Cells overexpressing CD43 and methods of use thereof

Abstract

Provided herein are engineered cells overexpressing a CD43 polypeptide. In some embodiments, the engineered cells can prevent and / or mitigate host cell-mediated rejection. In some embodiments, the engineered cells further overexpress one or more tolerogenic factors.

Description

[0001] Atorney Docket No 51624-0096WO 1 / LG-U2024141 WO

[0002] CELLS OVEREXPRESSING CD43 AND METHODS OF USE

[0003] THEREOF

[0004] CROSS REFERENCE TO RELATED APPLICATION

[0005] This application claims benefit of priority of U.S. Patent Application No. US 63 / 680.408 filed on August 07, 2024, the content of which is incorporated herein by reference in its entirety.

[0006] SEQUENCE STATEMENT

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named '‘ 1624-0096W01_SL_ST26.xml.’’ The XML file, created on August 5, 2025, is 30,201 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0008] TECHNICAL FIELD

[0009] This disclosure relates to cells overexpressing a CD43 polypeptide and methods of use thereof.

[0010] BACKGROUND

[0011] CAR-T cell therapy has achieved great success in the treatment of liquid tumor, but it also has limitations such as high cost, batch variations in manufacture, and longer preparation and waiting time. Unlike autologous CAR-T cell therapy, allogeneic off-the-shelf cell therapy can overcome these limitations. However, the vigorous host-versus-graft (HvG) immune response against the allogeneic cells prevents the expansion and persistence of allogeneic cells and mitigates the efficacy of these allogeneic cells. Thus, there is a need to prevent or reduce HvG reactions and at the same time, improve the efficacy of allogeneic cell therapies.

[0012] SUMMARY

[0013] The present disclosure relates to the prevention or mitigation of HvG reactions related to cell therapies (e.g., CAR-T therapies). Specifically, the disclosure relates to engineered cells that overexpress CD43 polypeptides that can dramatically prevent both NK cell- and primary PBMC-mediated rejection. Overexpression of CD43 polypeptides showed Atorney Docket No.: 51624-0096WO1 / LG-

[0014] U2024141WO comparable or even better protective functions than overexpression of the HLA-E trimeric construct described herein. The disclosure also relates to methods of increasing persistence and / or function of engineered cells (e.g., y5 T cells, a[3 T cells, or NK cells) by overexpressing CD43 polypeptides, either alone or in combination with one or more tolerogenic factors (e.g., the HLA-E trimeric construct and / or CD47).

[0015] In one aspect, the disclosure is related to an engineered cell that overexpresses a CD43 polypeptide. In some embodiments, the engineered cell further overexpresses one or more tolerogenic factors. In some embodiments, the one or more tolerogenic factors are selected from the group consisting of HLA-E, CD47, CD24, CD26, CD27, CD31, CD35, CD200, HLA-C, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL- 10, IL-35, FASL. DUX4. CCL21, MFGE8, SERPINB9. and any combination thereof. In some embodiments, the one or more tolerogfactors comprise HLA-E. In some embodiments, the HLA-E is a single-chain fusion HLA Class I protein. In some embodiments, the single-chain fusion HLA Class I protein comprises at least a portion of B2M protein and at least a portion of HLA-E heavy chain (e.g., HLA-E*01 :01 heavy chain or HLA-E*01:03 heavy chain). In some embodiments, the engineered cell further comprises a peptide antigen that is presented by the single-chain fusion HLA Class I protein on the cell surface; or in some embodiments, the single-chain fusion HLA Class I protein further comprises a peptide antigen that is presented by the single-chain fusion HLA Class I protein on the cell surface, optionally the peptide antigen is a peptide derived from HLA-G or HLA-C. In some embodiments, the single-chain fusion HLA Class I protein comprises an amino acid sequence set forth in SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the one or more tolerogenic factors comprise CD47 (e.g., human CD47) or a variant thereof. In some embodiments, the CD47 or the variant thereof comprises an amino acid sequence set forth in SEQ ID NO: 5, 6, or 7, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 5, 6, or 7.

[0016] In some embodiments, the engineered cell expresses endogenous Major Histocompatibility Complex (MHC). In some embodiments, the expression level of endogenous MHC Class I molecules and / or MHC Class II molecules are the same or comparable to that of a wildtype cell of the same type as the engineered cell. In some embodiments, the expression of endogenous MHC Class I molecules and / or MHC Class II Atorney Docket No.: 51624-0096WO1 / LG-

[0017] U2024141WO molecules are not eliminated or reduced in the engineered cell. In some embodiments, the beta-2-microglobulin (B2M) gene of the engineered cell is not genetically modified.

[0018] In some embodiments, the expression level of the CD43 polypeptide on the engineered cell is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60- fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 2000-fold, at least 5000-fold, or at least 10000-fold as compared to the expression level of CD43 (e.g., endogenous CD43) on a wildtype cell or a control cell. In some embodiments, the expression level of the CD43 polypeptide on the engineered cell is at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%. about 60% to about 70%. about 70% to about 80%, about 80% to about 90%, about 90% to about 1- fold, about 1-fold to about 2-fold, about 2-fold to about 3-fold, about 3-fold to about 4-fold, about 4-fold to about 5-fold, about 5-fold to about 6-fold, about 6-fold to about 7-fold, about 7-fold to about 8-fold, about 8-fold to about 9-fold, about 9-fold to about 10-fold, about 10- fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, about 600-fold to about 700-fold, about 700-fold to about 800-fold, about 800-fold to about 900-fold, about 900-fold to about 1000-fold, about 1000-fold to about 2000-fold, about 2000-fold to about 5000-fold, or about 5000-fold to about 10000-fold as compared to the expression level of CD43 (e.g., endogenous CD43) on a wildty pe cell or a control cell.

[0019] In some embodiments, the engineered cell is an allogeneic cell isolated from a donor for being administered to a subject.

[0020] In some embodiments, the CD43 polypeptide comprises an extracellular region of CD43, a transmembrane region of CD43, and / or an intracellular region of CD43. In some embodiments, the extracellular region of CD43 comprises an amino acid sequence set forth in SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, Atorney Docket No.: 51624-0096WO1 / LG-

[0021] U2024141WO

[0022] 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 16; in some embodiments, the transmembrane region of CD43 comprises an amino acid sequence set forth in SEQ ID NO: 17 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 17; in some embodiments, the intracellular region of CD43 comprises an amino acid sequence set forth in SEQ ID NO: 18 or a sequence that is at least 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the CD43 polypeptide comprises a full-length CD43 protein (e.g., human CD43). In some embodiments, the CD43 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0023] In some embodiments, the engineered cell is an immune cell (e.g., a T cell, a natural killer (NK) cell, a B cell, a monocyte, or a macrophage). In some embodiments, the immune cell is selected from the group consisting of a T cell, a NK cell, and a combination thereof. In some embodiments, the immune cell is selected from the group consisting of a natural killer T (NK-T) cell, a T cell, an aP T cell, a Treg cell, and a NK cell. In some embodiments, the immune cell is an aP T cell or a NK cell.

[0024] In some embodiments, the aP T cell or the NK cell has an eliminated or reduced expression of an endogenous CD43 ligand. In some embodiments, the endogenous CD43 ligand is sialic acid-binding Ig-like lectin 7 (Seglec-7). In some embodiments, the eliminated or reduced expression of endogenous CD43 ligand is achieved by disrupting an endogenous CD43 ligand gene of the engineered cells. In some embodiments, the disrupting endogenous CD43 ligand gene is achieved by using a gene editing method (e.g., clustered regularly interspaced short palindromic repeats / Cas9 protein (CRISPR / Cas9), transcription activatorlike (TAL) effector nucleases (TALENs), or Zinc finger nucleases (ZFNs)), RNA interference (RNAi) technology, homologous recombination, modifying one or more regulating elements (e.g., promoter) of endogenous CD43 ligand gene, knocking out a sequence encoding all or part of the endogenous CD43 ligand, and / or knocking in an exogenous sequence to replace all or part of endogenous CD43 ligand gene. In some embodiments, the expression level of the endogenous CD43 ligand in the engineered cell is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, Atorney Docket No.: 51624-0096WO1 / LG-

[0025] U2024141WO less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0. 1% as compared to that in a wildtype cell or a control cell.

[0026] In some embodiments, the immune cell is a y5 T cell. In some embodiments, the expression of an endogenous Seglec-7 is not eliminated or reduced in the y5 T cell.

[0027] In some embodiments, the engineered cell has an eliminated or reduced expression of endogenous MHC Class I molecules. In some embodiments, 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. In some embodiments, the engineered cell has an eliminated or reduced expression of endogenous MHC Class II molecules. In some embodiments, the eliminated or reduced expression of endogenous MHC Class II molecules is achieved by disrupting endogenous Class II major histocompatibility complex trans activator (OITA) gene of the engineered cells.

[0028] In some embodiments, the engineered cell further expresses an engineered receptor. In some embodiments, the engineered receptor is an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC) or a portion thereof. In some embodiments, the engineered receptor specifically targets a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30. CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD 138, 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, CD 123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta-like 3 (DLL3), CD70, CS-1, c-Met, Glycolipid F77, PD-L1, and PD-L2. In some embodiments, the engineered receptor is a CAR comprising an amino acid sequence set forth in SEQ ID NO: 4 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the engineered cell comprises a vector encoding the CD43 polypeptide and the engineered receptor, optionally the vector encodes one or more tolerogenic factors.

[0029] In some embodiments, overexpression of the CD43 polypeptide can prevent or reduce host rejection in a subject when the engineered cell is administered to the subject. Atorney Docket No.: 51624-0096WO1 / LG-

[0030] U2024141WO

[0031] In some embodiments, the CD43 polypeptide is glycosylated.

[0032] In some embodiments, the CD43 polypeptide is deglycosylated by N-glycosidase. In some embodiments, the N-glycosidase is selected from Peptide-N-Glycosidase F (PNGase F), Peptide-N-Glycosidase A (PNGase A), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase Fl (Endo Fl), Endoglycosidase F2 (Endo F2) and Endoglycosidase F3 (Endo F3), and any combination thereof, preferably the N- glycosidase is PNGase F. In some embodiments, the N-glycosidase is PNGase F.

[0033] In one aspect, the disclosure is related to a composition comprising the engineered cell described herein, and a pharmaceutically acceptable carrier.

[0034] In one aspect, the disclosure is related to a method of making the engineered cell described herein, comprising introducing a vector encoding the CD43 polypeptide into a cell, thereby making the engineered cell. In some embodiments, the vector is a retroviral vector or a lentiviral vector. In some embodiments, the method further comprises contacting the engineered cell with glycosidase. In some embodiments, the glycosidase is N-glycosidase. In some embodiments, the glycosidase is selected from Peptide-N-Glycosidase F (PNGase F), Peptide-N-Glycosidase A (PNGase A), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase Fl (Endo Fl), Endoglycosidase F2 (Endo F2) and Endoglycosidase F3 (Endo F3), and any combination thereof, preferably the glycosidase is PNGase F. In some embodiments, the N-glycosidase is PNGase F.

[0035] 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 or the composition described herein. In some embodiments, the disease or disorder is cancer, autoimmune disease, or infection.

[0036] In one aspect, the disclosure is related to a method of inhibiting immune clearance of an engineered cell in a subject, comprising: (a) overexpressing a CD43 polypeptide in the engineered cell, (b) administering the engineered cell from step (a) to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell. In some embodiments, the immune clearance of the engineered cell is through T cell-mediated cytotoxicity and / or NK. cell-mediated cytotoxicity.

[0037] In one aspect, the disclosure is related to a method of increasing the in vivo expansion of an engineered cell in a subject, comprising: (a) overexpressing a CD43 polypeptide in the engineered cell; (b) administering the engineered cell from step (a) to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell. Atorney Docket No.: 51624-0096WO1 / LG-

[0038] U2024141WO

[0039] In one aspect, the disclosure is related to a method of increasing persistence and / or function of an engineered cell in a subject, comprising: (a) overexpressing a CD43 polypeptide in the engineered cell; (b) administering the engineered cell from step (a) to a subject in need thereof, thereby increasing the persistence and / or function of the engineered cell.

[0040] In some embodiments, the method described above further comprising contacting the engineered cell with glycosidase between step (a) and step (b). In some embodiments, the glycosidase is N-glycosidase. In some embodiments, the glycosidase is selected from Peptide-N-Glycosidase F (PNGase F), Peptide-N-Glycosidase A (PNGase A), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S). Endoglycosidase D, Endoglycosidase Fl (Endo Fl), Endoglycosidase F2 (Endo F2) and Endoglycosidase F3 (Endo F3), and any combination thereof, preferably the glycosidase is PNGase F. In some embodiments, the N-glycosidase is PNGase F.

[0041] In some embodiments, the overexpressing the CD43 polypeptide in the engineered cells is achieved by introducing a vector expressing the CD43 polypeptide, using a gene editing system (e.g., clustered regularly interspaced short palindromic repeats / Cas9 protein (CRISPR / Cas9), transcription activator-like (TAL) effector nucleases (TALENs), or Zinc finger nucleases (ZFNs)), modifying one or more regulating elements (e.g., promoter) of endogenous CD43 gene, and / or knocking in a sequence encoding the CD43 polypeptide. In some embodiments, the CD43 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the engineered cell is a CAR-T cell. In some embodiments, the subject is a human subject.

[0042] Other features and advantages of the disclosure will be apparent from the following detailed description and figures, and from the claims.

[0043] DESCRIPTION OF DRAWINGS

[0044] FIG. 1A shows the expression of RV-CD43-BFP in K562 cells after a 3-day transduction.

[0045] FIG. IB shows the expression of RV-HLA-E in K.562 cells after a 3-day transduction.

[0046] FIG. 1C shows the expression of RV-CD47-BFP in K562 cells after a 3-day transduction. Atorney Docket No.: 51624-0096WO1 / LG-

[0047] U2024141WO

[0048] FIG. ID shows PBNK cell-mediated killing of untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43"). the HLA-E trimeric construct ("HLA-E"). or CD47 ("CD47"), without IL-2 after a 2-day co-culture. The PBNK cells were effector cells, and the K562 cells were target cells. The E:T ratio was 1:3.

[0049] FIG. IE shows PBNK cell-mediated killing of untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43"), the HLA-E trimeric construct ("HLA-E"), or CD47 ("CD47"). with 400U IL-2 after a 2-day co-culture. The PBNK cells were effector cells, and the K562 cells were target cells. The E:T ratio was 1 :3.

[0050] FIG. IF shows the cell number of viable untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43"), the HLA-E trimeric construct ("HLA-E"), or CD47 ("CD47") after a co-culture with PBNK cells for 2 days, without IL-2.

[0051] FIG. 1G shows the cell number of viable untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43"), the HLA-E trimeric construct ("HLA-E"), or CD47 ("CD47") after a co-culture with PBNK cells for 2 days, with 400U IL-2.

[0052] FIG. 2A shows primary’ NK cell-mediated killing of untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43") or the HLA-E trimeric construct ("HLA-E") without IL-2 after a 2-day co-culture. The primary NK cells were effector cells, and the K562 cells were target cells. The E:T ratio was 2:3.

[0053] FIG. 2B shows primary- NK cell-mediated killing of untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43") or the HLA-E trimeric construct ("HLA-E") with 400U IL-2 after a 2-day co-culture. The primary NK cells were effector cells, and the K562 cells yvere target cells. The E:T ratio yvas 2:3.

[0054] FIG. 2C shows the cell number of viable untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43") or the HLA-E trimeric construct ("HLA-E"), after a coculture with primary NK cells for 2 days, with or without 400U IL-2.

[0055] FIG. 3A sho vs that primary PBMCs inhibited K562 cell groyvth on Day 4 with an E:T ratio of 100: 1. "D807," "D987," and "D990" indicate that the primary- PBMCs were isolated from Donor 807, Donor 987, and Donor 990, respectively. "CD3-depleted D807" indicates CD3-depleted primary PBMCs isolated from Donor 807. Either untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43") or the HLA-E trimeric construct ("HLA-E") yvere used as target cells. The primary' PBMCs yvere used as effector cells. Atorney Docket No.: 51624-0096WO1 / LG-

[0056] U2024141WO

[0057] FIG. 3B shows that primary' PBMCs inhibited K562 cell grow th on Day 7 with an E:T ratio of 100: 1. "D807," "D987," and "D990" indicate that the primary PBMCs were isolated from Donor 807, Donor 987, and Donor 990, respectively. "CD3-depleted D807" indicates CD3-depleted primary PBMCs isolated from Donor 807. Either untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43") or the HLA-E trimeric construct ("HLA-E") were used as target cells. The primary PBMCs were used as effector cells.

[0058] FIGS. 3C-3F show the cell number of viable untransduced K562 cells ("K562") or K562 cells overexpressing CD43 ("CD43") or the HLA-E trimeric construct ("HLA-E") after a co- culture with primary PBMCs at different time points (Day 0, Day 4, and Day 7) with an E:T ratio of 100: 1.

[0059] FIG. 4A shows the knockout efficiency of B2M gene in gdT cells (left panel) and BCMA CAR-CD43 overexpression in gdT cells (right panel). UTD stands for untransduced cells.

[0060] FIG. 4B show s the PBNK killing capability of B2M KO gdT cells with or without overexpression of BCMA CAR-CD43 at different E:T ratios (1: 1, 1 :2, and 1 :3).

[0061] FIG. 4C shows the cell number of viable B2M KO gdT cells with or without overexpression of BCMA CAR-CD43 after a co-culture with expanded PBNK cells at different E:T ratios (1: 1, 1 :2, and 1 :3).

[0062] FIG. 5 A shows that primary PBMCs inhibited B2M KO gdT cell growth on Day 5 with two different E:T ratios (50: 1 or 20: 1). "D811" indicates that the B2M KO gdT cells were isolated from Donor 81 1 . "D298," "D807," and "D990" indicate that the primary PBMCs 'ere isolated from Donor 298, Donor 807, and Donor 990, respectively.

[0063] FIG. 5B show s that primary' PBMCs inhibited B2M KO gdT cell growth on Day 7 with two different E:T ratios (50: 1 or 20: 1 ). "D811 " indicates that the B2M KO gdT cells were isolated from Donor 81 1. "D298," "D807," and "D990" indicate that the primary PBMCs were isolated from Donor 298, Donor 807, and Donor 990, respectively.

[0064] FIG. 5C show s the cell number of viable untransduced B2MKO gdT cells ("UTD") or B2MKO gdT cells overexpressing BCMA-CAR and CD43 ("CD43") after a co-culture with primary PBMCs at different time points (Day 0. Day 5. and Day 7) with an E:T ratio of 50: 1. "D81 1" indicates that the B2M KO gdT cells were isolated from Donor 811. "D298," "D807," and "D990" indicate that the primary' PBMCs were isolated from Donor 298, Donor 807, and Donor 990. respectively. "Ctrl" indicates B2M KO gdT cells without co-culture of PBMCs. Atorney Docket No.: 51624-0096W01 / LG-

[0065] U2024141WO

[0066] FIG. 5D shows the cell number of viable untransduced B2MKO gdT cells ("UTD") or B2MKO gdT cells overexpressing BCMA-CAR and CD43 ("CD43") after a co-culture with primary PBMCs at different time points (Day 0, Day 5, and Day 7) with an E:T ratio of 20: 1. "D811" indicates that the B2M KO gdT cells were isolated from Donor 811. "D298," "D807," and "D990" indicate that the primary PBMCs were isolated from Donor 298, Donor 807, and Donor 990. respectively. "Ctrl" indicates B2MKO gdT cells without co-culture of PBMCs.

[0067] FIG. 6A shows the purity of BCMACAR B2MKO gdT cells (left panel) and BCMACAR-CD43 B2M KO gdT cells (right panel), respectively. CD3 and Vdelta2 are markers of gdT cells.

[0068] FIG. 6B shows the BCMA-CAR expression level in BCMACAR B2M KO gdT cells (left panel) and BCMACAR-CD43 B2MKO gdT cells (right panel), respectively.

