle, optionally a lentiviral particle or lentiviral-like particle.

[0042] In some embodiments, the viral particle is pseudotyped with a vesicular stomatitis virus envelope glycoprotein (VSV-G) or cocal virus glycoprotein (COV-G).

[0043] In some embodiments, the B3GNT2 polypeptide may be displayed on the surface of the particle via a transmembrane domain or a Glycosylphosphatidylinostol (GPI) membrane anchor.

[0044] In some embodiments, the viral particle further comprises a nucleic acid sequence.

[0045] In some embodiments, the nucleic acid sequence encodes an engineered receptor (e.g., chimeric antigen receptor (CAR), TCR, or TAC receptor.

[0046] In one aspect, the disclosure is related to a method of making a viral particle comprising B3GNT2 polypeptide, comprising (1) providing a producer cell that comprises a nucleic acid encoding B3GNT2 polypeptide; (2) culturing the cell under conditions that allow for production of a viral particle, and (3) separating, enriching, or purifying the particle from the cell.

[0047] In one aspect, the disclosure is related to a producer cell that overexpresses a B3GNT2 polypeptide.

[0048] In some embodiments, the producer cell is a HEK-293 cell or a derivative thereof, optionally a HEK-293T (e.g., free-style 293T cells) or a HEK-293 T-REx cell.

[0049] In some embodiments, the producer cell comprises a genetically disruption of endogenous MHC Class I molecules, wherein the disruption of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the producer cells.

[0050] In one aspect, the disclosure is related to a producer cell comprising (1) a viral nucleic acid(s) and (2) nucleic acid encoding an exogenous B3GNT2 polypeptide, optionally wherein the viral nucleic acid(s) are lentiviral nucleic acids.

[0051] In some embodiments, the viral nucleic acid(s) lacks one or more genes involved in viral replication.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0052] In one aspect, the disclosure is related to a viral particle produced by the method described herein.

[0053] In some embodiments, the nucleic acid sequence comprises one, two, three, four, five or more copies of a coding sequence of a B3GNT2 polypeptide; and / or wherein the nucleic acid sequence is operably linked to a non-native regulatory element.

[0054] In some embodiments, the B3GNT2 peptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.

[0055] In some embodiments, the B3GNT2 peptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0056] In some embodiments, the nucleic acid sequence encoding the engineered receptor and the nucleic acid sequence encoding the B3GNT2 polypeptide are separated by a third nucleic acid sequence encoding a cleavable linker.

[0057] In some embodiments, the cleavable linker comprises an amino acid sequence set forth in SEQ ID NO: 7, or a functional variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0058] In some embodiments, the engineered receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC) or a portion thereof.

[0059] In some embodiments, the engineered receptor is a CAR.

[0060] In one aspect, the disclosure is related to a vector comprising the nucleic acid of described herein.

[0061] In one aspect, the disclosure is related to an engineered cell comprising the nucleic acid described herein, or the vector described herein.

[0062] In one aspect, the disclosure is related to a composition comprising the engineered cell or the viral particle described herein, and a pharmaceutically acceptable carrier.

[0063] 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 the engineered cell described herein, the viral particle described herein, or the composition described herein.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0064] In some embodiments, the disease or disorder is cancer, autoimmune disease, or infection.

[0065] In one aspect, the disclosure is related to a method of inhibiting immune clearance of an engineered cell or a viral particle in a subject, comprising administering the engineered cell, the viral particle described herein, or the composition described herein to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell.

[0066] In some embodiments, the immune clearance of the engineered cell is through T cell- mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

[0067] In one aspect, the disclosure is related to a method of increasing the in vivo expansion of an engineered cell in a subject, comprising administering the engineered cell described herein, or the composition described herein to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell.

[0068] In one aspect, the disclosure is related to a method of reducing Host-versus-Graft (HvG) reactions in a subject, comprising administering the engineered cell described herein, the viral particle described herein, or the composition described herein to a subject in need thereof, thereby reducing HvG reactions in the subject.

[0069] In one aspect, the disclosure is related to a method of increasing the life of a particle in vivo in a mammal, method comprising administering the viral particle described herein, or the composition described herein to a mammalian subject wherein said administered particles have a longer half-life in said mammal than an otherwise similar particle that does not have B3GNT2 expressed thereon.

[0070] In one aspect, the disclosure is related to a method of delivering a nucleic acid sequence to a subject (e.g., a human subject), the method comprising administering to a subject the viral particle described herein, or the composition described herein, and the nucleic acid sequence is delivered to a target cell.

[0071] In some embodiments, the subject has undergone or will receive a cell or organ transplantation.

[0072] In some embodiments, the cell transplantation is a stem cell transplantation.

[0073] In some embodiments, the engineered cell is a CAR-T cell.

[0074] In some embodiments, the subject is a human subject.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0075] In one aspect, the disclosure is related to a method of producing the engineered cell described herein, the method comprising: (a) transfecting a producer cell with the vector described herein; (b) culturing the cell in a culture medium; and (c) harvesting the cell from the culture medium.

[0076] Other features and advantages of the disclosure will be apparent from the following detailed description and figures, and from the claims.DESCRIPTION OF DRAWINGS

[0077] FIGS. 1A-1D show that B3GNT2 protects K562 cells from NK cell-mediated killing.

[0078] FIG. 1A shows the expression of RV-B3GNT2-BFP in K562 cells 3 days after transduction, as indicated by the blue fluorescence protein (BFP) positive rate.

[0079] FIG. IB shows peripheral blood NK (PBNK) cell-mediated killing on K562 cells or B3GNT2-expressing K562 cells after a 2-day co-culture, as indicated by the amount of HLA-A2 negative cells. The effector cell (PBNK) to target cell ratio is 1 :3.

[0080] FIG. 1C shows peripheral blood NK (PBNK) cell-mediated killing on K562 cells or B3GNT2-expressing K562 cells after a 2-day co-culture in the presence of 400U IL2, as indicated by the amount of HLA-A2 negative cells. The effector cell (PBNK) to target cell ratio is 1 :3.

[0081] FIG. ID is generated based on the data in FIG. IB and FIG. 1C. FIG. ID shows the cell number of viable K562 cells or B3GNT2-expressing K562 cells after co-culture with PBNK cells for 2 days with or without 400U IL2.

[0082] FIGS. 2A-2D show that B3GNT2 promotes K562 cell growth in PBMC co-culture condition.

[0083] FIG. 2A shows the expression of BCMACAR in K562 cells 3 days after transduction.

[0084] FIG. 2B shows peripheral blood mononuclear cells (PBMC) cell-mediated growth inhibition on K562 cells or B3GNT2-expressing K562 cells after a 4-day co-culture, as indicated by the amount of HLA-A2 negative cells. The effector cell (PBMC) to target cell ratio is 100: 1.

[0085] FIG. 2C shows peripheral blood mononuclear cells (PBMC) cell-mediated growth inhibition on K562 cells or B3GNT2-expressing K562 cells after a 7-day co-culture, as indicated by the amount of HLA-A2 negative cells. The effector cell (PBMC) to target cell ratio is 100: 1.

[0086] FIG. 2D is generated based on the data in FIG. 2B and FIG. 2C. FIG. 2D shows the cell number of viable K562 cells or B3GNT2-expressing K562 cells after co-culture with PBMCAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO cells at different time points (DayO, Day4 and Day7). The effector cell (PBMC) to target cell ratio is 100: 1. PBMCs from one donor (D990) was used as effector cells.