[0069] FIG. 6C shows the knockout efficiency of B2M gene in BCMACAR gdT cells (left panel) and BCMACAR-CD43 gdT cells (right panel), respectively.

[0070] FIG. 6D shows the protective effect of CD43 overexpression from NK cell-mediated cytotoxicity. Expanded PBNK cells from two different donors (D15 (upper panel) and D398 (lower panel)) were used as effector cells. Either B2M KO gdT cells overexpressing BCMACAR ("BCMACAR") or B2M KO gdT cells overexpressing BCMA-CAR and CD43 ("BCMACAR-CD43") were used as target cells. "Mock" indicates untransduced gdT cells (target cells) co-cultured with the expanded PBNK cells (effector cells).

[0071] FIGS. 6E-6F show the cell number of survived B2MKO gdT cells after co-culture with PBNK cells. Expanded PBNK cells from two different donors (DI 5 (left panel) and D398 (right panel)) were used as effector cells. Either B2M KO gdT cells overexpressing BCMA-CAR ("BCMACAR") or B2MKO gdT cells overexpressing BCMA-CAR and CD43 ("BCMACAR-CD43") were used as target cells. "Mock" indicates untransduced gdT cells (target cells) co-cultured with the expanded PBNK cells (effector cells).

[0072] FIG. 7A shows that primary PBMCs inhibited B2MKO gdT cell growth on Day 4 at an E:T ratio of 20: 1. "Mock" indicates untransduced gdT cells (target cells) co-cultured with the primary PBMCs (effector cells).

[0073] FIG. 7B shows that primary PBMCs inhibited B2M KO gdT cell growth on Day 7 at an E:T ratio of 20: 1. "Mock" indicates untransduced gdT cells (target cells) co-cultured with the primary PBMCs (effector cells).

[0074] FIG. 7C show s the cell number of viable B2M KO gdT cells after co-culture with primary PBMCs at different time points (Day 4 or Day 7) with an E:T ratio of 20: 1. Atorney Docket No.: 51624-0096W01 / LG-

[0075] U2024141WO

[0076] FIG. 8A shows the purity of B2M KO gdT cells overexpressing BCMA-CAR ("BCMACAR"). B2MKO gdT cells overexpressing BCMA-CAR and the HLA-E trimeric construct ("BCMACAR-HLA-E"), B2MK0 gdT cells overexpressing BCMA-CAR and CD43 ("BCMACAR-CD43"), and B2M KO gdT cells overexpressing BCMA-CAR, CD43, and the HLA-E trimeric construct ("BCMACAR-CD43-HLA-E").

[0077] FIG. 8B shows the expression level of BCMA-CAR and the HLA-E trimeric construct in B2M KO gdT cells overexpressing BCMA-CAR ("BCMACAR"), B2MKO gdT cells overexpressing BCMA-CAR and the HLA-E trimeric construct ("BCMACAR-HLA- E"), B2MKO gdT cells overexpressing BCMA-CAR and CD43 ("BCMACAR-CD43"), and B2MKO gdT cells overexpressing BCMA-CAR, CD43, and the HLA-E trimeric construct ("BCM AC AR-CD43 -HLA-E" ).

[0078] FIG. 8C shows the knockout efficiency of B2M gene in B2MK0 gdT cells overexpressing BCMA-CAR ("BCMACAR"), B2M KO gdT cells overexpressing BCMACAR and the HLA-E trimeric construct ("BCMACAR-HLA-E"), B2MKO gdT cells overexpressing BCMA-CAR and CD43 ("BCMACAR-CD43"), and B2MKO gdT cells overexpressing BCMA-CAR, CD43, and the HLA-E trimeric construct ("BCMACAR-CD43- HLA-E").

[0079] FIG. 8D shows the protective effect of overexpression of the HLA-E trimeric construct, CD43, or a combination thereof fromNK cell-mediated cytotoxicity. Expanded PBNK cells from two different donors (DI 5 (upper panel) and D398 (lower panel)) were used as effector cells.

[0080] FIGS. 8E-8F show the cell number of survived B2MKO gdT cells after co-culture with PBNK cells. Expanded PBNK cells from two different donors (DI 5 (left panel) and D398 (right panel)) were used as effector cells.

[0081] FIG. 8G shows that primary PBMCs inhibited B2MKO gdT cell growth on Day 4 (upper panel) or Day 7 (lower panel) at an E:T ratio of 20: 1. PBMCs from donor D987 were used as effector cells.

[0082] FIG. 8H shows that primary PBMCs inhibited B2M KO gdT cell growth on Day 4 (upper panel) or Day 7 (lower panel) at an E:T ratio of 20: 1. PBMCs from donor D990 were used as effector cells.

[0083] FIGS. 8L8J show the cell number of survived B2MKO gdT cells after co-culture with PBMCs at different time points (Day 4 or Day 7) with an E:T ratio of 20:1. PBMCs from two different donors (D987 (left panel) and D990 (right panel)) were used as effector cells. Atorney Docket No.: 51624-0096W01 / LG-

[0084] U2024141WO

[0085] FIGS. 8K-8L show NKG2A and NKG2C expression in primary NK cells from PBMCs. PBMCs from two different donors (D987 (left panel) and D990 (right panel)) were used.

[0086] FIG. 9A shows MFI of ConA staining in CD43-overexpressing K562 cells.

[0087] FIG. 9B shows MFI of LEL staining in CD43 -overexpressing K562 cells. FIG. 9C shows MFI of SNA staining in CD43 -overexpressing K562 cells. FIG. 10A shows the number of surviving cells following co-culture with NK cells. FIG. 10B shows NK cell killing capability against K562 cells with or without enzyme treatment.

[0088] FIG. 11 lists sequences discussed in the disclosure.

[0089] 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] CD43 is a transmembrane protein expressed at high levels on all leukocytes except most resting B lymphocytes. The immune protection of CD43 maybe through the binding of an immune checkpoint receptor that is specific for CD43. e.g., Siglec-7, which need to be further validated. Here, the experiments show that overexpression of CD43 can prevent both allogeneic NK cell- and PBMC cell-mediated rejection in K562 tumor cells and human primary type II y5 T cells. The immune protection effect is comparable to or even better than overexpression of the HLA-E trimeric construct described herein. In addition, the experiments show that overexpressing both CD43 and the HLA-E trimeric construct can have a stronger protective function from NK cell-mediated killing as compared with overexpression of either the HLA-E trimeric construct or CD43 alone.

[0092] The present disclosure is based on. in part, that engineered allogeneic immune cells that overexpress a CD43 polypeptide can reduce NK cell- and PBMC-mediated killing, and can be used in allogeneic cell therapies. In one aspect, the disclosure provides engineered immune cells overexpressing a CD43 polypeptide.

[0093] In one aspect, the disclosure provides engineered cells that overexpress a CD43 polypeptide. The engineered cells can be allogeneic cell isolated from a donor. Atorney Docket No.: 51624-0096W01 / LG-

[0094] U2024141WO

[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 CD43 polypeptide in the engineered cell.

[0098] In one aspect, the disclosure provides methods of increasing the in vivo expansion of an engineered cell by overexpressing a CD43 polypeptide in the engineered cell.

[0099] In one aspect, the disclosure provides methods of increasing persistence and / or function of an engineered cell in a subject by overexpressing a CD43 polypeptide in the engineered cell.

[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 a 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 CD43 can have a sequence that is identical to the intracellular domain of CD43, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of the intracellular domain of CD43. Similarly, an extracellular domain derived from CD43 can have a sequence that is identical to the extracellular domain of CD43, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of the extracellular domain of CD43.

[0101] As used herein, the term “CD43 polypeptide” refers to a polypeptide derived from a wildtype CD43 or a functional variant thereof. The CD43 may be a wildtype CD43 (e.g.. human CD43). The CD43 may have one or more mutations (e.g.. insertions, deletions, and / or substitutions). The CD43 may be a human CD43. The CD43 may be a truncated CD43. The CD43 may include an extracellular domain of CD43, a transmembrane domain of CD43, and / or an intracellular domain of CD43. The CD43 may or may not include a signal peptide. Atorney Docket No.: 51624-0096W01 / LG-

[0102] U2024141WO

[0103] As used herein, the terms "extracellular domain"’ or “extracellular region"’ are used interchangeably herein to refer to the portion of a receptor that is outside the cell membrane. The extracellular domain can be the entire portion of a receptor that is outside the cell membrane, or just a part thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). The extracellular domain can be derived from the extracellular domain of a wildtype receptor or a functional variant thereof. The extracellular domain may have one or more mutations, including e.g., insertions, deletions, and / or substitutions.

[0104] As used herein, the terms “transmembrane domain” or “transmembrane region” are used interchangeably herein to refer to the portion of a receptor that is embedded in the cell membrane. The transmembrane region can be the entire portion of a receptor that is embedded in the cell membrane, or just a part thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). The transmembrane region can be derived from the transmembrane region of a wildtype receptor or a functional variant thereof. The transmembrane region may have one or more mutations, including e.g., insertions, deletions, and / or substitutions.

[0105] As used herein, the terms “intracellular domain”, “intracellular region” or “cytoplasmic region” are used interchangeably herein to refer to the portion of a receptor that is inside the cell. The intracellular domain can be the entire portion of a receptor that is inside the cell, or just a part thereof (e.g.. at least 50%. 60%. 70%. 80%. 85%. 90%. 95%. 96%. 97%, 98%, or 99% of the entire portion). The intracellular domain can be derived from the intracellular domain of a wildtype receptor or a functional variant thereof. The intracellular domain may have one or more mutations, including e.g., insertions, deletions, and / or substitutions.

[0106] As used herein, a “vector” is any construct capable of delivering one or more nucleic acids of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more nucleic acids of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the nucleic acid of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or a poly -A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the nucleic acid of interest such that the nucleic acid of interest will be translated in the host cell introduced with the expression vector. Atorney Docket No.: 51624-0096W01 / LG-

[0107] U2024141WO

[0108] 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.

[0109] 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 TCR5 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.

[0110] As used herein, the term “antigen binding domain” or “extracellular antigen binding domain” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. An antigen binding fragment may comprise at least one variable domain (e.g., a variable domain of a heavy chain, single domain antibody or VHH). Non-limiting examples of antibody fragments include, e.g., Fab, Fab', F(ab’)2, and Fv fragments.

[0111] 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 w ell as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of Atorney Docket No.: 51624-0096W01 / LG-

[0112] U2024141WO 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.

[0113] 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., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

[0114] 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 Atorney Docket No.: 51624-0096W01 / LG-

[0115] U2024141WO 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).

[0116] 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.”

[0117] As used herein, “glycosylated” is defined as a saccharide (or sugar) covalently attached, i.e. linked, to an amino acid. Specifically, the saccharide is linked to the side- chain of the amino acid. In one aspect, the glycosylated amino acid may comprise a saccharide O- linked to a natural amino acid. For instance, the saccharide is attached to the hydroxyl group of the side-chain of the amino acid, such as Ser, Thr, or Tyr. Examples of glycosylated peptides include, but are not limited to, glucosylated Serine, glucosylated Threonine, and lactosylated Serine. In another aspect, the glycosylated amino acid may comprise a saccharide N-linked to a natural amino. For example, the saccharide is attached to the amine group of the side-chain of the amino acid, such as Asn or Lys. In some embodiments, the saccharide may be a mono-, di-, tri- and poly- saccharides. Examples of saccharides include, but are not limited to, glucose, fructose, galactose, cellobiose, and lactose.

[0118] As used herein, "deglycosylated" CD43 refers to a form of CD43 in which one or more glycan chains (e.g. N-glycans) have been removed. Deglycosylation of CD43 may be effected by treatment with an enzyme such as peptide N-glycosidase (PNGase), e.g. PNGase F, which removes N- glycans.

[0119] 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 Atorney Docket No.: 51624-0096W01 / LG-

[0120] U2024141WO incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0121] HvG Reactions Against Engineered Immune Cells

[0122] 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.

[0123] 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.

[0124] Allogeneic cell therapy has huge potential in the treatment of cancer diseases. It also has faced big challenges. With the strong host rejection of allogeneic cells, the persistence and function of allogeneic cells can be dramatically affected. To solve this issue, several strategies have been used, as discussed below.

[0125] The first strategy is using immunosuppressive drugs to prevent host attack. The administration of immunosuppressive drugs to inhibit host immune response can also lead to the disability of allogeneic cells. The strong immune suppressive effect of drugs may cause severe side effects to the patients and make the patients more sensitive to infections.

[0126] The second strategy' is using adaptive defensive receptors (ADRs) to kill the host activated immune cells. The expression of 41BB-ADR in allogeneic cells can kill the activated host CD8+ T cells. However, the rejection mediated by other immune cells may still exist, e g., by CD4+ T cells. Another disadvantage is that the depletion of host activated CD8+ T cells may affect the CAR function, which requires the functional CD8+ T cells. Furthermore, the depletion of host CD8+ T cells makes the patient more sensitive to infections. Atorney Docket No.: 51624-0096W01 / LG-

[0127] U2024141WO

[0128] The third strategy is HLA Class I / II deletion and overexpression of HLA-G, HLA-E or CD47. The deletion of HLA can prevent host T cell attack, but it can also enhance the innate reaction such as NK cell-mediated rejection. Overexpression of HLA-G, HLA-E or CD47 can prevent NK cell-mediated rejection. However, this strategy faces challenges in the application owing to different cell source and expansion limitations. For instance, the expansion of primary’ cells itself is challenging, and gene engineering makes it even more difficult to achieve the expansion goal. Overexpression of genes such as CD47 may also affect the expansion and function of allogeneic cells.

[0129] Details of these strategies and their limitations can be found, e.g., in Mo, F., et al. "Engineered off-the-shelf therapeutic T cells resist host immune rejection." Nature Biotechnology 39.1 (2021): 56-63: Gomalusse, G.G., et al. "HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells." Nature Biotechnology 35.8 (2017): 765-772; and Deuse, T, et al. "The SIRPa-CD47 immune checkpoint in NK cells." Journal of Experimental Medicine 218.3 (2021): e20200839; each of which is incorporated herein by reference in its entirety.

[0130] The present disclosure provides methods of preventing, inhibiting, or reducing HvG reactions related to cell therapies (e.g., CAR-T therapies). In some embodiments, the methods include administering engineered cells overexpressing CD43 (e.g., any of the CD43 polypeptides described herein) to a subject in need thereof. Compared with other strategies to prevent host rejection, overexpression of CD43 in allogeneic cells is easy to manipulate and does not affect the proliferation and expansion of allogeneic cells. In addition, unlike HLA-E that has donor variation (the expression of inhibitory’ receptor that is specific for HLA-E, e.g., NKG2A, can be low in some populations), the overexpression of CD43 has universal protective effects with different donors (the expression of immune checkpoint receptor that is specific for CD43, e.g., Siglec-7, is usually high among different populations). For example, overexpression of the HLA-E trimeric construct in CAR-T cells may not be effective to mitigate the HvG reactions when administered to patients having a low expression of inhibitory’ receptor that is specific for HLA-E (e.g., NKG2A). By contrast, CD43- overexpressing CAR-T cells would not have this issue when administered to NKG2A- negative patients. Thus, methods described herein can be applied to more populations receiving cell therapies. Further, because Siglect-7 are widely expressed on both T cells and NK cells, CD43 overexpression can be used to mitigate both T cell- and NK cell-mediated rejections. Atorney Docket No.: 51624-0096W01 / LG-

[0131] U2024141WO

[0132] Therefore, the methods as described herein provide has significant clinical potential in both cell therapy and transplantation field.

[0133] CD43 and its Functions

[0134] Cluster of differentiation 43 (CD43, also know n as sialophorin or leukosialin), a sialoglycoprotein expressed on T cells, plays a crucial role as a ligand for E-Selectin on activated T cells. The 130-kDa glycoform of CD43. preferentially expressed on activated T cells, functions as an E-Selectin ligand under flow' conditions, mediating T cell rolling on the vascular endothelium and potentially facilitating T cell migration to sites of inflammation. CD43's glycosylation pattern, particularly the presence of sialic acid and specific glycosyltransferases, is essential for its binding to E-Selectin and subsequent cell rolling. Despite its antiadhesive properties in regulating T cell activation and adhesion, CD43's role as an E-Selectin ligand highlights its dual function in mediating leukocyte recruitment and migration.

[0135] CD43 plays a multifaceted role in immune cell interactions and can influence the susceptibility of target cells to CTL- or NK cell-mediated cytolysis through its glycosylation status and sialic acid content. Understanding the mechanisms by which CD43 affects immune responses can provide valuable insights for developing allogeneic platform and preventing host immune response.

[0136] Details of CD43 and its functions can be found, e.g., in Rosenstein. Y. et al. "CD43, a molecule with multiple functions." Immunologic Research 20 (1999): 89-99; van den Berg, T.K., et al. "Cutting edge: CD43 functions as a T cell counterreceptor for the macrophage adhesion receptor sialoadhesin (Siglec-1)." The Journal of Immunology 166.6 (2001): 3637- 3640; Manjunath, N., et al. "Negative regulation of T-cell adhesion and activation by CD43." Nature 377.6549 (1995): 535-539; McFarland, T.A., et al. "CD43 diminishes susceptibility to T lymphocyte-mediated cytolysis." Journal of immunology (Baltimore, Md.: 1950) 154.3 (1995): 1097-1104; Hasegawa, K., et al. "Glycosylation status of CD43 protein is associated with resistance of leukemia cells to CTL-mediated cytolysis." Pios One 11.3 (2016): eO 152326; Li, Y.Y., et al. "Targeting CD43 optimizes cancer immunotherapy through reinvigorating antitumor immune response in colorectal cancer." Cellular Oncology 46.3 (2023): 777-791; and Yoshimura, A., et al. "Identification and functional characterization of a Siglec-7 counter-receptor on K562 cells." Journal of Biological Chemistry 296 (2021): each of which is incorporated herein by reference in its entirety’. Atorney Docket No.: 51624-0096W01 / LG-

[0137] U2024141WO

[0138] Without wishing to be bound by theory', it is contemplated that the effects observed (e.g., the protective effects from NK cell or PBMC-mediated killing) by overexpressing CD43 are achieved by the multiple mechanisms. For example, the overexpressed CD43 may interact with one or more CD43 ligands (e.g., Siglec-7) expressed on host cells, to induce downstream signaling pathways that can inhibit the immune response from the host cells. In addition, overexpression of CD43 may weaken the formation of effective immunological synapses (ISs). which are at the interfaces between target cells and host immune cells (e.g., T cells, B cells, NK cells). With fewer and weaker immunological synapses, there could be a higher likelihood for the target cells to escape from attack from the host cells.

[0139] The CD43 Polypeptide

[0140] The present disclosure provides methods to preserve the efficacy of allogeneic cell therapy products and to overcome HvG in patients treated with a cell therapy, e.g., by overexpressing a CD43 polypeptide in the cell. In one aspect, the disclosure provides an allogeneic cell therapy product that overexpresses a CD43 polypeptide. The cell may not express an endogenous CD43. The cell may express an endogenous CD43.