[0087] FIG. 3A-3B show gdT Cell Purity and BCMACAR Expression in gdT Cells.

[0088] FIG. 4 shows the viable cell number of gdT cells (HLA-A2-) overexpressing BCMACAR or BCMACAR-B3GNT2 after a co-culture with primary PBMCs (HLA-A2+) at different time points (Day 4, and Day 9) with an E:T ratio of 20: 1. "D RG1754" indicates that the gdT cells were expanded from Donor RG1754. "D298," "D807," and "D987" indicate that the primary PBMCs were isolated from leukopak Donor 298, Donor 807, and Donor 987, respectively.

[0089] FIG. 5 lists some sequences described in the present disclosure.DETAILED DESCRIPTION

[0090] 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 these 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.

[0091] B3GNT2 encodes a member of the beta-1, 3-N-acetylglucosaminyltransferase family. It is a type II transmembrane protein with a short N-terminal cytosolic segment, a transmembrane region, and a C-terminal domain in Golgi lumen. It is involved in the biosynthesis of poly-N- acetyllactosamine chains. B3GNT2 encodes a poly-N-acetyllactosamine synthase that targets >10 ligands and receptors to disrupt interactions between tumor and T cells and reduce T cell activation.

[0092] The present disclosure demonstrated that the overexpression of B3GNT2 prevented both NK cell- and allogeneic PBMC cell-mediated killing. The overexpression of B3GNT2 can also promote the expansion and survival of cells expressing CAR. Therefore, the cells and methods described herein have various applications in allogeneic cell therapy and transplantation.

[0093] The present disclosure is based on, in part, that engineered allogeneic immune cells that overexpress a B3GNT2 polypeptide can reduce NK cell- and allogeneic PBMC cell-mediated killing, and can be used in allogeneic cell therapies. In one aspect, the disclosure provides engineered immune cells overexpressing a B3GNT2 polypeptide.

[0094] In one aspect, the disclosure provides engineered cells that overexpress a B3GNT2 polypeptide. The engineered cells can be allogeneic cell isolated from a donor.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0095] 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.

[0096] 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.

[0097] In one aspect, the disclosure provides methods of inhibiting immune clearance of an engineered cell by overexpressing a B3GNT2 polypeptide on the engineered cell.

[0098] In one aspect, the disclosure provides methods of increasing the in vivo expansion of an engineered cell by overexpressing a B3GNT2 polypeptide on the engineered cell.

[0099] In one aspect, the disclosure provides engineered cell that expresses a non-native B3GNT2 polypeptide, and also engineered cell that overexpresses a B3GNT2 polypeptide.

[0100] 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 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 particular protein. A domain derived from a particular protein can be from a natural or a synthetic source. For example, an intracellular domain derived from B3GNT2 can have a sequence that is identical to B3GNT2, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of B3GNT2.

[0101] As used herein, the term “B3GNT2 polypeptide” refers to a polypeptide derived from a wild-type B3GNT2 or a functional variant thereof. The B3GNT2 may be a wild-type B3GNT2 (e g., human B3GNT2). The B3GNT2 may have one or more mutations (e.g., insertions, deletions, or substitutions). The B3GNT2 may be a human B3GNT2. The B3GNT2 may be a truncated B3GNT2. The N-terminal cytosolic segment, the transmembrane region, or the C-terminal domain in Golgi lumen may be full-length or a portion thereof.

[0102] 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 anAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO 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.

[0103] As used herein, the term “producer cell” includes a cell that produces viral particles, after transient transfection, stable transfection, or vector transduction of all the elements necessary to produce the viral particles or any cell engineered to stably comprise the elements necessary to produce the viral particles.

[0104] The viral particle(s) referred to herein encompass replication-defective viruses, viral vectors derived therefrom, and which may or may not comprise a nucleotide or interest.

[0105] 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.

[0106] 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 TCRa polypeptide chain and a TCR(3 polypeptide chain. The TCR may comprise a TCRy polypeptide chain and a TCR8 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 that 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.

[0107] 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 oneAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO variable domain (e.g., a variable domain of a heavy chain, single domain antibody or VHH). Nonlimiting examples of antibody fragments include, e.g., Fab, Fab’, F(ab’)2, and Fv fragments.

[0108] 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.

[0109] 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 are 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.,Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

[0110] 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).

[0111] 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.”

[0112] As used herein, the term “retrovirus” as used herein is defined as an RNA virus of the Retroviridae family. Examples of retroviruses include, but are not limited to, members of the Lentivirus, Alpha-retrovirus, Beta-retrovirus, Gamma-retrovirus, Delta-retrovirus, Spumavirus and Epsilonretrovirus.

[0113] 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 byAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO reference in their entirety. Tn case of conflict, the present specification, including definitions, will control.HvG Reactions Against Engineered Immune Cells

[0114] 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.

[0115] The highly polymorphic HLA molecules in the population are the main cause of T cell- mediated HvG. Since P2-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.

[0116] 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 subj ect, 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 cellAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO 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

[0117] However, the above three strategies all have obvious and insurmountable drawbacks. 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.

[0118] 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 or the viral particle to a subject in need thereof, thereby reducing HvG reactions in the subject.

[0119] The present disclosure also provides methods of preventing, inhibiting, or reducing HvG reactions related to cell or organ transplantation. The subj ect 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.

[0120] 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 moreAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO times. Suitable methods of administering the engineered cell are described herein and are known in the art.The B3GNT2 Polypeptide

[0121] B3GNT2 encodes a member of the beta-1, 3-N-acetylglucosaminyltransferase family. It is a type II transmembrane protein with a short N-terminal cytosolic segment, a transmembrane region, and a C-terminal domain in Golgi lumen. It is involved in the biosynthesis of poly-N- acetyllactosamine chains. B3GNT2 encodes a poly-N-acetyllactosamine synthase that targets >10 ligands and receptors to disrupt interactions between tumor and T cells and reduce T cell activation.

[0122] A detailed review of B3GNT2 and its functions can be found in Joung, Julia, et al. "CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity." Nature Communications 13.1 (2022): 1606; and Hao, Yue, et al. "Structures and mechanism of human glycosyltransferase pi, 3-N-acetylglucosaminyltransferase 2 (B3GNT2), an important player in immune homeostasis." Journal of Biological Chemistry 296 (2021), each of which is incorporated by reference in its entirety.

[0123] The present disclosure provides a method of preserving the efficacy of allogeneic cell therapy products and to overcome HvG in patients treated with a cell therapy, e.g., by overexpressing a B3GNT2 polypeptide in the cell. In one aspect, the disclosure provides an allogeneic cell therapy product that overexpresses a B3GNT2 polypeptide. The cell may not express an endogenous B3GNT2. The cell may express an endogenous B3GNT2.

[0124] The present disclosure is related to engineered cells (e.g., CAR-T cells, CAR-NK cells, TCR-T cells) overexpressing a B3GNT2 polypeptide. The engineered cell may be an allogeneic cell isolated from a donor. The engineered cell may express an exogenous B3GNT2 polypeptide. The exogenous B3GNT2 polypeptide may protect the modified cells from NK cell- or T cell- mediated killing. The exogenous B3GNT2 polypeptide may be the only exogenous peptide that the modified cells express.