[0141] The present disclosure relates to a CD43 polypeptide comprising an extracellular domain of CD43, a transmembrane domain of CD43 and / or an intracellular domain of CD43. The present disclosure is related to engineered cells (e.g., CAR-T cells, CAR-NK cells, TCR- T cells) overexpressing a CD43 polypeptide. The engineered cell may be an allogeneic cell isolated from a donor. The engineered cell may express an exogenous CD43 polypeptide. The exogenous CD43 polypeptide may protect the engineered cells from NK cell- or T cell- mediated killing. In some embodiments, the engineered cells may be modified to overexpress the exogenous CD43 polypeptide from a vector (e.g., a viral vector). In some embodiments, the engineered cells may overexpress an endogenous CD43 polypeptide, e.g., by modifying one or more regulating elements (e.g., promoter) of endogenous CD43 gene. In some embodiments, overexpression of the CD43 polypeptide may be achieved by a gene editing system known in the art (e.g., clustered regularly interspaced short palindromic repeats / Cas9 protein (CRISPR / Cas9). transcription activator-like (TAL) effector nucleases (TALENs), or Zinc finger nucleases (ZFNs)). In some embodiments, overexpression of the CD43 polypeptide may be achieved by knocking in an exogenous sequence encoding the CD43 polypeptide. Atorney Docket No.: 51624-0096W01 / LG-

[0142] U2024141WO

[0143] The CD43 polypeptide may comprise a signal peptide of CD43. an extracellular domain of CD43, a transmembrane domain of CD43 and / or an intracellular domain of CD43. The full-length sequence of CD43 is shown in SEQ ID NO: 2. According to the UniProt database (UniProt ID: P16150), the signal peptide o CD43 corresponds to amino acids 1-19 of SEQ ID NO: 2; the extracellular domain of CD43 corresponds to amino acids 20-253 of SEQ ID NO: 2; the transmembrane domain of CD43 corresponds to amino acids 254-276 of SEQ ID NO: 2; and the intracellular domain of CD43 corresponds to amino acids 277-400 of SEQ ID NO: 2. The nucleic acid sequence encoding CD43 is set forth in SEQ ID NO: 1.

[0144] The CD43 polypeptide may comprise the entire sequence or any portion of the signal peptide of CD43. The CD43 polypeptide may comprise the entire sequence or any portion of the signal peptide of SEQ ID NO: 2. The signal peptide of the CD43 polypeptide may comprise an amino acid sequence of SEQ ID NO: 15 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: 15. The signal peptide of the CD43 polypeptide can have 1, 2, 3, 4, 5, 6, 7. 8, 9, 10 or more than 10 mutations. The mutation can be a deletion, an insertion, a substitution, or a combination thereof. The signal peptide of CD43 can have any suitable length and sequence.

[0145] The CD43 polypeptide may be replaced with a signal peptide form a heterogeneous protein. The heterologous protein can be, for example, CD8a. CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony stimulating factor (GM- CSF), GM-CSF receptor a (GM-CSF Ra), or an immunoglobulin (e.g., IgE or IgK). In some embodiments, the signal peptide is a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IE-2), or CD33), a serum albumin protein (e.g. HS A or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g. chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently express a protein by or on a cell.

[0146] The CD43 poly peptide may comprise the entire sequence or any portion of the extracellular domain of CD43. The CD43 polypeptide may comprise the entire sequence or any portion of the extracellular domain of SEQ ID NO: 2. The extracellular domain of the CD43 polypeptide may comprise an amino acid sequence of SEQ ID NO: 16 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: 16. The extracellular domain of the CD43 polypeptide can have 1, 2, 3. 4, 5, 6. Atorney Docket No.: 51624-0096W01 / LG-

[0147] U2024141WO

[0148] 7, 8, 9, 10 or more than 10 mutations. The mutation can be a deletion, an insertion, a substitution, or a combination thereof.

[0149] The CD43 polypeptide may comprise the entire sequence or any portion of the transmembrane domain of CD43. The CD43 polypeptide may comprise the entire sequence or any portion of the transmembrane domain of SEQ ID NO: 2. The transmembrane domain of the CD43 polypeptide may comprise an amino acid sequence of SEQ ID NO: 17 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: 17. The transmembrane domain of the CD43 polypeptide can have 1, 2, 3, 4, 5, 6, 7, 8. 9, 10 or more than 10 mutations. The mutation can be a deletion, an insertion, a substitution, or a combination thereof. The transmembrane domain of CD43 can have any suitable length and sequence.

[0150] The CD43 polypeptide may comprise the entire sequence or any portion of the intracellular domain of CD43. The CD43 polypeptide may comprise the entire sequence or any portion of the intracellular domain of SEQ ID NO: 2. The intracellular domain of the CD43 polypeptide may comprise an amino acid sequence of SEQ ID NO: 18 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: 18. The intracellular domain of the CD43 polypeptide can have 1, 2, 3, 4. 5, 6, 7, 8, 9, 10 or more than 10 mutations. The mutation can be a deletion, an insertion, a substitution, or a combination thereof.

[0151] The CD43 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 CD43 polypeptide may have an amino acid sequence that is identical to SEQ ID NO: 2. The CD43 polypeptide can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 mutations. The mutation can be a deletion, an insertion, a substitution, or a combination thereof.

[0152] The CD43 polypeptide may not be modified by any enzymes. The CD43 polypeptide may not be modified.

[0153] The CD43 polypeptide may be glycosylated. The glycosylation may be an O-linked glycosylation. The CD43 may be glycosylated and maintained its function to prevent NK cell Atorney Docket No.: 51624-0096W01 / LG-

[0154] U2024141WO mediated rejection. The CD43 may be glycosylated and increase its function to prevent NK cell mediated rejection.

[0155] The CD43 polypeptide may be deglycosylated. The deglycosylation of CD43 may enhance its function to prevent NK cell mediated rejection. The deglycosylation may remove an N-linked glycosylation. The CD43 polypeptide may be deglycosylated by N-glycosidase. The N-glycosidase may be selected from Peptide-N-Glycosidase F (PNGase F), Peptide-N- Glycosidase A (PNGase A). Endoglycosidase H (Endo H), Endoglycosidase S (Endo S). Endoglycosidase D, Endoglycosidase Fl (Endo Fl), Endoglycosidase F2 (Endo F2) and Endoglycosidase F3 (Endo F3), and any combination thereof. The N-glycosidase may be PNGase F. The CD43 may be deglycosylated by PNGase F and enhance its function to prevent NK cell mediated rejection.

[0156] Tolerogenic Factors

[0157] The engineered cells described herein may further overexpress one or more tolerogenic factors. As used herein, the term "tolerogenic factor" refers to an agent (e.g., a protein) that induces immunological tolerance. In some embodiments, the increased tolerance is reflected by a higher transplantation success rate, increased persistence of the grafted cells (e.g., any of the engineered cells described herein), reduced immune clearance of the grafted cells (e g., any of the engineered cells described herein), and / or a reduced level of host cell- mediated rejection.

[0158] The one or more tolerogenic factors may be selected from the group consisting of HLA Class I histocompatibility antigen, alpha chain E (HLA-E), CD47, CD24, CD26, CD27, CD31, CD35, CD200, human leukocyte antigen-C (HLA-C), human leukocyte antigen-G (HLA-G), programmed death-ligand 1 (PD-L1). indoleamine 2.3 -dioxygenase 1 (IDO1). CTLA4-Ig, Cl esterase inhibitor (Cl -inhibitor), IL- 10, IL-35, Fas ligand (FASL), double homeobox, 4 (DUX4), chemokine (C-C motif) ligand 21 (CCL21), milk fat globule-EGF factor 8 protein (MFGE8), serpin family B member 9 (SERPINB9), and any combination thereof. In some embodiments, the one or more tolerogenic factors include HLA-E (e.g., any of the HLA-E trimeric constructs described herein). In some embodiments, the one or more tolerogenic factors include CD47 (e.g., any of the CD47 or variants thereof described herein).

[0159] Overexpression of CD43 (e.g., any of the CD43 polypeptides described herein) and the one or more tolerogenic factors (e.g., HLA-E and / or CD47) can generate a synergistic effect. For example, overexpression of CD43 (e.g., any of the CD43 polypeptides described Atorney Docket No.: 51624-0096W01 / LG-

[0160] U2024141WO herein) and HLA-E (e.g., any of the single-chain fusion HLA Class I proteins described herein) can achieve a better tolerance of the engineered cells when administered to a subject, as compared to that when either CD43 or HLA-E is overexpressed. In some embodiments, overexpression of CD43 (e.g., any of the CD43 polypeptides described herein) and CD47 (e.g., any of the CD47 or its variants thereof described herein) can achieve a better tolerance of the engineered cells when administered to a subject, as compared to that when either CD43 or CD47 is overexpressed. In some embodiments, overexpression of CD43 (e.g.. any of the CD43 polypeptides described herein), HLA-E (e.g., any of the single-chain fusion HLA Class I proteins described herein), and CD47 (e.g., any of the CD47 or its variants thereof described herein) can achieve a better tolerance of the engineered cells when administered to a subject, as compared to that when either one or two of CD43, HLA-E, and CD47 are overexpressed. a. HLA-E and single-chain fusion HLA Class I proteins

[0161] HLA-E is a non-classical human leukocyte antigen. It has been found that HLA-E preferentially accommodates a signal peptide comprising residues 3-11 of MHC Class I leader sequences in its binding groove and that these peptides dominate the HLA-E-presented ligandome in the steady state. For example, HLA-E can bind and present 9-mer peptides derived from the leader peptides of HLA- A, B, C and G proteins. The peptide-bound HLA-E complexes constitute major ligands for heterodimeric inhibitory CD94-NKG2A and activating CD94-NKG2C receptors predominantly expressed on NK cells. The peptide-bound HLA-E-CD94 / NKG2A engagement has been shown to regulate NK cell-mediated lysis and represents an important component of immune homeostasis. In addition, HLA-E overexpression on B2M knockout allogeneic graft cells may protect the cells against allogeneic NK cell-mediated lysis, thereby overcoming the host-versus-graft response.

[0162] Some suitable HLA-E alleles include, but are not limited to, HLA-E*01 :01 (HLA- E*01:01:01:01), HLA- E*01:03(HLA-E*01:03:01:01), HLA-E*01:04, HLA-E*01:05, HLA- E* 01 : 06, HL A-E*01 : 07, HLA-E* 01 : 09, and HLA- E*01 : 10.

[0163] Details of HLA-E and its functions can be found, e.g.. in Borrego, F., et al. "Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94 / NKG2 confers protection from natural killer cell-mediated lysis." The Journal of Experimental Medicine 187.5 (1998): 813-818; Guo, Y., et al. "Mutant B2M-HLA-E and B2M-HLA-G fusion proteins protects universal chimeric antigen receptor-modified T cells from allogeneic NK cell-mediated lysis." Atorney Docket No.: 51624-0096W01 / LG-

[0164] U2024141WO

[0165] European Journal of Immunology 51.10 (2021): 2513-2521; and U.S. Patent Application Publication No. 2023 / 0014010 Al; each of which is incorporated herein by reference in its entirety.

[0166] The term “single-chain fusion HLA Class I protein,” “single-chain fusion HLA Class I molecule” or “single-chain fusion HLA Class I antigen” described herein refers to a fusion protein comprising at least a portion of the B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of an HLA-I a chain. In some embodiments, the term “HLA Class I protein,” “HLA Class I molecule” or “HLA Class I antigen” refers to a non-covalently associated heterodimer of B2M and an HLA a chain expressed on the surface of a wildtype cell. As used herein, the term “HLA Class I a chain” or “HLA-I heavy chain” refers to the a chain of the HLA Class I heterodimer. HLA Class I heavy chain includes without limitation HLA Class I a chains HLA- A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the HLA Class I a chain described herein is a HLA-E heavy chain.

[0167] In some embodiments, the single-chain fusion HLA Class I protein comprises at least a portion of B2M and at least a portion of HLA- A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G heavy chain (also referred to as a dimeric construct or a single-chain dimer (SCD) form). In some embodiments, the HLA a chain contained in the single-chain fusion HLA Class I protein does not contain the leader sequence (or signal sequence) of the HLA Class I a chain (leaderless HLA a chain). In some embodiments, the single-chain fusion HLA Class I protein comprises at least a portion of B2M and at least a portion of HLA-C, HLA-E or HLA- G heavy chain. In some embodiments, the single-chain fusion HLA Class I protein comprises at least a portion of B2M and at least a portion of HLA-E heavy chain. In some embodiments, the single-chain fusion HLA Class I protein comprises a leader sequence (or signal peptide) covalently linked to the at least a portion of B2M and at least a portion of an HLA a chain to ensure proper folding of the single-chain fusion HLA Class I protein on the cell surface. The leader sequence can be the leader sequence of the B2M protein, the leader sequence of an HLA a chain protein or the leader sequence of other secretary proteins. In some embodiments, the single-chain fusion HLA Class I protein comprises a B2M protein with its leader sequence removed. In some embodiments, the single-chain fusion HLA Class I protein comprises an HLA a chain protein with its leader sequence removed. Certain HLA Class I a chains are highly polymorphic. As will be understood by those of skill in the art, the human Atorney Docket No.: 51624-0096WO1 / LG-

[0168] U2024141WO cells and methods of the disclosure are applicable to any such HLA a chains and polymorphism thereof.

[0169] Single-chain fusion HLA Class I proteins comprising sequence variants and fragments of B2M and / or HLA a chains are contemplated by the present disclosure, wherein such single-chain fusion constructs nevertheless possess normal HLA Class I functions, e.g., forming proper secondary structure of the heterodimer on the cell surface, presenting peptides in the peptide binding cleft and engaging the inhibitory receptors on the surface of engineered cells. In some embodiments, the variants share at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with the naturally occurring HLA heavy chains and B2M sequences, wherein the variants possess normal HLA Class I functions. In some embodiments, the variants share at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with the sequences of B2M as shown in SEQ ID NO: 8 or HLA-E heavy chain as shown in SEQ ID NO: 9.

[0170] Natural killer (NK) cells are part of the innate immune response. Several pathogens can down regulate HLA Class I protein expression in infected cells. The NK cells monitor infection by recognizing and inducing apoptosis in cells that do not express HLA Class I proteins. The inhibitory receptors on the NK cell surface recognize HLA Class I a chain alleles thereby preventing NK-medicated apoptosis in uninfected normal cells. Thus, in certain particular embodiments, the single-chain fusion HLA-I protein inhibits NK cell- mediated killing of cells that do not express endogenous HLA Class I proteins by binding to the inhibitory receptors on the NK cells. For example, HLA-E is a ligand for the CD94 / NKG2 receptor of NK cells that inhibits NK cell-mediated apoptosis. Thus, in some embodiments, the engineered cell expresses the single-chain fusion HLA Class I protein comprising at least a portion of B2M and at least a portion of HLA-E heavy chain. In addition, HLA-G is normally expressed on the surface of placental cytotrophoblasts that do not express HLA- A, B or C, and it protects these cells from NK cell-mediated lysis by interacting with the inhibitory ILT2(LIR1) receptor on NK cells. Thus, in some embodiments, the engineered cell expresses the single-chain fusion HLA Class I protein comprising at least a portion of B2M and at least a portion of HLA-G.

[0171] In some embodiments, the single-chain fusion HLA Class I protein comprises at least a portion of B2M and at least a portion of HLA-E*01:01 heavy chain or HLA-E*01:03 heavy chain. Atorney Docket No.: 51624-0096W01 / LG-

[0172] U2024141WO

[0173] In some embodiments, the single-chain fusion HLA Class I protein also comprises a specific peptide antigen that occupies the peptide binding cleft of the single-chain fusion HLA Class I protein, wherein the peptide antigen is covalently linked to the single-chain fusion HLA Class I protein (also referred to as a trimeric construct or a single-chain trimer (SCT) form). For example, the trimer construct can comprise B2M and HLA-E heavy chain covalently linked to a peptide antigen (such as, but not limited to. a HLA-G peptide antigen (SEQ ID NO: 10) or a HLA-C peptide antigen (SEQ ID NO: 11)) designed to occupy the peptide binding cleft of the single-chain fusion HLA Class I protein. In some embodiments, the single-chain fusion HLA Class I protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids of SEQ ID NO: 19. In some embodiments, the single-chain fusion HLA Class I protein with signal peptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids of SEQ ID NO: 14. In some embodiments, the covalently linked peptide antigen is cleaved via a built in protease cleavage site, and the cleaved peptide antigen can bind to the peptide binding cleft of the single-chain fusion HLA-I protein for presentation. In some embodiments, the peptide antigen occupying the peptide binding cleft of the single-chain fusion HLA Class I protein is produced by the intracellular antigen processing pathway, in which the peptide antigen is produced by proteasome, transported to and loaded onto the single-chain fusion HLA Class I protein in the endoplasmic reticulum. In some embodiments, the peptide antigen comprises a peptide of a tumor antigen. In some embodiments, the peptide antigen comprises a peptide of a protein from a pathogen including without limitation a bacterium, a virus, a fungus and a parasite. In some embodiments, the peptide antigen comprises a peptide of a tumor antigen. In some embodiments, the engineered cell expresses a single-chain fusion HLA Class I protein that is covalently linked to a peptide that does not comprise an auto-antigen or neo-antigen to the patient. It is within the ability of a skilled person to design the single-chain fusion HLA Class I protein and the peptide antigen presented thereon to modulate the immune response that may be elicited in a recipient. The isolated engineered cell expressing a single-chain fusion HLA Class I protein comprising a specific peptide antigen either covalently or non-covalently bound to the single-chain fusion HLA Class I protein can be used, for example, for administration to a recipient to elicit an immune response.

[0174] The single-chain fusion HLA Class I protein can be expressed from an expression vector that allows either transient or optionally, stable expression of the protein in an Atorney Docket No.: 51624-0096W01 / LG-

[0175] U2024141WO engineered cell. Exemplary' suitable expression vectors are known in the art. One such example is a lentiviral or retroviral vector, which is capable of integrating into the cellular genome to provide long-term, stable expression of an exogenous gene. In some embodiments, the viral vector is derived from human foamy virus, a type of retrovirus. Other suitable viral vectors include without limitation vectors derived from retrovirus, adenoviral virus, adeno- associated virus, lentivirus, herpes simplex virus, vaccinia virus, and pox virus.

[0176] The polynucleotide capable of encoding a single-chain fusion HLA Class I protein can be integrated into the chromosome of the cells, optionally into the B2M or the HLA loci, for stable expression. Thus, in some embodiments, the B2M loci are disrupted by inserting in the B2M loci the polynucleotide capable of encoding a single-chain fusion HLA Class I protein to replace the expression of the endogenous wild type B2M protein. The result of such gene targeting disrupts normal B2M expression and precludes formation of wildtype HLA Class I proteins but permits expression of a predetermined single-chain fusion HLA Class I protein of choice on the surface of the otherwise B2M deficient cells. Other expression vectors are also contemplated and the selection of suitable expression vector is within the ability of one ordinary skill in the art.

[0177] According to the vector design, the polynucleotide capable of expressing a singlechain fusion HLA Class I protein is delivered to a cell by viral infection (when a viral vector is used) or by other delivery methods including without limitation transfection, electroporation, gene targeting or liposome-mediated DNA delivery.

[0178] More details of the design of single-chain fusion HLA Class I proteins can be found, e.g., in U.S. Patent No. 11813318B2 and PCT Application Publication No. W02004103149, each of which is incorporated herein by reference in its entirety.

[0179] Provided herein is a single-chain fusion HLA Class I protein comprising, optionally from N-terminus to C-terminus: a signal peptide (e.g., any of the signal peptide or leader sequences described herein), a peptide antigen (e.g., any of the peptide antigens described herein), optionally a first linker (e.g., a flexible linker), a human B2M protein (e.g., any of the human B2M proteins described herein, with or without signal peptide), optionally a second linker (e.g., a flexible linker), and a human HLA-E heavy chain (e.g.. any of the HLA-E heavy chains described herein, with or without signal peptide).