[0125] The B3GNT2 polypeptide may comprise one or more mutations. In some cases, the B3GNT2 polypeptide does not comprise any mutation in the following positions: A279, D247, H376, K149, D245, Y289, D332, and D333. In some embodiments, the B3GNT2 polypeptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0126] In one aspect, the present disclosure provides a B3GNT2 polypeptide that comprises a full-length N-terminal cytosolic segment, a transmembrane region, and a C-terminal domain in Golgi lumen. The B3GNT2 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. The B3GNT2 polypeptide may comprise a N-terminal cytosolic segment 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 amino acids 1-7 of SEQ ID NO: 2, a transmembrane region 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 amino acids 8-28 of SEQ ID NO: 2, and / or a C-terminal domain in Golgi lumen 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 amino acids 29-397 of SEQ ID NO: 2.

[0127] The B3GNT2 polypeptide may comprise or not comprise a signal peptide.

[0128] The B3GNT2 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 B3GNT2 polypeptide may have an amino acid sequence that is identical to SEQ ID NO: 2. The B3GNT2 polypeptide can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 mutations. The B3GNT2 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)

[0129] In one aspect, the present disclosure provides cells (e.g., immune cells) that express engineered receptor and the B3GNT2 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, 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-Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO 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).

[0130] 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.

[0131] 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 B3GNT2 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.

[0132] 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 0X40. The signaling domain of a naturally occurring molecule can comprise the entire intracellular or cytoplasmic portion, or the entire native intracellular signaling domain, of the molecule, or a fragment or derivative thereof.

[0133] 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 primaryAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO intracellular signaling domain may contain a signaling motif known as Immunoreceptor Tyrosinebased 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. CD3(^ 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.

[0134] 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 activation 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 0X40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11 a / CD 18), 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, CD 160, CD 19, CD4,Atorney Docket No.: 51624-0098WO1 / LG-U2024143WOCD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA- 1, ITGAM, CDl lb, ITGAX, CDl lc, 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, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83.

[0135] 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 CD3Q. 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 CD3Q 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), 0X40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specially bind to CD83.

[0136] 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 CD3(^ and a co-stimulatory signaling domain of CD28. The intracellular 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 CD3(^ and a co-stimulatory signaling domain of 4- 1BB.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0137] 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 CD3L / 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 CD3(^.

[0138] 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.

[0139] The tumor antigen may be selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD 19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD 10, 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-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, LI cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MARTI, 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, Deltalike 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.

[0140] 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 CARs can be or include the CD 19 binding fragment (e.g., FMC63, SJ25C1, orthose 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.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0141] 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, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), 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, CD8a, or CD28 transmembrane region. The transmembrane region of the CAR may comprise a transmembrane region of CD8a.

[0142] The extracellular domain may be connected to the transmembrane region by a hinge region. The hinge region may comprise a hinge region of CD8a.

[0143] The CAR may comprise a signal peptide (SP), such as a CD8a signal peptide.

[0144] 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.

[0145] The CAR may be a single CAR, dual CAR (or dual-targeting CAR), tandem CAR or split CAR.

[0146] 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: 4. The BCMA CAR may comprise an amino acid sequence of SEQ ID NO: 4. 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%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. The CAR may specifically bind to BCMA-positive tumor cells.

[0147] 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,Atorney Docket No.: 51624-0098WO1 / LG-U2024143WOCD33, CD38, CEA, CS1, CD138, CD123 / IL3Ra, c-Met, gplOO, 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, Claudinl8.2, 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.

[0148] The TCR receptor complex is an octomeric complex formed by variable TCR receptor a and chains (or y and 8 chains on case of y8 T cells) with three dimeric signaling modules CD38 / s, CDSy / s and CD247 (T-cell surface glycoprotein CD3 zeta chain) / or ^ / q. 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.

[0149] 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, 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 butAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO 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, CD 134, CD 137, CD 154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0150] 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.

[0151] 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 / IL3Ra, c-Met, gplOO, 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, Claudinl8.2, 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 a 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 aAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO transmembrane region and a cytosolic domain derived from CD4. The TAG receptor may comprise a transmembrane region and a cytosolic domain derived from CD8 (such as CD8a).

[0152] 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 coreceptor 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.

[0153] 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), P-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.

[0154] 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 gplOO 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 of 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 CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0155] 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.

[0156] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-l / 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, pl 5; 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, pl85erbB2, pl80erbB-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, M0V18, 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)

[0157] The present disclosure provides (i) nucleic acids (e.g., expression vectors) encoding the B3GNT2 polypeptide, and / or (ii) optionally nucleic acids (e.g., expression vectors) encoding an engineered receptor (e.g., CAR or TCR). The nucleic acids of the present disclosure can comprise a nucleic acid sequence encoding any one of the B3GNT2 polypeptide, CARs, and / or TCRs disclosed herein. The nucleic acid may encode both an engineered receptor and an B3GNT2 polypeptide.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0158] 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 multi ci stronic 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 B3GNT2 polypeptide described herein.

[0159] 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.

[0160] 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), and 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 asAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO“F2A,” “E2A,” “P2A,” and “T2A,” respectively. Those of skill in the art would be able to select the appropriate self-cleaving peptide.

[0161] 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: 5) and (GGGS)n (SEQ ID NO: 6), where n represents an integer of at least 1. The linker may be an SSGGGGS (SEQ ID NO: 7) linker. Those of skill in the art would be able to select the appropriate spacer sequence.

[0162] 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.

[0163] 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 Ncrl (p46) promoter; see, e.g., Eckelhart et al. Blood (2011) 117: 1565.

[0164] 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-la 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 expression is desired, or turning off the expression when expression is not desired. Examples of inducibleAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0165] A polynucleotide of the present disclosure may enable the production of (i) the B3GNT2 polypeptide described herein, and / or (ii) the engineered receptor described herein (e.g., in a mammalian cell). A polynucleotide of the present disclosure may enable replication of the polynucleotide.

[0166] 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-la promoter. The use of an EF-la promoter can increase the efficiency in expression of downstream transgenes (e.g., a CAR- or TCR-encoding polynucleotide). Physiologic promoters (e.g., an EF-la 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.

[0167] The polynucleotide may encode a naked CAR. The polynucleotide may comprise from the 5' end to the 3' end, a CD8a signal peptide, an antigen binding domain, a CD8a hinge region, a CD8a transmembrane region, a CD137 co-stimulatory signaling domain, a CD3(^ cytoplasmic domain.

[0168] The polynucleotide may encode a CAR and a B3GNT2 polypeptide. The polynucleotide may comprise from the 5' end to the 3' end, the coding sequences of a B3GNT2 polypeptide, a 2A cleavable linker, and a CAR. The polynucleotide may encode an amino acid sequence of B3GNT2 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 least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WOThe 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 examplee, 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

[0173] 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 mutagenesis, 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.

[0174] The B3GNT2 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 thatAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO comprises an expression cassette including the appropriate gene of active fragment thereof. The 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.

[0175] A construct encoding both a B3GNT2 polypeptide and a CAR can be introduced into the host cell using a lentiviral delivery system. A construct encoding both a B3GNT2 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 a.pT cells. The host cells can be yST cells. The host cells can be V81 yST cells. The host cells can be V82 yST cells. The host cells can be V83 y8T cells. The host cells can be tumor-infiltrating lymphocytes (TIL). The host cells can be NK cells.Engineered Cells

[0176] In one aspect, the present disclosure provides engineered cells comprising the B3GNT2 polypeptide described herein. The engineered cells comprising the B3GNT2 polypeptide described herein may further comprise an engineered receptor (e.g., CAR). The engineered cells comprising the B3GNT2 polypeptide described herein may further comprise a CAR (CAR armored with B3GNT2 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, a T cell, y8T cell, NK cell, NKT, tumor-infdtrating lymphocytes (TIL), peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, a mesenchymal stem cell, an embryonic stem cell, and a combination thereof.

[0177] In one aspect, the present disclosure provides engineered cells comprising a B3GNT2 polypeptide (e.g., an exogenous B3GNT2 polypeptide).

[0178] In one aspect, the present disclosure provides engineered cells that overexpress an endogenous B3GNT2 polypeptide. For example, the engineered cells may express a transcription activator for the endogenous B3GNT2 gene. In addition, the endogenous B3GNT2 gene may have a genetically modified regulatory element, e.g., a modified promoter. The engineered cells described herein may express one or more regulators that can up-regulate the expression of B3GNT2.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0179] 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) TCRa, TCRp, TCRy, TCR5, CD3y, CD38, CD3s and (^-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. 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.

[0180] The engineered cells expressing the B3GNT2 polypeptide described herein may express more than one engineered receptors, such as any combination of CAR, TCR, or TAC receptor.

[0181] The engineered cell expressing the B3GNT2 polypeptide described herein may be used to treat cancer.

[0182] Comparing to a cell without the expression of the B3GNT2 polypeptide described herein, the modified cell comprising the B3GNT2 polypeptide described herein may have a higher cytotoxicity against tumor cells. Comparing to a cell without the expression of the B3GNT2 polypeptide described herein, the modified cell comprising the B3GNT2 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.

[0183] In one aspect, the present disclosure provides engineered cells comprising (i) a B3GNT2 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 moreAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO polynucleotides encoding (i) a B3GNT2 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).

[0184] 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 aP T cells. The engineered cells may be yo T cells. The engineered cells may be V51 T cells. The engineered cells may be V82 T cells.

[0185] 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 P2- microglobulin to prevent the formation of functional Class I MHC molecules, or by inactivating Class II MHC molecules. The engineered cell expressing the B3GNT2 polypeptide described herein may have a disruption at the endogenous B2M gene, or has reduced expression or activity ofB2M.

[0186] 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.

[0187] 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.

[0188] The engineered cells may be from a cell line, e.g., a T2 human hybrid cell line (T and B lymphoblast).

[0189] Comparing to unmodified cells that do not comprise the B3GNT2 polypeptide, the engineered cells may have an enhanced proliferation. Comparing to unmodified cells that do not comprise the B3GNT2 polypeptide, the engineered cells may have an enhanced cytotoxicity toward tumor cells.

[0190] The engineered receptor may be a CAR. The CAR may comprise a signal peptide. The signal peptide may be derived from Ig molecule. The CAR may comprise a hinge region. The hinge region may be derived from FcR. The CAR may comprise a co-stimulatory domain. The co-Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO stimulatory domain may be derived from CD28. The CAR may comprise a cytoplasmic domain. The cytoplasmic domain may be derived from CD3 epsilon. The CAR may comprise from N- terminus to C-terminus: a CD8a signal peptide, an antigen binding domain, a CD8a hinge region, a CD8a transmembrane region, a CD137 co-stimulatory signaling domain, and a CD3(^ 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 may bind to a cancer specific antigen or a cancer associated antigen, or an autoimmune target antigen. 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: 4.

[0191] The B3GNT2 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.

[0192] Comparing to a cell without the overexpression of the B3GNT2 polypeptides, the engineered cell described herein may have an increased expression of B3GNT2 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 B3GNT2 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 B3GNT2 polypeptide.

[0193] The engineered cells may comprise both a B3GNT2 polypeptide and a CAR (CAR armored with B3GNT2 polypeptide). The engineered cells may be modified CAR-T cells (B3GNT2 polypeptide armored CAR-T cells). The expression of CAR and B3GNT2 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 thanAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO75%, less than 80%, or less than 90%. The engineered cells may have a CAR positive rate of 60%- 70%, 60%-80%, 60%-90%, 65%-85%, or 65%-90%.

[0194] 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%, less than 80%, or less than 90%. The engineered cells may have a purity of 90%-100%, 95%-100%, 98%-100%, or 98%-99%.

[0195] 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 rechallenge 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 than 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 B3GNT2 polypeptide, the cytotoxicity of the engineered cells comprising the B3GNT2 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 thanAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO500%, 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.[001961 The overexpression of B3GNT2 can protect the engineered cells described herein from NK cell-mediated killing (e.g., immune clearance), with or without IL2 stimulation. The effector cell to target cell ratio may be 1 :3, 1 :2, or 1 : 1. The incubation time may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. With the overexpression of the B3GNT2 polypeptide, the NK cell-mediated killing of the engineered cells (overexpressing the B3GNT2 polypeptide) may be reduced 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 the NK cell- mediated killing of control cells (without the overexpression of the B3GNT2 polypeptide). With the overexpression of the B3GNT2 polypeptide, the NK cell-mediated killing of the engineered cells (overexpressing the B3GNT2 polypeptide) may be reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more folds compared to control cells (without the overexpression of the B3GNT2 polypeptide). With the overexpression of the B3GNT2 polypeptide, the NK cell-mediated killing of the engineered cells (overexpressing the B3GNT2 polypeptide) may be further reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more folds in the presence of IL2 (e.g., 400 U / ml IL2) compared to control cells (without the overexpression of the B3GNT2 polypeptide).

[0197] The overexpression of B3GNT2 can protect the engineered cells described herein from allogeneic PBMC cell-mediated killing (e.g., immune clearance). The effector cell to target cell ratio may be 100: 1, 50: 1, or 25: 1. The incubation time may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. With the overexpression of the B3GNT2 polypeptide, the allogeneic PBMC cell-mediated killing of the engineered cells (overexpressing the B3GNT2 polypeptide) may be reduced 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 the allogeneic PBMC cell-mediated killing of control cells (without the overexpression of the B3GNT2 polypeptide). With the overexpression of the B3GNT2 polypeptide, the allogeneic PBMC cell-mediated killing of the engineered cells (overexpressing the B3GNT2 polypeptide) may be reduced by about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more folds compared to control cells (without the overexpression of the B3GNT2 polypeptide).Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0198] The overexpression of B3GNT2 may help preserve the expansion of the engineered cells in the presence of allogeneic PBMC cells (e.g., by protecting the cells from the killing of allogeneic PBMC cells). With the overexpression of the B3GNT2 polypeptide, the expansion of the engineered cells (overexpressing the B3GNT2 polypeptide) 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 the expansion of control cells (without the overexpression of the B3GNT2 polypeptide). The expansion of the engineered cells (overexpressing the B3GNT2 polypeptide) may be increased by about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more folds compared to control cells (without the overexpression of the B3GNT2 polypeptide). Cell expansion or proliferation can be measured by an in vitro cell proliferation assay. Comparing to control cells without the overexpression of the B3GNT2 polypeptide described herein, the modified cells overexpressing the B3GNT2 polypeptide described herein may have a viable 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 control cells without the overexpression of the B3GNT2 polypeptide described herein, the modified cells overexpressing the B3GNT2 polypeptide described herein may have a viable 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%.Allogeneic Cells

[0199] In one aspect, the present disclosure provides allogeneic cells, the terms “allogeneic cells”, “allogeneic immune cells” or “allogeneic engineered immune cells” are used interchangeably herein to refer to the cells are obtained from allogeneic donor. The allogeneic cells may be T cells or NK cells. The T cell may a Gamma delta (y5) T cells (or 76T cells, or gdT cells).