[0180] In some embodiments, the signal peptide described herein is a human B2M signal peptide. In some embodiments, the human B2M signal peptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids 1-20 of SEQ Atorney Docket No.: 51624-0096W01 / LG-

[0181] U2024141WO

[0182] ID NO: 12. In some embodiments, the human B2M protein described herein is a wildtype human B2M protein (e.g., NCBI Reference No. NP_004039. 1). In some embodiments, the human B2M protein without a signal peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids of SEQ ID NO: 8. In some embodiments, the human B2M protein with signal peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids of SEQ ID NO: 12. In some embodiments, the human HLA-E heavy chain signal peptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids 1-21 of SEQ ID NO: 13. In some embodiments, the human HLA-E heavy chain described herein is a wildtype human HLA-E heavy chain (e.g., NCBI Reference No. NP 005507.3). In some embodiments, the human HLA-E heavy chain without a signal peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids of SEQ ID NO: 9. In some embodiments, the human HLA-E heavy chain with signal peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to amino acids of SEQ ID NO: 13. In some embodiments, the first linker and / or the second linker described herein are flexible linkers. In some embodiments, the flexible linker is a GS linker. Details of flexible linkers can be found, e.g., in Chen, X., et al. "Fusion protein linkers: property7, design and functionality." Advanced Drug Dell very Reviews 65.10 (2013): 1357-1369. which is incorporated herein by reference in its entirety. In some embodiments, the peptide antigen described herein is a human HLA-G peptide antigen or a human HLA-C peptide antigen. In some embodiments, the HLA-G peptide antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 10. In some embodiments, the HLA-C peptide antigen comprises an amino acid sequence that is at least 80%. 85%. 90%. 95%, or 100% identical to SEQ ID NO: 11. In some embodiments, the single-chain fusion HLA Class I protein described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 19. In some embodiments, the singlechain fusion HLA Class I protein with signal peptide described herein comprise an amino acid sequence that is at least 80%. 85%. 90%. 95%. or 100% identical to SEQ ID NO: 14. b. CD 47 or variants thereof

[0183] CD47 is a ~50 kDa heavily glycosylated, ubiquitously expressed membrane protein of the immunoglobulin superfamily with a single IgV-like domain at its N-terminus, a highly Atorney Docket No.: 51624-0096W01 / LG-

[0184] U2024141WO hydrophobic stretch with five membrane-spanning segments and an alternatively spliced cytoplasmic C-terminus. Each of the four alternatively spliced cytoplasmic tails exists in vivo at different frequencies, but all lack a substantial signaling domain.

[0185] While CD47 was first identified as a membrane protein involved in P3 integrin- mediated signaling on leukocytes, it is now known to also interact with thrombospondin- 1, signal regulatory protein-alpha (SIRPa, also known as SIRPA, Sirpa. Sirpa, or CD172A), and others to regulate various cellular functions including cell migration, axon extension, cytokine production, and T cell activation.

[0186] Recent studies have focused most on CD47-SIRPa axis for its inhibitory role in phagocytosis. SIRPa, also known as Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 / brain Ig-like molecule with tyrosine-based activation motif / cluster of differentiation antigen-like family member A (SHPS-l / BIT / CD172a), is another membrane protein of the immunoglobulin superfamily that is particularly abundant in the myeloid- lineage hematopoietic cells such as macrophages and dendritic cells. The ligation of SIRPa on phagocytes by CD47 expressed on a neighboring cell results in phosphorylation of SIRPa cytoplasmic immunoreceptor tyrosine-based inhibition (ITIM) motifs, leading to the recruitment of SHP-1 and SHP-2 phosphatases. One resulting downstream effect is the prevention of myosin-IIA accumulation at the phagocytic synapse and consequently inhibition of phagocytosis. Thus, CD47-SIRPa interaction functions as a negative immune checkpoint to send a “don’t eat me” signal to ensure that healthy autologous cells are not inappropriately phagocytosed.

[0187] Overexpression of CD47 has been found in nearly all types of tumors, some of which include acute myeloid leukemia, non-Hodgkin’s lymphoma, bladder cancer, and breast cancer. While CD47 is implicated in the regulation of cancer cell invasion and metastasis, its most well-studied and important function related to tumor development is prevention of phagocytosis via ligating with SIRPa on the surrounding phagocytes. Also, CD47 expression on cancer stem cells (CSCs) implies its role in cancer recurrence. It can increase the chance of CSC survival, which in turn could repopulate a new tumor mass and cause a tumor relapse.

[0188] CD47 down-regulation is also involved in the clearance of red blood cells (RBCs) and platelets by splenic macrophages, which may cause hemolytic anemia and idiopathic thrombocytopenic purpura, respectively. Thus, when CD47 antagonists are used as therapies, it is also very important to assess its toxicities. Atorney Docket No.: 51624-0096W01 / LG-

[0189] U2024141WO

[0190] A detailed description of CD47 and its function can be found, e.g., in Liu, X., et al. "Is CD47 an innate immune checkpoint for tumor evasion?" Journal of Hematology & Oncology 10.1 (2017): 12; and Huang Y. et al. "Targeting CD47: the achievements and concerns of current studies on cancer immunotherapy." Journal of Thoracic Disease 9.2 (2017): El 68; which are incorporated by reference herein in the entirety.

[0191] In one aspect, the present disclosure provides an engineered cell (e.g., any of the engineered cells described herein) expressing CD47 (e.g.. human CD47) or its variant thereof. In some embodiments, the exogenous human CD47 is a wildtype human CD47 comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20. In some embodiments, the exogenous human CD47 variant is a mutated human CD47 comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21 or 22. In some embodiments, the exogenous human CD47 is a wildty pe human CD47 with signal peptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the exogenous human CD47 variant is a mutated human CD47 with signal peptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6 or 7.

[0192] Engineered Receptor

[0193] In one aspect, the present disclosure provides cells (e.g., immune cells) that express engineered receptor. 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 costimulatory 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). Atorney Docket No.: 51624-0096W01 / LG-

[0194] U2024141WO

[0195] 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.

[0196] 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 engineered cells comprising the CD43 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.

[0197] 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.

[0198] The intracellular signaling domain of a CAR may comprise a primary' intracellular signaling domain. “Primary7intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. The primary7intracellular signaling domain may' contain a signaling motif known as Immunoreceptor Tyrosine-based Activation Motif, or ITAM. The primary intracellular signaling domain may comprise a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, FcgammaRIIa. DAP10, and DAP12. The primary Atorney Docket No.: 51624-0096W01 / LG-

[0199] U2024141WO 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 primary7intracellular signaling domain.

[0200] 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-stimulatory7ligand, 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, CD5, ICAM-1, LFA-1 (CDl la / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such co- stimulatory molecules include GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160. CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma. IL-7R alpha, ITGA4. VLA1. CD49a, ITGA4. IA4, CD49D. ITGA6, VLA-6. CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la,, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM. Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, Atorney Docket No.: 51624-0096W01 / LG-

[0201] U2024141WO

[0202] 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.

[0203] The CAR may comprise a single co-stimulatory signaling domain. The CAR may comprise two or more co-stimulatory signaling domains. The intracellular signaling domain may comprise a functional primary’ intracellular signaling domain and one or more co- stimulatory signaling domains. The CAR may not comprise afunctional primary intracellular signaling domain (such as CD3 . 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.

[0204] The intracellular signaling domain of a CAR may comprise a co-stimulatory signaling domain derived from CD28. The intracellular signaling domain may comprise a cytoplasmic signaling domain of 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.

[0205] The intracellular signaling domain of the CAR may comprise a co-stimulatory signaling domain of CD28 and a co-stimulatory signaling domain of 4- IBB. The intracellular signaling domain may comprise a cytoplasmic signaling domain of CD3^, a co-stimulatory signaling domain of CD28, and a co-stimulatory signaling domain of 4- IBB. 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^. Atorney Docket No.: 51624-0096W01 / LG-

[0206] U2024141WO

[0207] 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.

[0208] The tumor antigen may be selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33. CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2. EGP-40. EpCAM. ERBB2, ERBB3, ERBB4. FBP, fetal acetylcholine receptor, folate receptor-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), ty rosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG- 72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR. EGFR-VIII, Claudin 18.2. Claudin 6, NKG2D, Delta-like 3 (DLL3). CD70, CS-1. c-Met, Glycolipid F77. PD-L1. and PD-L2, and other tumor antigens with clinical significance, and combinations thereof. The tumor antigen may be derived from an intracellular protein of tumor cells. The tumor antigen may be expressed on the surface of tumor cells. 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 histocompatibility7antigens), and breast cancer (e.g.. HER2, NY-BR1).

[0209] The transmembrane region of a CAR may comprise or be chosen from the transmembrane region of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 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, CD1 Id, ITGAE, CD 103, 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). CD160 (BY55), PSGL1. CDIOO (SEMA4D). Atorney Docket No.: 51624-0096W01 / LG-

[0210] U2024141WO

[0211] SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, 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.

[0212] The extracellular domain may be connected to the transmembrane region by a hinge region. The hinge region may be a hinge region derived from: CD8a. CD28, IgGl. IgG2, IgG3, or IgG4. The hinge region may comprise a hinge region of CD8a.

[0213] The CAR may comprise a signal peptide (SP). The signal peptide may be derived from a molecule selected from the group consisting of CD8a. GM-CSF receptor a, and IgGl heavy chain. The signal peptide may be a CD8a signal peptide.

[0214] 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 CD19 binding fragment (e g., FMC63, SJ25C1, or those disclosed in different patents such as WO 2022 / 012683, etc.). BCMA CARs also have been well described, related patents include but not limited to WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760, and WO 2018 / 028647, etc. The extracellular antigen binding domain of BCMA CARs may be or include BCMA binding fragment. The BCMA binding fragment may bind to one or more epitopes on BCMA. The BCMA CARs may be bivalent CARs comprising two anti-BCMA sdAbs targeting same or different BCMA epitopes.

[0215] The CAR may be a BCMA CAR. A wide variety7of antigen binding domain sequences can be used as the antigen binding domain of the CAR.

[0216] The CAR may be a single CAR. dual CAR, tandem CAR or split CAR.

[0217] The BCMA CAR may comprise a 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 CAR may specifically bind to BCMA-positive tumor cells. In some embodiments, the BCMA CAR described herein is encoded by a nucleic acid sequence set forth in SEQ ID NO: 3.

[0218] 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-L VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, Atorney Docket No.: 51624-0096W01 / LG-

[0219] U2024141WO

[0220] 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. 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.

[0221] The TCR receptor complex is an octomeric complex formed by variable TCR receptor a and P chains (or y and 5 chains on case of y3 T cells) with three dimeric signaling modules CD36 / 8, CD3y / s and CD247 (T-cell surface glycoprotein CD3 zeta chain) or tyq. 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.

[0222] 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 but not more than 20 modifications. The TFP may comprise a Atorney Docket No.: 51624-0096W01 / LG-

[0223] U2024141WO transmembrane region that comprises a transmembrane region of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0224] 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.

[0225] 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 Ankynn repeat (DARPin) polypeptide. The tumor antigen may be selected from the group consisting of CD 19, BCMA, NY-ESO-1, VEGFR2, MAGE- A3, VEGFR2, MAGE-A3. CD20. CD22, CD30, CD33. CD38, CEA. CS 1. CD138, CD123 / IL3Ra, c-Met, gplOO, MUC 1, 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 CD3a. 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 a transmembrane region and a cytosolic domain derived Atorney Docket No.: 51624-0096W01 / LG-

[0226] U2024141WO from CD4. The TAC receptor may comprise a transmembrane region and a cytosolic domain derived from CD8 (such as CD8a).

[0227] 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 subty pes 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.

[0228] 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.

[0229] 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 tissuespecific 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 CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Atorney Docket No.: 51624-0096W01 / LG-

[0230] U2024141WO

[0231] 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.

[0232] 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 embry onic 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 Ban 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-catemn. 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.

[0233] Engineered Cells

[0234] In one aspect, the present disclosure provides engineered cells comprising the CD43 polypeptide described herein. The engineered cells comprising the CD43 polypeptide described herein may further comprise an engineered receptor described herein (e.g.. CAR) and / or overexpress one or more of tolerogenic factors described herein. For example, the engineered cells comprising the CD43 polypeptide described herein may further comprise a CAR, and overexpress HLA-E (e.g., any of the single-chain fusion HLA Class I proteins described herein) and / or CD47 (e.g.. any of the CD47 or its variants thereof described Atorney Docket No.: 51624-0096W01 / LG-

[0235] U2024141WO herein). The cell can be an immune cell. The cell can be selected from a group consisting of T cell, aPT cell, yoT cell, NK cell, NKT, tumor-infiltrating lymphocytes (TIL), peripheral blood mononuclear cell (PBMC), hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, and a combination thereof.

[0236] In one aspect, the present disclosure provides engineered cells comprising a CD43 polypeptide (e.g., an exogenous CD43 polypeptide).

[0237] 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, TCR , TCRy, TCR5, CD3y, CD36, 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., byexpression 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.

[0238] The engineered cells expressing the CD43 polypeptide described herein may express more than one engineered receptors, such as any combination of CAR, TCR, or TAC receptor.

[0239] The engineered cell expressing the CD43 polypeptide described herein may be used to treat cancer.

[0240] Comparing to a cell without the expression of the CD43 polypeptide described herein, the engineered cell comprising the CD43 polypeptide described herein may have a higher cytotoxicity- against tumor cells. Comparing to a cell without the expression of the CD43 Atorney Docket No.: 51624-0096W01 / LG-

[0241] U2024141WO polypeptide described herein, the engineered cell comprising the CD43 polypeptide described herein may have a higher persistence and / or proliferation.

[0242] In one aspect, the present disclosure provides engineered cells comprising (i) a CD43 polypeptide described herein; (ii) one or more tolerogenic factors described herein; and / or (iii) an engineered receptor (e.g., CAR) described herein. The modified cell may be an immune cell. The modified cell may comprise one or more polynucleotides encoding (i) a CD43 polypeptide described herein, (ii) one or more tolerogenic factors desenbed herein; and / or (iii) 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, an engineered 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).

[0243] The engineered cells may be modified immune cells. The immune cells may be selected from the group consisting of T cells, natural killer (NK) cells, B cells, monocytes, or macrophages. The engineered cells may be T cells. The engineered cells may be NK cells. The engineered cells may be a T cells. The engineered cells may be yd T cells. The engineered cells may be V51 T cells. The engineered cells may be V52 T cells. The engineered cells may be Treg cells.

[0244] 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 2-microglobulin to prevent the formation of functional Class I MHC molecules, or by inactivating Class II MHC molecules. The engineered cell expressing the CD43 polypeptide described herein may have a disruption at the endogenous B2M gene, or has reduced expression or activity of B2M.

[0245] 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.

[0246] 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.

[0247] The engineered cells may be from a cell line, e.g., a K562 cell line (a leukemia cell line). Atorney Docket No.: 51624-0096W01 / LG-

[0248] U2024141WO

[0249] Comparing to unmodified cells that do not comprise the CD43 polypeptide, the engineered cells may have an enhanced proliferation. Comparing to unmodified cells that do not comprise the CD43 polypeptide, the engineered cells may have an enhanced cytotoxicity toward tumor cells.

[0250] The engineered receptor may be a CAR. 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 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.

[0251] The CD43 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.

[0252] Comparing to a cell without the overexpression of the CD43 polypeptides, the engineered cell described herein may have an increased expression of CD43 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 CD43 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 CD43 polypeptide.

[0253] In some embodiments, the expression level of the CD43 polypeptide (e.g., any of the CD43 polypeptides described herein) on the engineered cell (e.g., any of the engineered cells described herein) is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 2000-fold, at least 5000-fold, or at Atorney Docket No.: 51624-0096W01 / LG-

[0254] U2024141WO least 10000-fold as compared to the expression level of CD43 (e.g.. endogenous CD43) on a wildtype cell or a control cell (e.g., a cell not modified to overexpress CD43).

[0255] In some embodiments, the expression level of the CD43 polypeptide (e.g., any of the CD43 polypeptides described herein) on the engineered cell (e.g., any of the engineered cells described herein) is at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%. about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 1-fold, about 1-fold to about 2-fold, about 2-fold to about 3-fold, about 3-fold to about 4-fold, about 4-fold to about 5-fold, about 5-fold to about 6-fold, about 6-fold to about 7-fold, about 7-fold to about 8-fold, about 8-fold to about 9-fold, about 9-fold to about 10-fold, about 10-fold to about 20- fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, about 600-fold to about 700- fold, about 700-fold to about 800-fold, about 800-fold to about 900-fold, about 900-fold to about 1000-fold, about 1000-fold to about 2000-fold, about 2000-fold to about 5000-fold, or about 5000-fold to about 10000-fold as compared to the expression level of CD43 (e.g., endogenous CD43) on a wildtype cell or a control cell (e.g., a cell not modified to overexpress CD43).

[0256] In some embodiments, the expression level of the CD43 polypeptide (e g., any of the CD43 polypeptides described herein) on the engineered cell (e.g., any of the engineered cells described herein) is at least about 1-fold to about 10000-fold, about 10-fold to about 10000- fold, about 100-fold to about 10000-fold, about 1000-fold to about 10000-fold, about 1-fold to about 1000-fold, about 10-fold to about 1000-fold, about 100-fold to about 1000-fold, about 1-fold to about 100-fold, about 10-fold to about 100-fold, or about 1-fold to about 10- fold as compared to the expression level of CD43 (e.g., endogenous CD43) on a wildtype cell or a control cell (e.g., a cell not modified to overexpress CD43).

[0257] The engineered cells may comprise both a CD43 polypeptide and a CAR (CAR armored with CD43 polypeptide). The engineered cells may be modified CAR-T cells (CD43 polypeptide armored CAR-T cells). The expression of CAR and CD43 polypeptide by the engineered cells can be determined by flow cytometry (e.g., FACS). The engineered cells can have a CAR positive rate of more than 5%, more than 10%. more than 15%, more than 20%. Atorney Docket No.: 51624-0096W01 / LG-

[0258] U2024141WO more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%. more than 55%, more than 60%. more than 65%, more than 70%. more than 75%, more than 80%, or more than 90%. The engineered cells may have a CAR positive rate of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. The engineered cells may have a CAR positive rate of 70%-80%. 70%-90%. 75%-85%, or 75%-90%.

[0259] The purity of the engineered cells can be determined by flow cytometry (e.g., 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%.

[0260] The overexpression of CD43 can promote the expansion of the engineered cells (e.g., y5 T cells), e.g., in the presence of effector cells (e.g., NK cells or PBMCs). The expansion of the engineered cells may be increased by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%. more than 30%, more than 35%. more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 90%, more than 95%, more than 100%, more than 110%, more than 120%, more than 130%, more than 140%, more than 150%, more than 160%, more than 170%, more than 180%, more than 190%. or more than 200% compared to cells without the overexpression of the CD43 polypeptide. The expansion of the engineered cells may be increased by about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more folds compared to cells without the overexpression of the CD43 polypeptide. Cell expansion or proliferation can be measured by in vitro cell proliferation assays or any of the cytotoxicity assays described herein. Comparing to cells without the CD43 polypeptide described herein, the engineered cells comprising the CD43 polypeptide described herein has a cell number that is higher by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%. more than 70%, more than 75%. more than 80%, more than 90%, Atorney Docket No.: 51624-0096W01 / LG-

[0261] U2024141WO more than 95%, more than 100%, more than 110%, more than 120%, more than 130%, more than 140%. more than 150%, more than 160%, more than 170%, more than 180%, more than 190%, or more than 200%. Comparing to a cell without the CD43 polypeptide described herein, the engineered cells comprising the CD43 polypeptide described herein may have a cell number that is 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%. less than 90%. less than 95%, less than 100%, less than 110%, less than 120%, less than 130%, less than 140%, less than 150%, less than 160%, less than 170%, less than 180%, less than 190%, or less than 200%.

[0262] 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 10: 1, 9: 1, 8: 1, 7: 1, 6:1, 5: 1, 4: 1, 3:1, 2: 1, 1 : 1, 1:2, 1 :3, 1:4, 1 :5, 1:6, 1:7, 1 :8, 1:9, or 1:10. The in vitro cy totoxicity' 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: 1 or 1 :4. The engineered cells may have a cytotoxicity of more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%, after 1 round, 2 rounds, 3 rounds, 4 rounds, or 5 rounds of stimulation in a re-challenge assay. The engineered cells may have a cytotoxicity of less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less 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 CD43 polypeptide, the cytotoxicity of the engineered cells comprising the CD43 polypeptide may increase by more than 5%. more than 10%. more than 15%, more than 20%. more than 30%, more than 40%. more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%. more than 900%, or more than Atorney Docket No.: 51624-0096W01 / LG-

[0263] U2024141WO

[0264] 10,00%, after 1 round, 2 rounds, 3 rounds. 4 rounds, or 5 rounds of stimulation in a rechallenge assay.