[0200] In addition, y8 T cells have shown to possess the ability to bridge innate and adaptive immunity. The majority of yd T cells in adult human blood exhibit Vy9V52 T cell receptors and respond to small phosphorylated nonpeptide antigens, called phosphoantigens (pAgs), which areAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO commonly produced by malignant cells. Unlike conventional a.p T cells, y8 T cells do not recognize polymorphic classical major histocompatibility complex (MHC) molecules and are therefore free of graft-versus-host disease (GvHD) risk when adoptively transferred into an allogeneic host. Additionally, y5 T cells have several other unique features that make them ideal cellular carriers for developing off-the-shelf cellular therapy for cancer. These features include: 1) y5 T cells have roles in cancer immunosurveillance; 2) y8 T cells have the remarkable capacity to target tumors independent of tumor antigen- and major histocompatibility complex (MHC)- restrictions; 3) y6 T cells can employ multiple mechanisms to attack tumor cells through direct killing and adjuvant effects; and 4) y5 T cells express a surface receptor, FcyRIII (CD16), that is involved in antibody-dependent cellular cytotoxicity (ADCC) and can be potentially combined with monoclonal antibody for cancer therapy.

[0201] 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.

[0202] 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) TCRa, TCR[3, TCRy, TCR5, CD3y, CD36, CD3s and ij-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.

[0203] 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).

[0204] 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 subunitAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO of TCR orHLA. For example, the T cell orNK 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).Viral Particles

[0205] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. The viral particle(s) of the present invention may be vectors. A viral particle may comprise an outer lipid bilayer membrane. Numerous viral particles are known in the art, including retrovirus, herpes simplex virus, vaccina virus, hepadnavirus, togavirus, flavivirus, arenavirus, coronavitus, orthomyxovirus, paramyxovirus, bunyavirus, bornavirus, rhabdovirus and filovirus. The viral particle may be a virus-like particle (VLP).

[0206] In some aspects, the viral particle comprises a retroviral particle, a retroviral-like particle, a lentiviral particle, or a lentiviral-like particle.

[0207] In some aspects, the viral particle such as retrovirus or retrovirus-like particle, comprises one or more gag polyprotein, polymerase (e.g., pol), integrase (e.g., a functional or nonfunctional variant), protease and a fusogen.

[0208] In some embodiments, the retrovirus is a Gammretrovirus. In some embodiments the retrovirus is an Epsilonretrovirus. In some embodiments the retrovirus is an Alpha-retrovirus. In some embodiments the retrovirus is a Beta-retrovirus. In some embodiments the retrovirus is a Delta-retrovirus. In some embodiments the retrovirus is a Lentivirus. In some embodiments the retrovirus is a Spumaretrovirus. In some embodiments the retrovirus is an endogenous retrovirus.

[0209] Illustrative lentiviral particle include but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, HIV based vector backbones (i.e., HIV cisacting sequence elements) are used. In some embodiments, the virus particles are derived from lentivirus. In some embodiments, the lentiviral vector particle is Human Immunodeficiency Virus- 1 (HIV-1).

[0210] In some aspects, viral envelope protein of the viral particle includes a vesicular stomatitis virus envelope glycoprotein (VSV-G) envelope protein, a measles virus envelopeAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO protein, a nipha virus envelope protein, or a cocal virus glycoprotein (COV-G) envelope protein or a functional variant.Producer Cells

[0211] In some aspects, the invention provides the use of a producer cell for production of viral particles. The producer cell may comprise the viral genome. The viral genome is the nucleic acid sequence that is incorporated into the viral particle. The viral genome may be engineered to comprise a nucleotide of interest.

[0212] In some aspects, for use in producing viral particles, the producer cell may comprise the viral genome and subsequently cultured under conditions suitable for the production of the viral particles.

[0213] In some aspects, the producer cell may comprise nucleic acid sequences encoding some or all the structural proteins required for viral particle assembly.

[0214] In addition, the expression of MHC Class I molecules of the producer cell has been disrupted or decreased.

[0215] In one aspect, the cell may be transfected or transduced with or engineered to stably integrate by targeted integration a nucleic acid comprising the viral genome to enable production of enveloped viral particles which comprise the viral genome.

[0216] In some aspects, the producer cell of the invention comprises nucleic acid sequences encoding Gag, Gag / Pol, and / or Env proteins, or functional substitutes thereof. The cell may optionally comprise nucleic acid sequences encoding additional proteins that may be required for retroviral vector particle assembly, for example Rev protein.

[0217] In addition, the viral particle producer cells can be of any suitable cell type that is capable of producing or packaging enveloped viral particles. The cells are preferably mammalian cells, particularly human cells. For example, the enveloped viral particles producer cell may be derived from a parental HEK-293 cell.Methods of Treatment

[0218] The methods described herein, the B3GNT2 polypeptide described herein, the polynucleotides described herein and the modified cells or the viral particles described herein can be used in a variety of experimental, therapeutic and commercial applications.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0219] In one aspect, the disclosure provides a method of modulating an immune response comprising administering an effective amount of modified cells or viral particles described herein to a subject in need thereof.

[0220] 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 or the viral particle described herein.

[0221] 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 B3GNT2 polypeptide into the engineered cell, thereby overexpressing the B3GNT2 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.

[0222] 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 B3GNT2 polypeptide into the engineered cell, thereby overexpressing the B3GNT2 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.

[0223] The term “effective amount” as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired results.

[0224] In another aspect, the present disclosure provides a method for treating cancer comprising administering an effective amount of the modified cells or the viral particles 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.

[0225] The disclosure further includes the use of the modified cells or the viral particles 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.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0226] The modified cells can also be used in experimental models, for example, to further study and elucidate the function of the cells.

[0227] One or more of the modified cells or the viral particles 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.

[0228] 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 or the viral particles. 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.

[0229] The modified cells or viral particles described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the modified cells or the viral particles 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 any 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 anAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO 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.

[0230] 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.

[0231] 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 be genetically similar. The second subject may express the same HLA class or supertype as the first subject.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0232] 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.

[0233] 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.

[0234] 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.

[0235] The modified cells or the viral particles 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 or the viral particles 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 also included. The cancer can be a solid tumor orAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO 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.

[0236] 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).

[0237] 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.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WO

[0238] 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.

[0239] The modified cells (e.g., immune cells, T cells, or NK cells) or the viral particles 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.

[0240] 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.

[0241] The modified cells or the viral particles 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 multidose containers with a preservative.EXAMPLESThe disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.Attorney Docket No.: 51624-0098WO1 / LG-U2024143WOExample 1. Materials and MethodsAntibodies

[0242] The antibodies used in the below examples are listed below in Table 2. Table 2: Antibody informationPlasmid

[0243] The B3GNT2 DNA sequence (SEQ ID NO: 1) and BCMA-CAR DNA sequence (SEQ ID NO: 3) were inserted into the GFP section of Sin-CMV plasmid (Biovec pharma).Cell lines

[0244] 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.

[0245] K562 cells (ATCC) were cultured using RPMI1640 (Gibco) medium completed with10% Fetal Bovine Serum (Gibco) at 37°C in a humidified atmosphere with 5% CO2.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WOHealthy blood donors

[0246] 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. yd T cell expansion

[0247] PBMCs were expanded under a condition using zoledronic acid (Sigma) and IL-2 (Life Technology) in the CTS™ OpTmizer™ T Cell Expansion medium (Fisher Scientific). On Day 0, Day 2, and Day 9, the cell number of PBMCs was determined using a Vi-CELL Cell Counter (Beckman Coulter). On Day 0 and Day 9, PBMCs were immunophenotyped for y5 T cell (CD3+Vy9+V52+) and natural killer (NK) cell content (CD3-CD56+) using a Cytek® Aurora 5L (UV-V-B-YG-R) cytometer. Unprocessed data were exported and analyzed in FlowJo™ (vl0.8). All information regarding antibodies for immunophenotyping is provided in table 2.PBNK cell expansion

[0248] PBNK cells were expanded using feeder free protocol.Primary NK cell activation

[0249] Primary NK cells were isolated from PBMCs using NK cell isolation kit (Stemcell Technologies). They were activated by 1000U IL2 (Gibco) for three days.Retroviral packaging and transduction

[0250] 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 293 Vec-BaEV cells using Lipofectamine 3000 (Life Technology) for 6 h at 37°C in 5% CO2 according to the manufacturer's protocol. Following 48-72 h, supernatant containing retroviral particles was harvested and filtered through a 0.45-pm filter (EMD Millipore) to remove cell debris. The supernatants were concentrated by adding Retro-Atorney Docket No.: 51624-0098WO1 / LG-U2024143WOX 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 l>< 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 / pl) = (% gene+ cells x number of cells transduced) / virus volume.

[0251] For retroviral transduction, K562 cells or 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=I The cell and virus mixture were spin inoculated for 2hr at 2,500 rpm at 32°C. After centrifugation, the plate was put back into the incubator overnight. The medium was replaced on the 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

[0252] yb T cells (or gdT cells) and NK cells were characterized with different markers by flow cytometry. Detailed information of the antibodies used is provided in Table 2. Specifically, yb T cells or NK cells were washed with FACS staining buffer (BioLegend). Cells were resuspended in FACS staining buffer containing Fc Receptor Block™ Reagents (BD Biosciences) and incubated for 10 minutes at room temperature. Immediately following the incubation, cells were stained with the antibody panel (Table 2) for 45 minutes at 4°C. After staining, cells were washed twice with FACS staining buffer and immediately analyzed for cell surface marker expression using a Cytek® Aurora 5L (UV-V-B-YG-R) cytometer. Data analysis was performed using FlowJo™ (vl0.8).Flow cytometry-based cytotoxicity assay

[0253] Target cells (e.g. K562 or primary ybT cells, with or without modification) were harvested and re-suspended in RPMI media containing 10% FBS and counted. Effector cells (expanded NK cells) were harvested and re-suspended in RPMI media (Gibco) containing 10% FBS (Gibco) and counted. The effector: target ratio is 1:3. After target cell seeding, the effector cells were added to target cells for 48hr. After the two-day co-culture, cells were harvested and counted. The cell mixture is washed and stained with HLA-A2 for 45 mins at 4 °C. ImmediatelyAtorney Docket No.: 51624-0098WO1 / LG-U2024143WO prior to analysis, the cells were added with 7-AAD (BD Biosciences) 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. mixed lymphocyte reaction (MLR) assay

[0254] Allogeneic primary PBMCs (HLA-A2+) were freshly thawed, counted and resuspended in RPMI media containing 10% FBS. Candidate gene over-expressing K562 cells or gdT cells (HLA-A2-) were harvested, counted and re-suspended in RPMI media containing 10% FBS. The primary PBMCs were co-cultured with candidate gene over-expressing K562 cells or gdT cells(HLA-A2-) at 100: 1 50: 1, or 20: 1 ET ratio for 7 days or 9 days. Cell medium was changed every two or three days. On day 4 , day 7 or day 9, cells were taken out for counting and flow cytometry analysis.Example 2: B3GNT2 Expressing K562 cells are Resistant to NK cell Mediated Killing

[0255] K562 cells are HLA-A2 negative cells and very sensitive to NK cell mediated killing owing to the disfunction of B2M. K562 cells were used as target cells to determine whether the overexpression of B3GNT2 can prevent both NK cell mediated killing.

[0256] Retrovirus (RV) was used to transduce the K562 cells and enhance the expression of B3GNT2. Blue fluorescence protein (BFP) was used as a marker to detect the expression of B3GNT2. RV-B3GNT2-BFP was introduced into K562 cells by transduction. The expression of B3GNT2 in K562 cells was above 95%, as indicated by the blue fluorescent protein (BFP) positive rate (FIG. 1A). B3GNT2 expressing K562 cells or control K562 cells were co-cultured with expanded PBNK cells for two days with or without 400U IL2. After two-day co-culture, NK cells showedB strong killing capability on K562 cells even at 1 :3 ET ratio, as indicated by the amount of HLA-A2 negative cells (FIGS. 1B-1D). The cell number of B3GNT2 expressing K562 cells is dramatically increased compared with control K562 cells with or without IL2 condition (FIG. ID). This indicates that B3GNT2 overexpression protected K562 cells from NK cell mediated killing.Atorney Docket No.: 51624-0098WO1 / LG-U2024143WOExample 3: B3GNT2 Reverse the Inhibitory Effects of PBMCs on K562 Cell Growth

[0257] The primary PBMCs (from Donor 990) were obtained from HLA-A2 positive donors. D990 primary PBMCs were freshly thawed, counted and co-cultured with BCMA-CAR expressing K562 cells or BCMA-CAR-B3GNT2 expressing K562 cells for 7 days at 100: 1 ET ratio without normalization of BCMA-CAR expression. The expression of BCMA-CAR in K562 cells showed some variations. Three days after transduction, the expression of BCMA-CAR in BCMA-CAR-B3GNT2 expressing K562 cells was a little lower (65%) compared to BCMA-CAR expressing K562 cells (93%) (FIG. 2A). PBMCs inhibited K562 cell growth at both Day 4 and Day 7, as indicated by the amount of HLA-A2 negative cells (FIGS. 2B-2D). On Day 4, the inhibition of K562 cell growth by PBMCs was stronger than at Day 7 (FIGS. 2B-2D). The expression of BCMA-CAR-B3GNT2 in K562 cells helped preserve the growth of K562 cells compared to BCMA-CAR expressing K562 cells (FIGS. 2B-2D).Example 4: B3GNT2 Prevent gdT Cells from Allogeneic PBMC Mediated Rejection

[0258] To expanse yd T (gdT) cell, 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, 9, PBMCs cell number were determined using Vi-CELL Cell Counters (Beckman Coulter). On days 0 and day9, PBMCs were immunophenotyped for y5 T cell (CD3+Vy9+V52+) and natural killer (NK) cell content (CD3-CD56+) using a Cytek Aurora 5 L (UV, V, B, YG, R) cytometer. Unmixed data were exported and analyzed in FlowJo (vl0.8). All information regarding antibodies for immunophenotyping is provided in table 2.