[0265] The overexpression of CD43 (e.g., any of the CD43 polypeptides described herein) can protect the engineered cells described herein from host cell-mediated killing (e.g., immune clearance). The host cells can be T cells, NK cells, or a combination thereof. The host cells can be PBMCs. The host cell-mediated killing of the engineered cells may be decreased by more than 5%. more than 10%, more than 15%. more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 90% compared to cells without the overexpression of the CD43 polypeptide. The host cell-mediated killing of the engineered cells may be decreased by about 2, 3, 4. 5, 6, 7, 8, 9, 10 or more folds compared to cells without the overexpression of the CD43 polypeptide.

[0266] In some embodiments, overexpression of CD43 (e.g., any of the CD43 polypeptides described herein) can increase the cell number of the engineered cells described herein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, al least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to a wildtype cell or a control cell (e.g., a cell not modified to overexpress CD43 or an untransduced cell), when the engineered cells are co-cultured with host cells (e.g., T cells, NK cells, or PBMCs) for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some embodiments, the E:T ratio is about 1: 10, 1 :9. 1:8, 1 :7, 1 :6. 1:5, 1 :4.5, 1 :4, 1 :3.5, 1 :3, 1:2.5, 1 :2, 1 : 1.5, 1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10:1. In some embodiments, the E:T ratio is about 500:1, 400: 1, 300: 1, 200: 1, 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30:1, 20:1, or 10: 1.

[0267] In some embodiments, the engineered cells and host cells are co-cultured in the presence of one or more cytokines (e.g.. IL-2). In some embodiments, the engineered cells and host cells are co-cultured in the absence of any cytokines (e.g., IL-2). In some embodiments, the concentration of the one or more cytokines is about 10U, about 20U, about 50U, about 100U, about 200U, about 300U, about 400U, about 500U, about 600U, about Atorney Docket No.: 51624-0096W01 / LG-

[0268] U2024141WO

[0269] 700U, about 800U. about 900U, about 1000U, about 2000U, about 3000U, about 4000U, about 5000U, or about 10000U.

[0270] In some embodiments, the engineered cells are HLA-A2 negative. In some embodiments, the host cells are HLA-A2 positive. In some embodiments, the NK cells described herein are PBNK cells, primary NK cells. In some embodiments, the PBMCs described herein are PBMCs isolated from HLA-A2 positive donors. In some embodiments, the host cells contain at least 10%. at least 20%. at least 30%. at least 40%. at leas 50%, at least 60%, at least 70%, or at least 80% CD3-positive cells.

[0271] In some embodiments, the engineered cells are cell line cells (e.g., K562 cells). In some embodiments, the engineered cells have a normal expression level (e.g., at least 50%, 60%. 70%. 80%. 90%. 100%, 110%, 120%. 130%, 140% or 150% as compared to that of a wildtype cell) of endogenous HLA Class I and / or HLA Class II molecules. In some embodiments, the control cell is a cell overexpressing HLA-E (e.g., any of the single-chain fusion HLA Class I proteins described herein) and / or CD47 (e.g., any of the CD47 or variants thereof).

[0272] In some embodiments, the engineered cells are gdT cells. In some embodiments, the gdT cells are B2MKO gdT cells described herein. In some embodiments, the knockout efficiency of B2M gene is at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95%. In some embodiments, the control cell is a cell overexpressing a CAR (e.g., any of the CARs described herein) or an untransduced cell.

[0273] In some embodiments, overexpression of CD43 (e.g., any of the CD43 polypeptides described herein) and a tolerogenic factor (e.g., HLA-E (e.g., any of the single-chain fusion HLA Class I proteins described herein) or CD47 (e.g., any of the CD47 or variants thereof described herein)) can increase the cell number of the engineered cells described herein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to a wildty pe cell or a control cell, when the engineered cells are co-cultured with host cells (e.g., T cells, NK cells, or PBMCs) for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In some embodiments, the engineered cells further overexpress an engineered receptor (e.g., any of the CARs described herein). In some Atorney Docket No.: 51624-0096W01 / LG-

[0274] U2024141WO embodiments, the control cell is a cell overexpressing the CAR. In some embodiments, the control cell is a cell overexpressing the CAR, and either one of CD43 or the tolerogenic factor. In some embodiments, the E:T ratio is about 1: 10, 1:9, 1 :8, 1:7, 1:6, 1 :5, 1 :4.5, 1:4, 1 :3.5, 1 :3, 1 :2.5, 1 :2, 1: 1.5, 1 : 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5:1, 4: 1, 4.5: 1, 5: 1, 6:1, 7: 1, 8: 1, 9:1, or 10: 1. In some embodiments, the E:T ratio is about 500:1, 400: 1, 300: 1, 200: 1, 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, or 10: 1. In some embodiments, the expression level of the CAR is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% among all engineered cells. In some embodiments, the expression level of the tolerogenic factor is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%. at least 12%. at least 13%. at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% among all engineered cells. a. Gamma delta (yd) T cells

[0275] Gamma delta (y6) T cells (or y5T cells, or gdT cells) are a subgroup of T cells with distinct T cell receptors (TCRs) y and 8 chains on their surface, which account for 0.5-5% of all T-lymphocytes. This small subset of cells was first found in 1987, after the accidental discovery of third chain of the TCR (y chain) in 1984. In contrast, the most T cells in normal human body are a0 T cells (65-70%) with TCR composed of two glycoprotein chains called a and P TCR chains. These cells are generally simply referred to as “T cells”. Although, y8 T cells are much less common than a T cells, they are at their highest abundance in the gut mucosa, within a population of lymphocytes known as intraepithelial lymphocytes. Unlike classical aP T cells that recognize specific peptide antigens presented by major histocompatibility complex (MHC) molecules, y6 T cells can recognize generic determinants expressed by cells that have become dysregulated as a result of either malignant transformation or viral infection. Consequently, y6 T cells have the innate ability to recognize and kill a broad spectrum of tumor cell types, in a manner that does not require the existence of conventional tumor-specific antigens.

[0276] As outstanding research on y8 T cells ever since, these immune cells have gained close attention than ever before. Their features include non-MHC restricted antigen recognition and an abundant cytokine secretion capacity, suggesting that they possess a high antitumor capability. These attractive features have raised expectations for their application in Atorney Docket No.: 51624-0096W01 / LG-

[0277] U2024141WO cancer adoptive immunotherapy. Up until now, clinical trials have been conducted in numerous cancers, such as renal cell carcinoma, malignant leukemia, and advanced lung cancer, as well as others, with the majority of trials showing them to be well tolerated and safe.

[0278] In addition, y5 T cells have shown to possess the ability to bridge innate and adaptive immunity. The majority of y5 T cells in adult human blood exhibit Vy9V52 T cell receptors and respond to small phosphorylated nonpeptide antigens, called phosphoantigens (pAgs). which are commonly produced by malignant cells. Unlike conventional o.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 earners 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) y5 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.

[0279] Details of y5 T cells can be found, e.g., in Lepore. M.. et al. "The conventional nature of non-MHC-restricted T cells." Frontiers in Immunology’ 9 (2018): 393121 ; Zou, C., et al. "y5 T cells in cancer immunotherapy." Oncotarget 8.5 (2017): 8900; and Zhao, Y., et al. "Gamma-delta (y8) T cells: friend or foe in cancer development?" Journal of Translational Medicine 16 (2018): 1-13; each of which is incorporated herein by reference in its entirety. b. Engineered cells with maintained expression of endogenous B2M

[0280] In some embodiments, the engineered cells described herein express endogenous Major Histocompatibility Complex (MHC), e.g., endogenous MHC Class I molecules and / or MHC Class II molecules. In some embodiments, the expression level of endogenous MHC (e.g., all or part of endogenous MHC Class I molecules and / or MHC Class II molecules) is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% as compared to that in a wildtype cell or a control cell (e.g., a cell not modified to overexpress CD43). Atorney Docket No.: 51624-0096W01 / LG-

[0281] U2024141WO

[0282] In some embodiments, the expression of endogenous MHC Class I molecules and / or MHC Class II molecules are not eliminated or reduced in the engineered cell. For example, the endogenous B2M gene loci are not disrupted (e.g., genetically modified). In some embodiments, the engineered cells described herein contain wildtype B2M gene loci. In some embodiments, the engineered cells described herein do not have a B2M" ' background. c. Disruption of genes encoding CD43 ligands (e.g. Siglec-7)

[0283] In some embodiments, the engineered cells described herein o.p T cells and / or NK cells, which has an eliminated or reduced expression of an endogenous CD43 ligand. In some embodiments, the endogenous CD43 ligand is an immune checkpoint receptor that is specific for CD43. In some embodiments, the endogenous CD43 ligand is sialic acid-binding Ig-like lectin 7 (Seglec-7). Siglec-7 is highly expressed on activated CD8 cells, monocytes and NK cells.

[0284] In some embodiments, the endogenous CD43 ligand gene in the engineered cells described herein is disrupted (e.g., knocked out). In some embodiments, the engineered cells described herein have a Siglec-7- ’ background.

[0285] In some embodiments, the endogenous CD43 ligand gene is disrupted by using a gene editing method (e.g., clustered regularly interspaced short palindromic repeats / Cas9 protein (CRISPR / Cas9), transcription activator-like (TAL) effector nucleases (TALENs), or Zinc finger nucleases (ZFNs)), RNA interference (RNAi) technology, homologous recombination, modifying one or more regulating elements (e.g., promoter) of endogenous CD43 ligand gene, knocking out a sequence encoding all or part of the endogenous CD43 ligand, and / or knocking in an exogenous sequence to replace all or part of endogenous CD43 ligand gene. Any suitable technique for disrupting the endogenous CD43 ligand gene can be used; exemplary techniques are disclosed throughout the application and are within the level of skill in the art based on the teachings herein and the teachings known in the art. Exemplary other techniques can be found, for example, in U.S. Patent Application Publication No. US2008 / 0219956, which is incorporated by reference herein in its entirety.

[0286] In some embodiments, the expression level of the endogenous CD43 ligand is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or Atorney Docket No.: 51624-0096W01 / LG-

[0287] U2024141WO less than 0.1% as compared to that in a wildtype cell or a control cell (e.g., a cell not modified to overexpress CD43).

[0288] In some embodiments, the engineered cells described herein are not y5 T cells.

[0289] In some embodiments, the engineered cells described herein are y5 T cells, whose expression of an endogenous CD43 ligand is not eliminated or reduced as compared to a wildtype cell or a control cell (e.g., a y5 T cell whose endogenous CD43 ligand gene is not disrupted). In some embodiments, the expression of an endogenous Seglec-7 is not eliminated or reduced in the y5 T cell.

[0290] Eliminating or reducing expression of endogenous MHC Class I / II molecules

[0291] A protein that plays the most important role in distinguishing between self cells and non-self cells is a cell surface protein called a human leukocyte antigen (HLA) or a major histocompatibility complex (MHC). In humans, MHC is referred to as human leukocyte antigen (HLA). Mismatched HLA proteins that are presented to T cells as foreign antigens activate this allo-immune response.

[0292] MHC genes can be subdivided into Class I and Class II. MHC Class I molecules, which include three classical (HLA-A, HLA-B and HLA-C) and three non-classical (HLA-E, HLA-F and HLA-G) a chains, are expressed on the surface of all somatic cells. The HLA Class I (HLA-I) is expressed on all nucleated cells and consists of an HLA Class I heavy chain (or a chain) and P-2 microglobulin (B2M) to form a functional heterodimer. Among these, HLA-A, HLA-B, and HLA-C have particularly great sequence diversity among individuals and play a major role in identifying self cells and non-self cells in transplantation immunity. The MHC Class I heavy chains can form a functional heterodimer with 2- Microglobulin (B2M) to be expressed on a cell surface, and presenting intracellular peptides to CD8 T cells to induce cytotoxic lymphocyte activation and killing of host cells. In contrast, MHC Class II (e.g., HLA-DR, -DQ, -DP) presents extracellular derived antigens to CD4 T cells and are generally expressed on professional antigen presenting cells (APCs, e.g., dendritic cells, macrophages), activating CD4 T cells help to drive a B-cell mediated antibody response to host antigens.

[0293] The cells of the disclosure may have eliminated or reduced expression of endogenous MHC Class I molecules and / or endogenous MHC Class II molecules. Reduction of MHC I and / or MHC II expression can be accomplished, for example, by one or more of the following: (1) targeting the polymorphic HLA alleles (HLA-A, HLA-B, HLA-C) and MHC- Atorney Docket No.: 51624-0096W01 / LG-

[0294] U2024141WO

[0295] II genes directly; (2) removal of B2M and / or TAPI, which will prevent surface trafficking of all MHC-I molecules; (3) removal of CIITA, which will prevent surface trafficking of all MHC-II molecules; and / or (4) deletion of components of the MHC enhanceosomes, such as NLRC5, RFX5, RFXANK, RFXAP, IRF1, NF-Y (including NFY-A, NFY-B, NFY-C), and CIITA that are critical for HLA expression.

[0296] In one aspect, the present disclosure provides y5 T cells having an eliminated or reduced expression of endogenous MHC Class I molecules. For example, the yo T cells comprise a genetically engineered disruption in a beta-2 microglobulin (B2M) gene. In some embodiments, the y5 T cells comprise genetically engineered disruptions of all copies of the B2M gene. In some embodiments, the genetic disruptions in the B2M gene result in defective or no expression of the endogenous B2M protein. Since B2M is a common component of all HLA Class I proteins, the disruptions preclude the expression of all natural HLA Class I proteins on the cell surface. The B2M protein sequence with signal peptide is shown in SEQ ID NO: 12. There may be many single nucleotide polymorphisms (SNPs) in the gene; as will be understood by those of skill in the art, the human cells and methods of the disclosure are applicable to any such B2M gene and SNPs.

[0297] The cells of these embodiments of the disclosure can be used, for example, as donor cells for transplantation in a recipient in need thereof. B2M deficient cells encompass cells that comprise a B2M’ ' genetic background (referred to as B2M" ' cells). The term "B2M" ’ cells’7refers to primate cells, optionally human cells, that comprise genetically engineered disruptions in all copies of the B2M gene. The B2M" ‘ cells can serve as “universal donor cells” in that they are immunologically compatible to all or a significant percentage of recipients in a population. As used herein, a recipient or patient refers to a primate, and optionally a human. In some embodiments, the cell is a human cell and the patient is a human.

[0298] The cells of the disclosure can be engineered to disrupt the B2M gene such that no functional endogenous B2M protein is produced from the disrupted genetic loci. In some embodiments, the disruption results in expression of non-functional B2M proteins, including but not limited to truncations, deletions, point mutations and insertions. In some embodiments, the disruption results in no protein expression from the B2M gene.

[0299] Cells deficient in B2M expression are unable to express HLA Class I proteins on the cell surface. HLA Class I-deficiency provides further benefits; for example, cells without Atorney Docket No.: 51624-0096W01 / LG-

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[0301] HLA Class I expression cannot present auto-antigens that would otherwise prevent successful cell therapies for autoimmune diseases such as diabetes and rheumatoid arthritis.

[0302] Any suitable technique for disrupting one, two or all copies of the B2M gene can be used; exemplary techniques are disclosed throughout the application and are within the level of skill in the art based on the teachings herein and the teachings known in the art. Exemplary7other techniques can be found, for example, in U.S. Patent Application Publication No. US2008 / 0219956, which is incorporated by reference herein in its entirety. These techniques may optionally include steps to remove non-human DNA sequences from the cells after B2M gene disruption.

[0303] An exemplary7embodiment of this method is using an adeno-associated virus gene targeting vector, optionally7including removing the transgene used for targeting via techniques such as those described below, or by removing the transgene used for targeting by Cre-mediated loxP recombination, or other suitable recombination techniques. See Khan, I.F., et al. "AAV -mediated gene targeting methods for human cells." Nature Protocols 6.4 (2011): 482-501, which is incorporated by reference in its entirety7. Exemplary targeting vectors and exemplary vector diagrams are also disclosed herein. It is w ithin the level of those of skill in the art, based on the teachings herein and known in the art, to utilize a variety7of techniques for making the B2M" ' cells, optionally human cells.

[0304] In certain embodiments, the cell genome of the B2M" ' cells may comprise no more than 100. no more than 50 or no more than 30 nucleotides of non-human DNA sequences. In some embodiments, the cell genome may comprise 6, 5, 4, 3, 2, 1 , or 0 nucleotides of non- human DNA sequences.

[0305] In some embodiments, expression of the endogenous MHC Class I molecules is eliminated or reduced by disrupting endogenous B2M gene(s). In some embodiments, the disruption is achieved by using a gene editing system (e.g., clustered regularly interspaced short palindromic repeats / Cas9 protein (CRISPR / Cas9)). In some embodiments, the gene editing system comprises a guide RNA (gRNA) targeting the endogenous B2M gene(s).

[0306] In one aspect, the disclosure provides a method of producing an engineered cell (e.g., a modified y6 T cell), which has reduced or eliminated expression of MHC Class I molecules relative to a starting cell, the method comprising disrupting expression of one or more genes encoding endogenous MHC Class I molecules (e.g., endogenous B2M gene) of a starting cell by targeting a nucleotide sequence on B2M gene through a gene silencing method. Exemplary gene silencing methods include, but not limited to, CRISPR / Cas9, RNA Atorney Docket No.: 51624-0096W01 / LG-

[0307] U2024141WO interference (RNAi) technology, transcription activator-like (TAL) effector nucleases (TALENs) and Zinc finger nucleases (ZFNs).

[0308] The methods may involve knocking out the [32 microglobulin (B2M) gene. B2M is a component of MHC Class I molecules. Without being bound by theory, knocking out the B2M gene can increase the immune compatibility of the engineered cells.

[0309] In one aspect, the disclosure provides a method of producing an engineered cell (e.g., a modified y6 T cell), the method comprising disrupting one or more genes encoding endogenous MHC Class I molecules (e g., endogenous B2M gene) of a starting cell by a gene editing system. The gene editing system can comprise a universal gRNA and a RNA-guided nuclease or a base editor, wherein the universal gRNA targets one or more genes encoding endogenous MHC Class I molecules. The modified cell may have reduced or eliminated expression of MHC Class I molecules. The method may further comprise reducing or eliminating the expression of Class II major histocompatibility complex transactivator (CIITA) in the modified cell.

[0310] The RNA-guided nuclease described herein may be a Cas9 nuclease. The RNA- guided nuclease described herein may be an inactivated Cas9 nuclease with a cytosine base editor or an adenine base editor.

[0311] The cells for engineering can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. The subject from which the cell is isolated can be one having the disease or condition or in need of a cell therapy (autologous cells). The subject from the which the cell is isolated can be a different subject other than the one having the disease or condition or in need of a cell therapy (e.g., allogeneic cells).

[0312] The cells may be primary cells, e.g., primary' human cells. The samples may include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and / or incubation. The biological sample may be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to. body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0313] The engineered cells may express a decreased level of endogenous MHC Class I molecules as compared to that in the starting cell. The modified cell may elicit no or reduced GvHD and / or HvG response in a histoincompatible individual as compared to the GvHD Atorney Docket No.: 51624-0096W01 / LG-

[0314] U2024141WO and / or HvG response elicited by a primary T cell isolated from the donor of the starting T cell from which the modified cell is derived.

[0315] Primary T cells can be isolated from human PBMC. The primary T cells can be activated with anti-CD3 / CD28 beads and cultured in RPMI1640 medium supplemented with IL-2. Cas9 protein and gRNAs can be introduced into the activated T cells by electroporation. The knocking out efficiency can be determined by flow cytometry analysis, by detecting the cell surface expression of one or more genes encoding endogenous MHC Class I molecules (e.g., endogenous B2M gene).