[0259] On day 9, the purity of gdt cells and BCMAC AR transduction efficiency were analyzed by flow cytometry. The purity of gdt cells is 80% or above which were CD3+ V82+ (FIG. 3A). The BCMACAR expression with B3GNT2 is lower (39.2%) compared to the BCMACAR transduced gdt cells (76.6%) (FIG. 3B). Three different HLA-A2+ allogeneic PBMCs (D298, D807, D987) were co-cultured with day 9 harvested human gdT cells (D RG1754, HLA-A2-) at 20: 1 effector-to-target (E: T) ratio for 9 days.

[0260] On day 4 and day 9, small portions of cells were taken out for counting and analysis of viable HLA-A2- gdT cells. Compared with BCMACAR gdt cells, the leftover B3GNT2 overexpressed gdt cells was dramatically increased (FIG. 4). These findings indicate thatB3GNT2 has a protective effect against allogeneic PBMCs mediated rejection.Attorney Docket No.: 51624-0098WO1 / LG-U2024143WOOTHER EMBODIMENTS

[0261] 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.: 51624-0098WO1 / LG-U2024143WOWHAT IS CLAIMED IS:

1. An engineered cell that overexpresses a Beta-1,3-N-Acetylglucosaminyltransferase 2 (B3GNT2) polypeptide.

2. The engineered cell of claim 1, wherein the engineered cell expresses an exogenous B3GNT2 polypeptide.

3. The engineered cell of claim 1, wherein the engineered cell has an enhanced transcriptional activity of an endogenous B3GNT2 polypeptide.

4. An engineered cell, comprising an exogenous nucleic acid sequence that encodes a B3GNT2 polypeptide.

5. The engineered cell of any one of claims 1-4, wherein the B3GNT2 polypeptide comprises a mutation, wherein the B3GNT2 retains or enhances the poly-N-acetyl-lactosamine synthase activity of wildtype B3GNT2.

6. The engineered cell of any one of claims 1-5, wherein the B3GNT2 polypeptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.

7. The engineered cell of any one of claims 1-6, wherein the B3GNT2 peptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 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: 2.

8. The engineered cell of any one of claims 1-7, wherein the engineered cell is an allogeneic cell isolated from a donor for being administered to a subject or an autologous cell.

9. The engineered cell of any one of claims 1-8, wherein the engineered cell is a T cell, a NK cell, a Treg cell, a B cell, a monocyte, a macrophage cell, a dendritic cell, a peripheral bloodAttorney Docket No.: 51624-0098WO1 / LG-U2024143WO mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, a mesenchymal stem cell, or an embryonic stem cell.

10. The engineered cell of claim 9, wherein the T cell is a natural killer T (NK-T) cell, a y8 T cell, or an aP T cell.

11. The engineered cell of any one of claims 1-10, wherein the expression level of the B3GNT2 in the engineered cell is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a wild-type cell or the control cell.

12. The engineered cell of any one of claims 1-11, wherein the engineered cell expresses an engineered receptor (e.g., chimeric antigen receptor (CAR), TCR, or TAC receptor.

13. The engineered cell of claim 12, wherein the engineered receptor is a CAR, and the CAR specifically targets an antigen.

14. The engineered cell of claim 13, wherein the antigen is a tumor antigen, preferably, the tumor antigen is 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-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, LI cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MARTI, 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- Ll, or PD-L2.

15. The engineered cell of any one of claims 1-14, wherein:(1) the HLA (MHC Class I and MHC Class II molecules) gene of the engineered cell is not genetically modified;Attorney Docket No.: 51624-0098WO1 / LG-U2024143WO(2) only the expression of endogenous MHC Class I molecules is eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the engineered cells;(3) only the expression of endogenous MHC Class II molecules is eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class II molecules is achieved by disrupting endogenous Class II major histocompatibility complex transactivator (CIITA) gene of the engineered cells; or(4) both the expression of endogenous MHC Class I molecules and MHC Class II molecules are eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the engineered cells, and wherein the eliminated or reduced expression of endogenous MHC Class II molecules is achieved by disrupting endogenous Class II major histocompatibility complex transactivator (CIITA) gene of the engineered cells.

16. The engineered cell of any one of claims 1-15, wherein the expression of the B3GNT2 polypeptide confers resistance to T cell-mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

17. A method of reducing immunogenicity of an engineered cell, comprising one of the following:(1) inhibiting immune clearance of an engineered cell;(2) inhibiting resistance of an engineered cell to T cell-mediated cytotoxicity and / or NK cell-mediated cytotoxicity;(3) increasing the in vivo expansion of an engineered cell in a subject; or(4) reducing Host-versus-Graft (HvG) reaction of an engineered cell in a subject; wherein the method comprising overexpressing a B3GNT2 polypeptide or expressing an exogenous B3GNT2 polypeptide in the engineered cell.

18. The method of claim 17, wherein the method comprises introducing a nucleic acid encoding a B3GNT2 polypeptide into a cell.Attorney Docket No.: 51624-0098WO1 / LG-U2024143WO19. The method of claim 18, wherein the nucleic acid comprises a non-native regulatory element (e.g., a promoter).

20. The method of any one of claims 17-19, wherein the method comprises introducing a nucleic acid encoding a transcription activator that enhances the transcriptional activity of an endogenous B3GNT2 polypeptide.

21. The method of any one of claims 17-20, wherein the B3GNT2 polypeptide comprises a mutation, wherein the B3GNT2 retains or enhances the poly-N-acetyl-lactosamine synthase activity of wildtype B3GNT2.

22. The method of any one of claims 17-21, wherein the B3GNT2 polypeptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.

23. The method of any one of claims 17-22, wherein the B3GNT2 peptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 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: 2.

24. The method of any one of claims 17-23, wherein the engineered cell is an allogeneic cell isolated from a donor for being administered to a subject or an autologous cell.

25. The method of any one of claims 17-24, wherein the engineered cell is a T cell, aNK cell, a Treg cell, a B cell, a monocyte, a macrophage cell, a dendritic cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, a mesenchymal stem cell, or an embryonic stem cell.

26. The method of claim 25, wherein the T cell is a natural killer T (NK-T) cell, a y8 T cell, or an ct,|3 T cell.Attorney Docket No.: 51624-0098WO1 / LG-U2024143WO27. The method of any one of claims 17-26, wherein the expression of the B3GNT2 on the engineered cell is increased by 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 method of any one of claims 17-27, wherein the engineered cell expresses an engineered receptor (e.g., chimeric antigen receptor (CAR), TCR, or TAC receptor).

29. The method of any one of claims 17-28, wherein:(1) the HLA (MHC Class I and MHC Class II molecules) gene of the engineered cell is not genetically modified;(2) only the expression of endogenous MHC Class I molecules is eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the engineered cells;(3) only the expression of endogenous MHC Class II molecules is eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class II molecules is achieved by disrupting endogenous Class II major histocompatibility complex transactivator (CIITA) gene of the engineered cells; or(4) both the expression of endogenous MHC Class I molecules and MHC Class II molecules are eliminated or reduced, wherein the eliminated or reduced expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the engineered cells, and wherein the eliminated or reduced expression of endogenous MHC Class II molecules is achieved by disrupting endogenous Class II major histocompatibility complex transactivator (CIITA) gene of the engineered cells.

30. The method of any one of claims 17-29, wherein the expression of the B3GNT2 polypeptide confers resistance to T cell-mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

31. An engineered cell prepared by the method of any one of claims 17-30.

32. A nucleic acid, comprising one or more nucleic acid sequences encoding:Attorney Docket No.: 51624-0098WO1 / LG-U2024143WO(a), an engineered receptor, and(b). a B3GNT2 polypeptide.

33. The nucleic acid of claim 32, wherein the nucleic acid sequence comprises one, two, three, four, five or more copies of a coding sequence of a B3GNT2 polypeptide; and / or wherein the nucleic acid sequence is operably linked to a non-native regulatory element.

34. The nucleic acid of claim 32 or claim 33, wherein the B3GNT2 peptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 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: 2.

35. The nucleic acid of any one of claims 32-34, wherein the nucleic acid sequence encoding the engineered receptor and the nucleic acid sequence encoding the B3GNT2 polypeptide are separated by a third nucleic acid sequence encoding a cleavable linker.

36. The nucleic acid of claim 35, wherein the cleavable linker comprises an amino acid sequence set forth in SEQ ID NO: 7, or a functional variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 7.

37. The nucleic acid of any one of claims 32-36, wherein the engineered receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC) or a portion thereof.

38. The nucleic acid of claim 37, wherein the engineered receptor is a CAR.

39. A vector comprising the nucleic acid of any one of claims 32-38.

40. An engineered cell comprising the nucleic acid of any one of claims 32-38, or the vector of claim 39.Attorney Docket No.: 51624-0098WO1 / LG-U2024143WO41 . A composition comprising the engineered cell of any one of claims 1-16, 31, and 40, and a pharmaceutically acceptable carrier.

42. A viral particle comprising B3GNT2 polypeptide, wherein the viral particle displays B3GNT2 polypeptide on the surface of the particle.

43. The viral particle of claim 42, wherein the viral particle is a virus particle or a viruslike particle (VLP), optionally a retroviral particle or a retroviral-like particle, optionally a lentiviral particle or a lentiviral-like particle.

44. The viral particle of claim 43, wherein the particle is pseudotyped with a vesicular stomatitis virus envelope glycoprotein (VSV-G) or cocal virus glycoprotein (COV-G).

45. The viral particle of any one of claims 42-44, wherein the B3GNT2 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 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: 2.

46. The viral particle of any one of claims 42-45, wherein the B3GNT2 polypeptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.

47. The viral particle of any one of claims 42-46, wherein the B3GNT2 polypeptide is displayed on the surface of the particle via a transmembrane domain or a Glycosylphosphatidylinostol (GPI) membrane anchor.

48. A method of making a viral particle comprising B3GNT2 polypeptide, comprising:(1) providing a producer cell that comprises a nucleic acid encoding B3GNT2 polypeptide;(2) culturing the cell under conditions that allow for production of a viral particle, and(3) separating, enriching, or purifying the particle from the cell.Attorney Docket No.: 51624-0098WO1 / LG-U2024143WO49. The method of claim 48, wherein the B3GNT2 polypeptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.

50. The method of claim 48 or 49, wherein the B3GNT2 peptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 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: 2.

51. A producer cell that overexpresses a B3GNT2 polypeptide.

52. The producer cell of claim 51, wherein the producer cell is a HEK-293 cell or a derivative thereof, optionally a HEK-293 T or a HEK-293 T-REx cell.

53. The producer cell of claim 51 or 52, wherein the producer cell comprises a genetically disruption of endogenous MHC Class I molecules, wherein the disruption of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the producer cells.

54. A producer cell comprising (1) a viral nucleic acid(s) and (2) nucleic acid encoding an exogenous B3GNT2 polypeptide, optionally wherein the viral nucleic acid(s) are lentiviral nucleic acids.

55. The producer cell of any one of claims 51-54, wherein the viral nucleic acid(s) lacks one or more genes involved in viral replication.

56. The producer cell of any one of claims 51-55, wherein the B3GNT2 polypeptide comprises one or more mutations at A279, D247, H376, K149, D245, Y289, D332, and / or D333, or any combination thereof.Attorney Docket No.: 51624-0098WO1 / LG-U2024143WO57. The producer cell of any one of claims 51 -56, wherein the B3GNT2 peptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 50%, 60%, 70%, 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: 2.

58. The viral particle of any one of claims 42-47, further comprising a nucleic acid sequence.

59. The viral particle of claim 58, wherein the nucleic acid sequence encodes an engineered receptor (e.g., chimeric antigen receptor (CAR), TCR, or TAC receptor.

60. A viral particle produced by the method of any one of claim 48-50.

61. A composition comprising the viral particle of any one of claims 42-47, and 58-60, and a pharmaceutically acceptable carrier.

62. A method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of (1) the engineered cell of any one of claims 1- 16, 31, and 40, or the composition of claim 41; or (2) the viral particle of any one of claims 42-47, and 58-60, or the composition of claim 61.

63. The method of claim 62, wherein the disease or disorder is cancer, autoimmune disease, or infection.

64. A method of inhibiting immune clearance of an engineered cell or a viral particle in a subject, comprising administering (1) the engineered cell of any one of claims 1-16, 31, and 40, or the composition of claim 41; or (2) the viral particle of any one of claims 42-47, and 58-60, or the composition of claim 61 to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell.Attorney Docket No.: 51624-0098WO1 / LG-U2024143WO65. The method of claim 64, wherein the immune clearance of the engineered cell is through T cell-mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

66. 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 1-16, 31, and 40, or the composition of claim 41 to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell.

67. A method of reducing Host-versus-Graft (HvG) reactions in a subject, comprising administering (1) the engineered cell of any one of claims 1-16, 31, and 40, or the composition of claim 41 ; or (2) the viral particle of any one of claims 42-47, and 58-60, or the composition of claim 61 to a subject in need thereof, thereby reducing HvG reactions in the subject.

68. The method of claim 67, wherein the subject has undergone or will receive a cell or organ transplantation.

69. The method of claim 68, wherein the cell transplantation is a stem cell transplantation.

70. The method of any one of claims 62-69, wherein the engineered cell is a CAR-T cell.

71. The method of any one of claims 62-70, wherein the subject is a human subject.

72. The method of increasing the life of a particle in vivo in a mammal, method comprising administering the viral particle of any one of claims 42-47, and 58-60, or the composition of claim 61 to a mammalian subject wherein said administered particles have a longer half-life in said mammal than an otherwise similar particle that does not have B3GNT2 expressed thereon.

73. A method of delivering a nucleic acid sequence to a subject (e.g., a human subject), the method comprising administering to a subject the viral particle of any one of claims 42-47, andAttorney Docket No.: 51624-0098WO1 / LG-U2024143WO58-60, or the composition of claim 61 , wherein the nucleic acid sequence is delivered to a target cell.

74. A method of producing the engineered cell of any one of claims 1-16, 31, and 40, the method comprising:(a) transfecting a producer cell with the vector of claim 39;(b) culturing the cell in a culture medium; and(c) harvesting the cell from the culture medium.

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