[0316] Additional gRNAs may be used to knock out the CIITA gene. The method described herein may further comprise disrupting the expression of endogenous TCR.

[0317] The methods described herein can knock out one or more genes encoding endogenous MHC Class I molecules (e.g., endogenous B2M gene) with a knocking out efficiency 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 methods described herein can knock out one or more genes encoding endogenous MHC Class I molecules (e.g., endogenous B2M gene) with a knocking out efficiency 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 methods described herein can knock out HLA-A genes with a knocking out efficiency of 10%-90%, 30%-90%, or 50%-90%.

[0318] Disclosed herein are methods for disrupting a target position in a gene or locus (e.g., a B2M gene or locus). Disrupting the target position can be achieved, e.g., by disrupting one or more locus or allelic variants in the gene. Disrupting the target position can be achieved, e.g., by: (1) knocking out a gene: (a) insertion or deletion (e.g., NHEJ-mediated insertion or deletion) of one or more nucleotides in the gene, or (b) deletion (e.g., NHEJ-mediated deletion) of a genomic sequence including at least a portion of the gene, or (2) knocking down a gene mediated by enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9-fusion protein (e.g., fused to a transcriptional repressor) by targeting the promoter region of the gene. Both approaches give rise to disruption of the gene.

[0319] The method may comprise introducing an insertion or deletion of one more nucleotides within a locus (e.g., an endogenous MHC Class I locus or the coding region Atorney Docket No.: 51624-0096W01 / LG-

[0320] U2024141WO thereof). As described herein, in one embodiment, the method comprises the introduction of one or more breaks (e.g.. single strand breaks or double strand breaks) within the locus. NHEJ-mediated repair of the break(s) allows for the NHEJ-mediated introduction of an indel within the locus.

[0321] The method may comprise introducing a deletion of a genomic sequence comprising at least a portion (e.g., a portion within a coding region, an early coding region, or a portion within a non-coding region (e.g., a promoter, an enhancer, an intron, a 3’UTR, and / or a polyadenylation signal)) of a locus (e g., an endogenous MEIC Class I locus or the coding region thereof). In some embodiments, the method may comprise the introduction of two double stand breaks — one 5' and the other 3’ to (i.e., flanking) a position (e.g., within a coding region, an early coding region, or within a non-coding region (e.g., a promoter, an enhancer, an intron, a 3’ UTR, and / or a poly adenylation signal)) of an endogenous MHC Class I locus or the coding region thereof. Two gRNAs, e.g., unimolecular (or chimeric) or modular gRNA molecules, may be configured to position the two double strand breaks on opposite sides of a position (e.g., within a coding region, an early coding region, or within a non-coding region (e.g., a promoter, an enhancer, an intron, a 3’UTR, and / or a polyadenylation signal)) of an endogenous MHC Class I locus or the coding region thereof.

[0322] A single strand break may be introduced (e.g., positioned by one gRNA molecule) within a locus, e.g., an endogenous MHC Class I locus or the coding region thereof. A single gRNA molecule (e.g., with a Cas9 nickase) may be used to create a single strand break within the locus. The break can be positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.

[0323] A double strand break can be introduced (e.g., positioned by one gRNA molecule) within a locus, e.g., an endogenous MHC Class I locus or the coding region thereof. A single gRNA molecule (e.g., with a Cas9 nuclease other than a Cas9 nickase) can be used to create a double strand break within the locus, e.g., the gRNA molecule is configured such that the double strand break is positioned either upstream or dow nstream of a position within the locus. The break can be positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g.. an Alu repeat.

[0324] Humans are diploid organisms containing two pairs of homologous chromosome that share the same genes but vary in alleles. Only one allele of a gene can be present at the gene loci of a single chromosome, as a result a pair of homologous chromosome contain two Atorney Docket No.: 51624-0096W01 / LG-

[0325] U2024141WO alleles of a gene. In some embodiments, the gRNA can target both alleles of a given gene in a cell. In some embodiments, the gRNA can target both alleles of a given gene in a cell.

[0326] A targeted knockdow n approach reduces or eliminates expression of functional gene product, e.g., a functional endogenous MHC Class I gene product. A targeted knockdown can be mediated by targeting an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fused to a transcription repressor domain or chromatin modifying protein to alter transcription, e.g., to block, reduce, or decrease transcription, of the gene.

[0327] The methods and composition described herein may also include additional non-HLA genetic modifications to donor cells. The method may further involve knocking out the Class II major histocompatibility complex transactivator (CIITA) gene. CIITA controls the expression of HLA Class II genes. Without being bound by theory, knocking out the CIITA gene can further increase the immune compatibility of the engineered cells. The method may further involve overexpressing the HLA-E gene (e.g., in a single-chain trimer (SCT) form). HLA-E has a very specialized role in cell recognition by natural killer cells (NK cells). Without being bound by theory, overexpressing HLA-E can protect the engineered cells from NK cell mediated cell killing. The methods may further involve disrupting the expression of an endogenous TCR.

[0328] Nucleic Acids (Polynucleotides)

[0329] The present disclosure provides (i) nucleic acids (e.g.. expression vectors) encoding (i) a CD43 polypeptide described herein; (ii) nucleic acids (e.g., expression vectors) encoding one or more tolerogenic factors described herein; and / or (iii) nucleic acids (e.g., expression vectors) encoding an engineered receptor (e.g., CAR or TCR) described herein. The nucleic acids of the present disclosure can comprise a nucleic acid sequence encoding any one or more of the CD43 polypeptides, tolerogenic factors, and engineered receptors (e.g., CARs or TCRs) disclosed herein. The nucleic acid may encode both an engineered receptor and a CD43 polypeptide described herein. The nucleic acid may encode a CD43 polypeptide and one or more of the tolerogenic factors (e.g., HLA-E and / or CD47) described herein. The nucleic acid may encode an engineered receptor, a CD43 polypeptide, and one or more of the tolerogenic factors (e g., HLA-E and / or CD47) described herein.

[0330] A polynucleotide of the present disclosure may comprise a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequences can be separated by a linker. A linker for use in the present disclosure allows for Atorney Docket No.: 51624-0096W01 / LG-

[0331] U2024141WO multiple proteins to be encoded by the same nucleic acid sequence (e.g., a multi cistronic 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 CD43 polypeptide described herein. The first polynucleotide sequence may encode one or more of the tolerogenic factors (e.g., HLA-E and / or CD47) and the second polynucleotide sequence may encode a CD43 polypeptide described herein.

[0332] A polynucleotide of the present disclosure may comprise a first polynucleotide sequence, a second polynucleotide sequence, and a third polynucleotide sequence. The first, second, and third polynucleotide sequences can be separated by one or more linkers. A linker for use in the present disclosure allows for multiple proteins to be encoded by the same nucleic acid sequence (e.g., a multicistronic or bicistronic sequence), which are translated as a polyprotein that is dissociated into separate protein components. The polynucleotide may comprise from 5’ to 3’ the first polynucleotide sequence, a first linker, the second polynucleotide sequence, a second linker, and the third polynucleotide sequence. The first, second, and third polynucleotide sequences may encode an engineered receptor (e.g., CAR), a CD43 polypeptide, and one or more of the tolerogenic factors (e.g.. HLA-E and / or CD47) described herein. The first and second linkers can be any linkers described herein.

[0333] 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. Atorney Docket No.: 51624-0096W01 / LG-

[0334] U2024141WO

[0335] 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 Picomaviridae 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 as “F2A,” “E2A.” “P2A,” and “T2A,” respectively. Those of skill in the art would be able to select the appropriate self-cleaving peptide.

[0336] 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: 23) and (GGGS)n (SEQ ID NO: 24), where n represents an integer of at least 1 (e.g., 1, 2, 3. 4, 5, 6, 7, or 8). The linker may be an SSGGGGS linker (SEQ ID NO: 25). Those of skill in the art would be able to select the appropriate spacer sequence.

[0337] 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.

[0338] 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.

[0339] 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 Atorney Docket No.: 51624-0096W01 / LG-

[0340] U2024141WO early promoter, a Rous sarcoma virus promoter, the elongation-factor- 1 -alpha promoter (EF-1 alpha promoter, EF-1 a 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 inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0341] A polynucleotide of the present disclosure may enable the production of (i) a CD43 polypeptide described herein, (ii) one or more of the tolerogenic factors (e.g., HLA-E and / or CD47) described herein, and / or (iii) an engineered receptor (CAR or TCR) described herein (e.g., in a mammalian cell). A polynucleotide of the present disclosure may enable replication of the polynucleotide.

[0342] An expression vector (e.g., a retroviral vector or 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 retroviral vector or a lentiviral vector) of the present disclosure can comprise a polynucleotide encoding for a CAR or a TCR. The expression vector (e.g., the retroviral vector or the 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-1 a 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 are known to those of skill in the art and can be incorporated into a vector of the present disclosure. The vector may further comprise a non-requisite cis acting sequence that can improve titers and gene expression.

[0343] 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 Atorney Docket No.: 51624-0096W01 / LG-

[0344] U2024141WO hinge region, a CD8a transmembrane region, a CD137 co-stimulatory signaling domain, a CD3^ cytoplasmic domain.

[0345] The polynucleotide may encode a CAR and a CD43 polypeptide. The polynucleotide may comprise from the 5' end to the 3' end, the coding sequences of a CD43 polypeptide, a 2A cleavable linker, and a CAR. The polynucleotide may comprise from the 5' end to the 3' end, the coding sequences of a CAR, a 2A cleavable linker, and a CD43 polypeptide described herein.

[0346] The polynucleotide may encode a CD43 polypeptide and one ore more of the tolerogenic factors described herein. The polynucleotide may comprise from the 5' end to the 3' end, the coding sequences of a CD43 polypeptide, a 2A cleavable linker, and a single-chain fusion HLA Class I protein described herein. The polynucleotide may comprise from the 5' end to the 3' end, the coding sequences of a single-chain fusion HLA Class I protein, a 2A cleavable linker, and a CD43 polypeptide described herein. The polynucleotide may comprise from the 5' end to the 3' end, the coding sequences of a CD43 polypeptide, a 2A cleavable linker, and a CD47 or its variant thereof described herein. The polynucleotide may comprise from the 5' end to the 3' end, the coding sequences of a CD47 or its variant thereof, a 2A cleavable linker, and a CD43 polypeptide described herein.

[0347] The polynucleotide may encode a CAR, a CD43 polypeptide, and one ore more of the tolerogenic factors described herein. The polynucleotide may comprise, the coding sequences of a CD43 polypeptide, a first 2A cleavable linker, a tolerogenic factor, a second 2A cleavable linker, and a CAR described herein. The polynucleotide may comprise, the coding sequences of a CD43 polypeptide, a first 2A cleavable linker, a CAR, a second 2A cleavable linker, and a tolerogenic factor described herein. The polynucleotide may comprise, the coding sequences of a tolerogenic factor, a first 2A cleavable linker, a CD43 polypeptide, a second 2A cleavable linker, and a CAR described herein. The polynucleotide may comprise, the coding sequences of a tolerogenic factor, a first 2A cleavable linker, a CAR, a second 2A cleavable linker, and a CD43 polypeptide described herein. The polynucleotide may comprise, the coding sequences of a CAR, a first 2A cleavable linker, a CD43 polypeptide, a second 2A cleavable linker, and a tolerogenic factor described herein. The polynucleotide may comprise, the coding sequences of a CAR, a first 2A cleavable linker, a tolerogenic factor, a second 2A cleavable linker, and a CD43 polypeptide described herein.

[0348] The polynucleotide may encode an amino acid sequence of CD43 that is at least 70%, 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. Atorney Docket No.: 51624-0096W01 / LG-

[0349] U2024141WO

[0350] 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0351] 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.

[0352] 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.

[0353] 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.

[0354] 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 Atorney Docket No.: 51624-0096W01 / LG-

[0355] U2024141WO amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4. and a frameshift gap penalty of 5.

[0356] Introduction of Polynucleotides into Host Cells

[0357] The polynucleotides (e.g., vectors) described herein can be introduced as one or more polynucleotides or constructs, optionally comprising a marker that will allow for selection of host cells that contain the construct(s). The genes and regulatory regions can be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing. Particularly, using PCR, individual fragments including all or portions of a functional unit can be isolated, where one or more mutations can be introduced using "primer repair", ligation, in vitro mutagensis, etc. as appropriate. The polynucleotides obtained and demonstrated to have the appropriate sequences can then be introduced into the host cell by any convenient means. The polynucleotides can be integrated and packaged into nonreplicating. 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.

[0358] The CD43 polypeptide, the one ore more tolerogenic factors, and the engineered receptor described herein can be introduced into the engineered 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. Atorney Docket No.: 51624-0096W01 / LG-

[0359] U2024141WO

[0360] Introduction of constructs into the cell's genome can occur via transposons. An example of a synthetic transposon for use is the Sleeping Beauty transposon that comprises an expression cassette including the appropriate gene of active fragment thereof. 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.

[0361] A construct encoding one or more of a CD43 polypeptide, one or more of the tolerogenic factors, and a CAR described herein can be introduced into the host cell using a lentiviral delivery system. A construct encoding one or more of a CD43 polypeptide, one or more of the tolerogenic factors, and a CAR described herein can be introduced into the host cell using a retroviral delivery system.

[0362] The host cells described herein 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 aPT cells. The host cells can be y5T cells. The host cells can be V51 70T cells. The host cells can be V52 yoT cells. The host cells can be V53 y5T cells. The host cells can be tumorinfiltrating lymphocytes (TIL). The host cells can be NK cells (e.g., primary NK cells). The human NK cells can be purified from commercialized PBMCs. The host cells can be PBNK cells. The host cells can be expanded and / or activated before use. The host cells can be PBMCs isolated from healthy donors.

[0363] Allogeneic Cell or Allogeneic Cells

[0364] In one aspect, the present disclosure provides allogeneic cells (or allogenic 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 y5 T cell or an 0 T cell.

[0365] 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. In some embodiments, the allogeneic T cell has a normal expression level (e.g., at least 50%, 60%, 70%, 80%, 90%, 100%. 110%, 120%, 130%, 140% or 150% as compared to that of a wildtype T cell) of endogenous HLA Class I and / or HLA Class II molecules.

[0366] 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 Atorney Docket No.: 51624-0096W01 / LG-

[0367] U2024141WO 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. CD36, CD3e and / or y-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.

[0368] In some aspects, the T cell or NK cell described herein can be, e.g., engineered such that it does not express a functional HL A 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).

[0369] In some aspects, the T cell or NK cell described herein can express a functional HLA on its surface. For example, the expression of cell surface HLA, e.g., HLA Class I and / or HLA Class II, are not eliminated or reduced in the engineered cells. In some embodiments, the beta-2-microglobulin (B2M) gene of the engineered cell is not genetically modified.

[0370] In some aspects, the cell can lack a functional TCR and a functional HLA, e.g., HLA Class I and / or HLA Class II. Engineered cells that lack expression of a functional TCR and / or HLA can be obtained by any suitable means, including a knockout or knock down of one or more subunit of TCR or HLA. For example, the T cell or NK cell can include a knock down of TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription-activator like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0371] Methods of Treatment

[0372] The CD43 polypeptide described herein, the polynucleotides described herein and the engineered cells described herein can be used in a variety of experimental, therapeutic and commercial applications.

[0373] In one aspect, the disclosure provides a method of modulating an immune response comprising administering an effective amount of engineered cells described herein to a subject in need thereof. Atorney Docket No.: 51624-0096W01 / LG-

[0374] U2024141WO

[0375] In one aspect, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject, an effective amount of the engineered cell described herein.

[0376] In one aspect, the disclosure provides a method of inhibiting immune clearance of an engineered cell in a subject, comprising: (a) overexpressing a CD43 polypeptide (e.g., human CD43 or a portion thereof) in the engineered cell (e.g., introducing a vector expressing an exogenous CD43 polypeptide into the engineered cell); and (b) administering the engineered cell from step (a) to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell.

[0377] In one aspect, the disclosure provides a method of increasing the in vivo expansion of an engineered cell in a subject, comprising: (a) overexpressing a CD43 polypeptide (e.g., human CD43 or a portion thereof) in the engineered cell (e.g., introducing a vector expressing an exogenous CD43 polypeptide into the engineered cell); and (b) administering the engineered cell from step (a) to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell.

[0378] In one aspect, the disclosure provides a method of increasing persistence and / or function of an engineered cell in a subject, comprising: (a) overexpressing a CD43 polypeptide (e.g., human CD43 or a portion thereof) in the engineered cell (e.g., introducing a vector expressing an exogenous CD43 polypeptide into the engineered cell); and (b) administering the engineered cell from step (a) to a subject in need thereof, thereby- increasing the persistence and / or function of the engineered cell.

[0379] The method may further comprise contacting the engineered cell with glycosidase between step (a) and step (b). The glycosidase may be N-glycosidase. The glycosidase is selected from Peptide-N-Glycosidase F (PNGase F), Peptide-N-Glycosidase A (PNGase A), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase Fl (Endo Fl), Endoglycosidase F2 (Endo F2) and Endoglycosidase F3 (Endo F3), and any combination thereof. The glycosidase may be PNGase F.

[0380] The term “effective amount" as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired results.

[0381] In another aspect, the present disclosure provides a method for treating cancer comprising administering an effective amount of engineered cells described herein to a subject in need thereof. Examples of cancer that can be treated include, but are not limited to, leukemias including chronic lymphocytic leukemia, chronic myelogenous leukemia, acute Atorney Docket No.: 51624-0096W01 / LG-

[0382] U2024141WO 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.

[0383] The disclosure further includes the use of the engineered cells described herein in the manufacture of a medicament or pharmaceutical composition to modulate an immune response, to treat an infection or to treat cancer as described hereinabove.

[0384] The engineered cells can also be used in experimental models, for example, to further study and elucidate the function of the cells.

[0385] One or more of the engineered cells described herein can be administered to a subject in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions or injections, or subcutaneous injections. In some cases, the engineered cells can expand within a subject's body, in vivo, after administration to a subject. The engineered cells can be frozen to provide cells for multiple treatments with the same cell preparation. The engineered 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 engineered cells and compositions comprising the same.

[0386] In one aspect, the present disclosure provides a method of treatment that comprises administering to a subject a therapeutically-effective amount of the engineered cells.

[0387] The cells can be administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell ty pes. Thus, the dosage of cells may be based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types. The dosage of cells may be based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell ty pes. The dosage may7be based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations. The engineered cells described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the engineered cells can vary. For example, the engineered cells can be used as a prophylactic and can be administered continuously to subjects with a propensity7to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The engineered cells can be administered to a subject during or as soon as possible Atorney Docket No.: 51624-0096W01 / LG-

[0388] U2024141WO after the onset of the symptoms. The administration of the engineered 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 engineered cells of the disclosure can be an intravenous administration. One or multiple dosages of the engineered cells can be administered as soon as is practicable after the onset of a cancer or an infectious disease, and for a length of time necessary for the treatment of the disease, such as. for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. For the treatment of cancer, one or multiple dosages of the engineered cells can be administered years after onset of the cancer and before or after other treatments. The engineered 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.

[0389] Methods for administration of engineered cells for adoptive cell therapy are know n 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. Atorney Docket No.: 51624-0096W01 / LG-

[0390] U2024141WO

[0391] 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. The second subject may express a high level of NK.G2C and / or a low level of NKG2A.

[0392] 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.

[0393] 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.

[0394] 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.

[0395] The engineered cells described herein can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the engineered cells can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the engineered 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. Atorney Docket No.: 51624-0096W01 / LG-

[0396] U2024141WO

[0397] 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 or a hematological tumor. The cancer can be a carcinoma. The cancer can be a sarcoma. The cancer can be a leukemia. The cancer can be a solid tumor.

[0398] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different ty pes 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).

[0399] 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, sw eat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, Atorney Docket No.: 51624-0096W01 / LG-

[0400] U2024141WO 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.

[0401] 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.

[0402] The engineered cells (e.g., immune cells, T cells, or NK cells) described herein can be included in a composition for immunotherapy. The composition can include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the engineered cells can be administered.

[0403] The engineered 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.

[0404] The engineered cells disclosed herein can be formulated in unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage forms comprise additional lymphocytes. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative or without a preservative. In some examples, the pharmaceutical composition does not comprise a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

[0405] EXAMPLES

[0406] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Atorney Docket No.: 51624-0096WO1 / LG-

[0407] U2024141WO

[0408] Materials and methods

[0409] Plasmids

[0410] Nucleic acid sequences encoding human CD43 (SEQ ID NO: 2), human CD47 (SEQ ID NO: 5), HLA-E trimeric construct (SEQ ID NO: 14), and / or BCMA-CAR (SEQ ID NO: 4) were inserted into the GFP section of the Sin-CMV plasmid (Biovec Pharma).

[0411] Cell lines

[0412] The 293Vec-BaEV stable cell line was obtained from Biovec Pharma. The 293 Vec- BaEV cells were cultured in the DMEM (Gibco™) medium supplemented with 10% Fetal Bovine Serum (FBS; Gibco™) at 37°C in a humidified atmosphere with 5% CO2.

[0413] K562 cells (ATCC) were cultured using the RPMI 1640 (Gibco™) medium supplemented with 10% FBS (Gibco™) at 37°C in a humidified atmosphere with 5% CO2.

[0414] Healthy blood donors

[0415] Peripheral blood mononuclear cells (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 the CryoStor® CS5 Cell Freezing Medium (Stemcell Technologies) and stored in a liquid nitrogen tank. yb T cell expansion

[0416] 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 yo 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 1 below.

[0417] Table 1. Atorney Docket No.: 51624-0096WO1 / LG-

[0418] U2024141WO

[0419] PBNK cell expansion

[0420] The PBNK cells (peripheral blood natural killer cells) were expanded in a feeder-free system.

[0421] Primary NK cell activation

[0422] Primary’ NK cells were isolated from PBMCs using an NK cell isolation kit (Stemcell Technologies). The isolated NK cells were activated by 1000U IL-2 (Gibco™) for three days.

[0423] Retroviral packaging and transduction

[0424] The 293Vec-BaEV cells were seeded in a T225 culture flask (Falcon®) at a density of 2 x io7cells the day before transfection. The 293Vec-BaEV cells were transfected with the retroviral plasmid Sin-CMV containing one or more immune tolerance sequences (e.g., nucleic acid sequences encoding human CD43, human CD47, and / or the HLA-E trimeric construct) and / or one or more nucleic acid sequences encoding the BCMA-CAR using Lipofectamine™ 3000 (Life Technology) for 6 hours at 37°C in 5% CO2 according to the manufacturer's protocol. After 48-72 hours, supernatants containing retroviral particles were harvested and filtered through a 0.45 pm filter (EMD Millipore) to remove cell debris. The supernatants were concentrated using a Retro-X concentrator (Takara) according to the manufacturer's protocol. The retroviral particle pellet was re-suspended in cold PBS and stored at -80°C. The viral titers of the concentrated retroviral particles were measured by Atorney Docket No.: 51624-0096W01 / LG-

[0425] U2024141WO infecting primary T cells seeded at a density of 1 x 106cells / well in a 24-well plate (VWR) with viral serial dilutions. Three days later, gene expression (e.g.. expression of human CD43, CD47, HLA-E trimeric construct, and / or BCMA-CAR) was detected by flow cytometry and the viral titer w as calculated using the following equation: viral titer (Tu / pl) = (percentage of gene+ cells x number of cells transduced) / virus volume.

[0426] For retroviral transduction. K562 cells or PBMCs stimulated with zoledronic acid and IL-2 were seeded at 5 x io6cells in 2 ml Gibco™ CTS™ OpTmizer™ T Cell Expansion SFM (Gibco™, Cat#: A1048501) in 6-well plates (VWR), and the concentrated retroviral particles were added at MOI (multiplicity of infection) = 1. The cell and virus mixture were spin-inoculated for 2 hours at 2,500 rpm and 32°C. After centrifugation, the plate was put back into an incubator (37°C, 5% CO2) overnight. The medium was replaced on the second day. After a 3-day culture thereafter, the cells were collected, and expression of the immune tolerance sequences and / or sequences encoding the BCMA-CAR was detected by flowcytometry, as described above.

[0427] Immunophenotyping yo 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 1. Specifically, y5 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 1) 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).

[0428] Flow cytometry-based cytotoxicity assays

[0429] Target cells (K562 cells or primary’ 76T cells, with or without any of the modifications described herein) were harvested and re-suspended in RPMI media containing 10% FBS and counted. Effector cells (expanded NK cells or activated primary' NK cells) were harvested and re-suspended in RPMI media (Gibco™) containing 10% FBS (Gibco™) and counted. After target cell seeding, the effector cells w ere added and incubated with the target cells for 48 hours. After the 2-day co-culture. cells were harvested and counted. The cell mixture was Atorney Docket No.: 51624-0096W01 / LG-

[0430] U2024141WO washed and stained with antibodies targeting HLA-A2 for 45 minutes at 4°C. Immediately prior to analysis, 7-AAD (BD Biosciences) was added to the cells to differentiate live cells and dead cells. After 5 minutes of incubation, samples were analyzed for viable target cells using a Cytek" Aurora 5L (UV-V-B-YG-R) cytometer. Cytotoxicity was calculated by subtracting background cell death of each target cell type from the experimental samples.

[0431] MLR (mixed lymphocyte reaction) assays

[0432] Allogeneic primary PBMCs (HLA-A2+) were freshly thawed, counted and resuspended in RPMI media containing 10% FBS. K562 cells or B2M KO gdT cells (HLA-A2- ) that expressed CD43. CD47, the HLA-E trimeric construct, and / or BCMA-CAR were harvested, counted and re-suspended in RPMI media containing 10% FBS. The primary PBMCs were co-cultured with the K562 cells or B2M KO gdT cells at an E:T ratio of 100: 1, 50: 1, or 20: 1 for 7 days. The cell medium was changed every two or three days. On Day 4 (or Day 5) or Day 7, cells were sampled for counting and flow cytometry analysis.

[0433] Example 1. CD43-overexpressing K562 cells are resistant to NK cell-mediated killing

[0434] K562 cells are HLA-A2 negative cells and very sensitive to NK cell-mediated killing owing to the low expression levels of MHC Class I molecules. Here, we transduced K562 cells with retroviral particles RV-CD43-BFP, RV-CD47-BFP, and RV -HLA-E that express CD43, CD47, and the HLA-E trimeric construct, respectively. The expression of all constructs in K562 cells was above 95% (FIGS. 1A-1C) CD43 -overexpressing K562 cells or other immune tolerance protein-overexpressing K562 cells (e.g., K562 cells expressing CD43, CD47, or the HLA-E trimeric construct) were co-cultured with expanded PBNK cells for 2 days, with or without 400U IL-2. After the 2-day co-culture, NK cells exhibited a strong killing capability on K562 cells even at an E:T ratio of 1 :3 (FIGS. 1D-1G). It has been reported that CD47 needs a high cell surface expression level (e.g., a high MFI) to achieve good protective effects. As shown in FIG. 1C, the MFI of CD47 in CD47-overexpressing K562 cells was about 21 -fold higher as compared to that of untransduced K562 cells. As shown in FIGS. ID- IE, overexpression of CD47 showed a protective effect (e.g., about 50% in FIG. ID) from PBNK cell-mediated killing as compared to untransduced K562 cells, while overexpression of the HLA-E trimeric construct and CD43 dramatically improved the protective effect as compared to the overexpression of CD47. The cell number of K562 cells overexpressing the HLA-E trimeric construct and CD43 dramatically increased as compared Atorney Docket No.: 51624-0096W01 / LG-

[0435] U2024141WO to that of K562 cells overexpressing CD47, especially under the condition with IL-2 (FIGS. 1F-1G). Under the condition with IL-2 (FIG. 1G), there was a minimal protective effect by overexpressing CD47, whereas overexpression of the HLA-E trimeric construct and CD43 still showed a dramatic protective effect on NK cell-mediated killing. As shown in FIG. IE, the cell number of K562 cells overexpressing the HLA-E trimeric construct and CD43 after co-culture with PBNK cells without IL-2 for 2 days increased as compared to the initial cell seeding density (0.6 x io6cells / well). In addition, as shown in FIG. 1G, the cell number of CD43 -overexpressing K562 cells after co-culture with PBNK cells with 400U IL-2 for 2 days also increased as compared to the initial cell seeding density (0.6 x io6cells / well). Therefore, overexpression of the HLA-E trimeric construct and CD43 are resistant to PBNK-mediated killing at an E:T ratio of 1 :3. and CD43-overexpressing K562 cells showed an improved protective effect as compared to the HLA-E trimeric construct-o verexpressing K562 cells in the presence of IL-2.

[0436] Example 2. CD43-overexpressing K562 cells are resistant to primary NK cell-mediated killing

[0437] Primary NK cells (from Donor 990, HLA-A2+) were activated with 1000U IL-2 for 3 days. The activated primary NK cells were harvested, counted and co-cultured with CD43- overexpressing K562 cells or the HLA-E trimeric construct-overexpressing K562 cells for 2 days with or without 400U IL-2 at an E:T ratio of 2:3. As shown in FIGS. 2A-2C, overexpression of the HLA-E trimeric construct and CD43 exhibited a dramatic protective effect from primary NK cell-mediated killing as compared to untransduced K562 cell, either with or without IL-2. The cell number of K562 cells overexpressing the HLA-E trimeric construct or CD43 dramatically increased as compared to that of untraduced K562 cells (FIG. 2C). With respect to the co-culture with primary NK cells, no significant difference w as observed on the cell number of the HLA-E trimeric construct- or CD43-overexpressing K562 cells with or without IL-2 (FIG. 2C). Further, the cell number of the HLA-E trimeric construct- and CD43-overexpressing K562 cells either increased or maintained as compared to the initial cell seeding density (0.6 x io6cells / well). The results showed that the protective effect of CD43 overexpression is comparable to overexpression of the HLA-E trimeric construct (FIGS. 2A-2C). The results indicate that overexpression of the HLA-E trimeric Atorney Docket No.: 51624-0096W01 / LG-

[0438] U2024141WO construct and CD43 can confer resistance to primary NK cell-mediated killing an E:T ratio of 2:3.

[0439] Example 3. CD43 overexpression reverses the inhibitory effects of PBMCs on K562 cell growth

[0440] Three batches of primary PBMCs were isolated from HLA-A2 positive donors (from Donor 807. Donor 990, and Donor 987, respectively). These primary PBMCs were freshly thawed, counted and co-cultured with the HLA-E trimeric construct- or CD43-overexpressing K562 cells for 7 days at an E:T ratio of 100:1. For PBMCs from Donor 807, two groups of cells were prepared: one group contained PBMCs directly isolated from the donor, while the other group only contained CD3-depleted PBMCs. At the E:T ratio of 100: 1. PBMCs inhibited growth of K562 cells in different degrees owing to the donor-specific variation of immune cell composition (FIGS. 3A-3F). On Day 4, the inhibition of K562 cell growth by PBMCs was stronger than that on Day 7. Interestingly, the inhibitory effect of CD3-depleted D807 PBMCs (FIG. 3D) was weaker as compared to that of D807 PBMCs (FIG. 3C). The results indicate that CD3-positive T cells may play an important role in the inhibition of K562 cell growth.

[0441] In addition, overexpression of CD43 and the HLA-E trimeric construct in K562 cells reversed the inhibitory effect of PBMCs and promoted the growth of K562 cells. In particular, overexpression of CD43 achieved a similar or even better effect on K562 cell growth as compared to overexpression of the HLA-E trimeric construct (FIGS. 3C-3F).

[0442] Example 4. CD43 overexpression protects gdT cells from NK cell-mediated killing

[0443] Human ty pe II gdT cells were expanded under a condition using zoledronic acid (Sigma) and IL-2 (Life Technology) in the CTS™ OpTmizer™ T Cell Expansion medium (Fisher Scientific). Specifically, the gdT cells were transduced to overexpress BCMA-CAR and CD43, respectively. The retrovirus particles used for transduction were obtained by transfecting 293Vec-BaEV cells with a plasmid encoding BCMA-CAR and CD43. under control of the same promoter. Sequences encoding BCMA-CAR and CD43 were linked by a sequence encoding a P2A peptide. Thus, the expression level of BCMA-CAR can indicate CD43 expression.

[0444] As shown in FIG. 4A, the expression levels of BCMA-CAR and CD43 in gdT cells (from Donor 811, HLA-A2-) were very high (around 90%). It has been shown that gdT cells Atorney Docket No.: 51624-0096W01 / LG-

[0445] U2024141WO are not sensitive to NK cell-mediated killing. To improve the sensitivity, B2M gene was knocked out in gdT cells. As shown in FIG. 4A, the knockout (KO) efficiency (as reflected by the low expression of HLA-ABC) of B2M gene in gdT cells was very high (above 89%) in each individual sample. B2MKO gdT cells with or without overexpression of BCMA CAR and CD43 were co-cultured with expanded PBNK cells at different E:T ratios for 2 days (FIG. 4B). After the 2-day co-culture (i.e., Day 4), cells were harvested, counted and analyzed by flow cytometry to determine the killing capability of PBNK cells.

[0446] As shown in FIG. 4C, overexpression of BCMA CAR and CD43 can protect gdT cells from NK cell-mediated killing.

[0447] Example 5. CD43 overexpression prevents gdT cells from allogeneic PBMC-mediated rejection

[0448] Three different HLA-A2+ allogeneic PBMCs (from Donor 298, Donor 807, and Donor 990) were co-cultured with human B2M KO gdT cells (from Donor 811, HLA-A2-) overexpressing BCMA-CAR and CD43 as described in Example 4 at two different E:T ratios (50: 1 or 20: 1) for 7 days. On Day 5 and Day 7, small quantities of cells were sampled for cell number counting and analysis of viable HLA-A2- gdT cells (FIGS. 5A-5D). The protective effect from CD43 overexpression was more obvious when the E:T ratio was 20: 1 as compared to that when the E:T ratio was 50: 1. In addition, the allo-rej ection was stronger after the 7-day co-culture, especially in the co-culture with D807 PBMCs (FIG. 5B) at the E:T ratio of 50: 1.

[0449] Donor variations were observed in the MLR response. In particular, the immune rejection was stronger when the human B2M KO gdT cells were co-cultured with D807 PBMCs. Compared with the UTD (untransduced) control, overexpression of BCMA-CAR and CD43 dramatically increased the number of viable gdT cells in all three allogeneic donor co-culture system at both E:T ratios of 50:1 and 20: 1.

[0450] Example 6. CD43 overexpression protects gdT cells from NK cell-mediated killing

[0451] Human type II gdT cells (from donor RG1754) were expanded according to methods described above. As shown in FIG. 6A, the purity of gdT cells (as reflected by the percentage of CD3+ / Vdelta2+ cells) was above 90%. As shown in FIG. 6B, the expression levels of BCMA-CAR in the BCMACAR B2MKO gdT cells (expressing BCMA-CAR only) and BCMACAR-CD43 B2M KO gdT cells (expressing BCMA-CAR and CD43 from the same Atorney Docket No.: 51624-0096W01 / LG-

[0452] U2024141WO vector) were similar. It has been shown that gdT cells are not sensitive to NK cell-mediated killing. To improve the sensitivity, B2M gene was knocked out in gdT cells. As shown in FIG. 6C, the knockout (KO) efficiency (as reflected by the low expression of HLA-ABC) of B2M gene in gdT cells was very high (above 87%) in each individual sample. BCMA-CAR- expressing B2M KO gdT cells with or without overexpression of CD43 were co-cultured with in vitro expanded PBNK cells (from donor DI 5 or D398) at an E:T ratio of 1:3 for 2 days. After the 2-day co-culture. cells were harvested, counted and analyzed by flow cytometry to determine the killing capability of PBNK cells. As shown in FIGS. 6D-6F, BCMACAR- CD43 B2M KO gdT cells showed a higher survived cell number as compared to B2M KO gdT cells that only expressed BCMA-CAR. Further, only the cell number of the BCMACAR- CD43 B2MKO gdT cells increased as compared to the initial cell seeding density (0.6 x io6cells / well). The results indicate that overexpression of CD43 can protect B2MK0 gdT cells from NK cell-mediated killing.

[0453] Example 7. CD43 overexpression prevents gdT cells from allogeneic PBMC-mediated rejection

[0454] The HLA-A2+ allogeneic PBMCs (from donor 990 (D990)) were co-cultured with human B2M KO gdT cells (from donor RG1754, HLA-A2-) as described in Example 6 at an E:T ratio of 20: 1 for 7 days. On Day 4 and Day 7, small quantities of cells were sampled for cell number counting and analysis of viable HLA-A2- gdT cells (FIGS. 7A-7B). Compared with BCMACAR B2M KO gdT cells or B2M KO Mock cells, more survived target cells (HLA-A2-) were detected when BCMACAR-CD43 B2MKO gdT cells were co-cultured with PBMCs (FIG. 7C). The results indicate that CD43 expression confers a protective effect against allogeneic PBMC-mediated rejection. Donor variations in the MLR response were observed (data not shown).

[0455] Example 8. CD43 overexpression enhances the protective function of the HLA-E trimeric construct

[0456] B2MKO gdT cells were transduced with retroviral particles BCMACAR-HLA-E, BCMACAR-CD43 and BCMACAR-CD43-HLA-E, respectively. The obtained cells were named BCMACAR-HLA-E B2MKO gdT cells (overexpressing BCMA-CAR and the HLA- E trimeric construct), BCMACAR-CD43 B2M KO gdT cells (overexpressing BCMA-CAR Atorney Docket No.: 51624-0096W01 / LG-

[0457] U2024141WO and CD43), and BCMACAR-CD43-HLA-E B2MKO gdT cells (overexpressing BCMA- CAR, CD43. and the HLA-E trimeric construct), respectively.

[0458] As shown in FIG. 8A, the purity of B2M KO gdT cells was above 80% after the expression of each individual construct. The expression levels of BCMA-CAR and the HLA- E trimeric construct were also measured. As shown in FIG. 8B, the expression level of BCMA-CAR was above 60%, and the expression level of the HLA-E trimeric construct was around 15%. The expression level of BCMA-CAR in BCMACAR-CD43 B2M KO gdT cells was slightly higher than that of other cells. The expression level of BCMA-CAR in BCMACAR B2MKO gdT cells was about 62%, similar to that of BCMACAR-HLA-E B2M KO gdT cells. As shown in FIG. 8C, the knockout efficiency of B2M gene was above 87%.

[0459] Two groups of in vitro expanded NK cells were used to evaluate the protective function of overexpression of the HLA-E trimeric construct, CD43, or their combination in B2MKO gdT cells. Specifically, B2M KO gdT cells expressing the HLA-E trimeric construct, CD43, or their combination were co-cultured with two groups of in vitro expanded PBNK cells (from donor D15 or D398) at an E:T ratio of 1 :3 for 2 days. After the 2-day coculture, cells were harvested, counted and analyzed by flow cytometry to determine the killing capability of PBNK cells. As shown in FIGS. 8D-8F, BCMACAR-HLAE-CD43 B2M KO gdT cells showed more survived cells as compared to BCMACAR-HLAE or BCMACAR-CD43 B2MKO gdT cells. The results indicate that cells overexpressing both the HLA-E trimeric construct and CD43 may have a stronger protective function from NK cell- mediated killing as compared with those expressing either the HLA-E trimeric construct or CD43.

[0460] To determine whether overexpression of both the HLA-E trimeric construct and CD43 has better protective effects from primary PBMC-mediated rejection as compared to overexpression of either one of them, tw o HLA-A2+ allogeneic PBMCs (from donors D987 or D990) w ere co-cultured with human B2M KO gdT cells (from donor RG1754, HLA-A2-) overexpressing the HLA-E trimeric construct, CD43, or their combination at an E:T ratio of 20: 1 for 7 days. On Day 4 and Day 7, small quantities of cells were sampled for cell number counting and analysis of viable HLA-A2- B2MKO gdT cells. In D990 co-culture condition (FIG. 8H), compared with B2M KO gdT cells overexpressing either the HLA-E trimeric construct or CD43, more survived target cells (HLA-A2-) were detected when BCMACAR- HLA-E-CD43 B2M KO gdT cells were co-cultured with PBMCs. In D987 co-culture condition (FIG. 8G), B2M KO gdT cells overexpressing the HLA-E trimeric construct did Atorney Docket No.: 51624-0096W01 / LG-

[0461] U2024141WO not exhibit any protective effects against PBMC-mediated rejection, whereas cells overexpressing CD43 showed good protective effects against PBMC-mediated rejection. Overexpression of both the HLA-E trimeric construct and CD43 also exhibited some protective effects against PBMC-mediated rejection, as compared with cells overexpressing the HLA-E trimeric construct only. As shown in FIGS. 81-8 J, the protective effect of the HLA-E trimeric construct showed donor variations. Overexpression of CD43 enhanced the protective effect of the HLA-E trimeric construct, especially when the HLA-E trimeric construct failed to exhibit any protective effects.

[0462] HLA-E predominantly presents peptides derived from HL A Class I signal peptides and is the ligand for the C-type lectin NKG2 / CD94 receptor family on NK cells. Regular HLA Class I expression can therefore be monitored by NK cells. In particular, HLA-E is recognized by either the inhibitory NKG2A / CD94 or the stimulatory NKG2C / CD94 receptor and, therefore, plays a critical role in the balance between cell protection and attack. It has been found that the effectiveness of overexpression of the HLA-E trimeric construct in the suppression of NK cell rejection is highly correlated with the expression of NKG2A and NKG2C on the host NK cells. As shown in FIGS. 8K-8L, primary NK cells isolated from different donors (D987 and D990, respectively) showed different expression levels of NKG2A and NKG2C. Generally, host NK cells with high NKG2C expression can rapidly recognize and kill allogeneic CAR-T cells even when the HLA-E trimeric construct is overexpressed. However, co-overexpressing the HLA-E trimeric construct and CD43 as disclosed herein can synergistically overcome this challenge, providing significant protection regardless of host NK cells' NKG2C expression level. More specifically, in hosts with a high NKG2C expression level, overexpression of the HLA-E trimeric construct may have little protective effect against host rejection, but overexpression of both the HLA-E trimeric construct and CD43 can significantly enhance this protection. In hosts with high NKG2A and low NKG2C levels, HLA-E may be effective in preventing host rejection, and simultaneous overexpression of CD43 can have a degree of synergistic effect. Thus, overexpression of both the HLA-E trimeric construct and CD43 can overcome differences in the protective effect Atorney Docket No.: 51624-0096W01 / LG-

[0463] U2024141WO against host rejection caused by the difference of NKG2A and / or NKG2C expression levels in different hosts.

[0464] In conclusion, the results indicate that CD43 can enhance the protective function of the HLA-E trimeric construct from both PBNK- and allogeneic PBMC-mediated rejection.

[0465] Example 9. Changes in lectin staining mean fluorescence intensity (MFI) in CD43- overexpressing K562 cells following glycosidases treatment

[0466] Five different enzymes were employed in the experiment to investigate their effects on glycoprotein glycosylation: O-Glycosidase, which catalyzes the cleavage of Core 1 and Core 3 O-linked disaccharide moieties from glycoproteins; PNGase F, a highly efficient amidase that removes nearly all N-linked oligosaccharides by cleaving the amide bond between the innermost N-acetylglucosamine (GlcNAc) residue and asparagine, acting on high-mannose, hybrid, and complex glycan structures; Neuraminidase (Sialidase), which exerts hydrolytic activity toward terminal sialic acid residues on glycoproteins; Galactosidase, which facilitates the hydrolytic degradation of galactoside linkages; and Acetyl Hexosaminidase, which catalyzes the cleavage of terminal 0-D-N-acetyl- galactosamine and glucosamine residues from oligosaccharide chains.

[0467] CD43-overexpressing K562 cells were seeded in 96-well plates and treated overnight with gradient concentrations of the five enzymes. Following incubation, cells were harvested, washed thrice with phosphate-buffered saline (PBS), and subjected to staining with three lectins exhibiting distinct glycan-binding specificities: Concanavalin A (ConA), which binds to mannose residues on glycoproteins; Lycopersicon esculentum Lectin (LEL), which exhibits affinity' for N-acetylglucosamine (GlcNAc) oligomers; and Sambucus Nigra Agglutinin (SNA), which specifically recognizes sialic acid residues, with preferential binding to a(2,6)-linked sialic acids attached to terminal galactose.

[0468] Enzymatic treatment induced significant alterations in glycosylation patterns of CD43 -overexpressing K562 cells, as evidenced by changes in lectin binding mean fluorescence intensity (MFI). PNGase F treatment resulted in a pronounced reduction in MFI across all three lectins, consistent with its role in removing nearly all N-linked oligosaccharides (FIGS. 9A-C). For ConA staining, MFI remained unchanged following treatment with O-Glycosidase, Neuraminidase, and Galactosidase, indicating these enzymes do not affect mannose residues targeted by ConA, while reductions were observed only with PNGase F and Acetyl Hexosaminidase (FIG. 9A). In LEL staining, dramatic MFI decreases Atorney Docket No.: 51624-0096W01 / LG-

[0469] U2024141WO occurred with PNGase F, Galactosidase, and Acetyl Hexosaminidase treatments, while O- Glycosidase and Neuraminidase had no significant effect (FIG. 9B). For SNA staining, PNGase F treatment caused a modest MFI reduction, whereas Neuraminidase induced a substantial MFI increase, and the remaining enzymes (O-Glycosidase, Galactosidase, Acetyl Hexosaminidase) did not elicit significant changes in SNA binding (FIG. 9C).

[0470] Example 10. The effect of de-glycosylation on CD43 functional activity

[0471] Following enz me treatment, CD43-overexpressing K562 cells exhibited altered glycosylation patterns. To investigate whether these modifications influence CD43 function, CD43 -overexpressing K562 cells were treated overnight with three selected enzymes (PNGase F, Neuraminidase, or Acetyl hexosaminidase). Post-treatment, cells were washed three times with RPMI medium supplemented with 10% fetal bovine serum (FBS) and cocultured for 4 hours with peripheral blood mononuclear cell (PBMC)-expanded natural killer (NK) cells (day 15, HLA-A2+) at varying effector-to-target (E:T) ratios. CD43-mediated protective function was subsequently analyzed via flow cytometry.

[0472] K562 cells were susceptible to NK cell-mediated cytotoxicity in the absence of glycosidases treatment, whereas CD43 overexpression significantly impaired NK cell killing capacity in the absence of glycosidases treatment (FIGS. 10A-B). Notably, PNGase F treatment enhanced CD43's protective function, as evidenced by increased survival of CD43- overexpressing K562 cells and further inhibition of NK cell cytotoxicity (FIGS. 10A-B). Conversely, Neuraminidase treatment completely abrogated CD43-mediated protection: survival rates of CD43-overexpressing K562 cells and NK cell killing capacity were comparable to those observed in K562 cells (FIGS. 10A-B). Acetyl hexosaminidase treatment did not affect CD43 function (FIGS. 10A-B). These findings indicate that cleavage of sialic acid residues from CD43 by Neuraminidase abolishes its protective role in NK cell- mediated cytotoxicity assays, whereas PNGase F enhances the protective function of CD43.

[0473] OTHER EMBODIMENTS

[0474] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An engineered cell that overexpresses a CD43 polypeptide.

2. The engineered cell of claim 1, wherein the engineered cell further overexpresses one or more tolerogenic factors.

3. The engineered cell of claim 2, wherein the one or more tolerogenic factors are selected from the group consisting of HLA-E, CD47, CD24, CD26, CD27, CD31, CD35, CD200, HLA-C, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl -Inhibitor, IL- 10, IL-35, FASL, DUX4, CCL21, MFGE8, SERPINB9, and any combination thereof.

4. The engineered cell of claim 2 or 3, wherein the one or more tolerogenic factors comprise HLA-E.

5. The engineered cell of claim 4, wherein the HLA-E is a single-chain fusion HLA Class I protein.

6. The engineered cell of claim 5, wherein the single-chain fusion HLA Class I protein comprises at least a portion of B2M protein and at least a portion of HLA-E heavy chain (e.g., HLA-E*01 :01 heavy chain or HLA-E*01 :03 heavy chain).

7. The engineered cell of claim 5 or 6, wherein the engineered cell further comprises a peptide antigen that is presented by the single-chain fusion HLA Class I protein on the cell surface; or wherein the single-chain fusion HLA Class I protein further comprises a peptide antigen that is presented by the single-chain fusion HLA Class I protein on the cell surface, optionally the peptide antigen is a peptide derived from HLA-G or HLA-C.

8. The engineered cell of any one of claims 5-7, wherein the single-chain fusion HLA Class I protein comprises an amino acid sequence set forth in SEQ ID NO: 14 or a sequence that is atAtorney Docket No.: 51624-0096WO1 / LG-U2024141WO least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 14.

9. The engineered cell of any one of claims 1-8, wherein the one or more tolerogenic factors comprise CD47 (e g., human CD47) or a variant thereof.

10. The engineered cell of claim 9, wherein the CD47 or the variant thereof comprises an amino acid sequence set forth in SEQ ID NO: 5, 6, or 7, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 5, 6, or 7.

11. The engineered cell of any one of claims 1-10, wherein the engineered cell expresses endogenous Major Histocompatibility Complex (MHC).

12. The engineered cell of any one of claims 1-11, wherein the expression level of endogenous MHC Class I molecules and / or MHC Class II molecules are the same or comparable to that of a wildtype cell of the same type as the engineered cell.

13. The engineered cell of any one of claims 1-12, wherein the expression of endogenous MHC Class I molecules and / or MHC Class II molecules are not eliminated or reduced in the engineered cell.

14. The engineered cell of any one of claims 1-13, wherein the beta-2-microglobulin (B2M) gene of the engineered cell is not genetically modified.

15. The engineered cell of any one of claims 1-14, wherein the expression level of the CD43 polypeptide on the engineered cell is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2- fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, atAtorney Docket No.: 51624-0096WO1 / LG-U2024141WO least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 2000-fold, at least 5000-fold, or at least lOOOO-fold as compared to the expression level of CD43 (e.g., endogenous CD43) on a wildtype cell or a control cell.

16. The engineered cell of any one of claims 1-15, wherein the expression level of the CD43 polypeptide on the engineered cell is at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 1-fold, about 1-fold to about 2-fold, about 2-fold to about 3 -fold, about 3 -fold to about 4- fold, about 4-fold to about 5-fold, about 5-fold to about 6-fold, about 6-fold to about 7-fold, about 7-fold to about 8-fold, about 8-fold to about 9-fold, about 9-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40- fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600- fold, about 600-fold to about 700-fold, about 700-fold to about 800-fold, about 800-fold to about 900-fold, about 900-fold to about 1000-fold, about 1000-fold to about 2000-fold, about 2000-fold to about 5000-fold, or about 5000-fold to about 10000-fold as compared to the expression level of CD43 (e.g., endogenous CD43) on a wildtype cell or a control cell.

17. The engineered cell of any one of claims 1-16, wherein the engineered cell is an allogeneic cell isolated from a donor for being administered to a subject.

18. The engineered cell of any one of claims 1-17, wherein the CD43 polypeptide comprises an extracellular region of CD43, a transmembrane region of CD43, and / or an intracellular region of CD43.

19. The engineered cell of claim 18, wherein the extracellular region of CD43 comprises an amino acid sequence set forth in SEQ ID NO: 16 or a sequence that is at least 80%, 85%,Atorney Docket No.: 51624-0096WO1 / LG-U2024141WO90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 16; wherein the transmembrane region of CD43 comprises an amino acid sequence set forth in SEQ ID NO: 17 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 17; wherein the intracellular region of CD43 comprises an amino acid sequence set forth in SEQ ID NO: 18 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 18.

20. The engineered cell of any one of claims 1-19, wherein the CD43 polypeptide comprises a full-length CD43 protein (e.g., human CD43).

21. The engineered cell of any one of claims 1-20, wherein the CD43 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

22. The engineered cell of any one of claims 1-21, wherein the engineered cell is an immune cell (e g., a T cell, a natural killer (NK) cell, a B cell, a monocyte, or a macrophage).

23. The engineered cell of claim 22, wherein the immune cell is selected from the group consisting of a T cell, a NK cell, and a combination thereof.

24. The engineered cell of claim 22 or 23, wherein the immune cell is selected from the group consisting of a natural killer T (NK-T) cell, a yd T cell, an a T cell, a Treg cell, and a NK cell.

25. The engineered cell of any one of claims 22-24, wherein the immune cell is an a T cell or a NK cell.Atorney Docket No.: 51624-0096WO1 / LG-U2024141WO26. The engineered cell of claim 25, wherein the a. T cell or the NK cell has an eliminated or reduced expression of an endogenous CD43 ligand.

27. The engineered cell of claim 26, wherein the endogenous CD43 ligand is sialic acid-binding Ig-like lectin 7 (Seglec-7).

28. The engineered cell of claim 26 or 27, wherein the eliminated or reduced expression of endogenous CD43 ligand is achieved by disrupting an endogenous CD43 ligand gene of the engineered cells.

29. The engineered cell of claim 28, wherein the disrupting endogenous CD43 ligand gene is achieved by using a gene editing method (e.g., clustered regularly interspaced short palindromic repeats / Cas9 protein (CRISPR / Cas9), transcription activator-like (TAL) effector nucleases (TALENs), or Zinc finger nucleases (ZFNs)), RNA interference (RNAi) technology, homologous recombination, modifying one or more regulating elements (e.g., promoter) of endogenous CD43 ligand gene, knocking out a sequence encoding all or part of the endogenous CD43 ligand, and / or knocking in an exogenous sequence to replace all or part of endogenous CD43 ligand gene.

30. The engineered cell of any one of claims 26-29, wherein the expression level of the endogenous CD43 ligand in the engineered cell is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% as compared to that in a wildtype cell or a control cell.

31. The engineered cell of any one of claims 22-24, wherein the immune cell is a y8 T cell.

32. The engineered cell of claim 31, wherein the expression of an endogenous Seglec-7 is not eliminated or reduced in the y8 T cell.Atorney Docket No.: 51624-0096WO1 / LG-U2024141WO33. The engineered cell of any one of claims 1-10, and 15-32, wherein the engineered cell has an eliminated or reduced expression of endogenous MHC Class I molecules.

34. The engineered cell of claim 33, 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.

35. The engineered cell of any one of claims 1-10 and 15-34, wherein the engineered cell has an eliminated or reduced expression of endogenous MHC Class II molecules.

36. The engineered cell of claim 35, 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.

37. The engineered cell of any one of claims 1-36, wherein the engineered cell further expresses an engineered receptor.

38. The engineered cell of claim 37, wherein the engineered receptor is an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC) or a portion thereof.

39. The engineered cell of claim 37 or 38, wherein the engineered receptor specifically targets a tumor antigen.

40. The engineered cell of claim 39, wherein the tumor antigen is selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-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,Atorney Docket No.: 51624-0096WO1 / LG-U2024141WO h5T4, PSCA, PSMA, R0R1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, Delta-like 3 (DLL3), CD70, CS-1, c- Met, Glycolipid F77, PD-L1, and PD-L2.

41. The engineered cell of any one of claims 37-40, wherein the engineered receptor is a CAR comprising an amino acid sequence set forth in SEQ ID NO: 4 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 4.

42. The engineered cell of any one of claims 37-41, wherein the engineered cell comprises a vector encoding the CD43 polypeptide and the engineered receptor, optionally the vector encodes one or more tolerogenic factors.

43. The engineered cell of any one of claims 1-42, wherein overexpression of the CD43 polypeptide can prevent or reduce host rejection in a subject when the engineered cell is administered to the subject.

44. The engineered cell of any one of claims 1-43, wherein the CD43 polypeptide is glycosylated.

45. The engineered cell of any one of claims 1-43, wherein the CD43 polypeptide is deglycosylated by N-glycosidase.

46. The engineered cell of claim 45, wherein the N-glycosidase is selected from Peptide-N- Glycosidase F (PNGase F), Peptide-N-Glycosidase A (PNGase A), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase Fl (Endo Fl), Endoglycosidase F2 (Endo F2) and Endoglycosidase F3 (Endo F3), and any combination thereof, preferably wherein the glycosidase is PNGase F.

47. A composition comprising the engineered cell of any one of claims 1-46, and a pharmaceutically acceptable carrier.Atorney Docket No.: 51624-0096WO1 / LG-U2024141WO48. A method of making the engineered cell of any one of claims 1-46, comprising introducing a vector encoding the CD43 polypeptide into a cell, thereby making the engineered cell.

49. The method of claim 48, wherein the vector is a retroviral vector or a lentiviral vector.

50. The method of claim 48 or 49, wherein the method further comprising contacting the engineered cell with glycosidase.

51. A method of treating a disease or disorder in a subject, the method comprising administering to the subject, an effective amount of the engineered cell of any one of claims 1-46, or the composition of claim 47.

52. The method of claim 51, wherein the disease or disorder is cancer, autoimmune disease, or infection.

53. A method of inhibiting immune clearance of an engineered cell in a subject, comprising:(a) overexpressing a CD43 polypeptide in the engineered cell,(b) administering the engineered cell from step (a) to a subject in need thereof, thereby inhibiting immune clearance of the engineered cell.

54. The method of claim 53, wherein the immune clearance of the engineered cell is through T cell-mediated cytotoxicity and / or NK cell-mediated cytotoxicity.

55. A method of increasing the in vivo expansion of an engineered cell in a subject, comprising:(a) overexpressing a CD43 polypeptide in the engineered cell;(b) administering the engineered cell from step (a) to a subject in need thereof, thereby increasing the in vivo expansion of the engineered cell.

56. A method of increasing persistence and / or function of an engineered cell in a subject, comprising:Atorney Docket No.: 51624-0096WO1 / LG-U2024141WO(a) overexpressing a CD43 polypeptide in the engineered cell;(b) administering the engineered cell from step (a) to a subject in need thereof, thereby increasing the persistence and / or function of the engineered cell.

57. The method of any one of claims 53-56, wherein the method further comprising contacting the engineered cell with glycosidase between step (a) and step (b).

58. The method of claim 50 or 57, wherein the glycosidase is N-glycosidase.

59. The method of claim 50, 57 or 55, wherein the glycosidase is selected from Peptide-N- Glycosidase F (PNGase F), Peptide-N-Glycosidase A (PNGase A), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase Fl (Endo Fl), Endoglycosidase F2 (Endo F2) and Endoglycosidase F3 (Endo F3), and any combination thereof, preferably wherein the glycosidase is PNGase F.

60. The method of any one of claims 53-59, wherein the overexpressing the CD43 polypeptide in the engineered cells is achieved by introducing a vector expressing the CD43 polypeptide, using a gene editing system (e.g., clustered regularly interspaced short palindromic repeats / Cas9 protein (CRISPR / Cas9), transcription activator-like (TAL) effector nucleases (TALENs), or Zinc finger nucleases (ZFNs)), modifying one or more regulating elements (e.g., promoter) of endogenous CD43 gene, and / or knocking in a sequence encoding the CD43 polypeptide.

61. The method of any one of claims 53-60, wherein the CD43 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a sequence that is at least 80%, 85%, 90%, 91%, 92%>, 93%, 94%, 95%, 96 ), 97%o, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2.

62. The method of any one of claims 53-61, wherein the engineered cell is a CAR-T cell.

63. The method of any one of claims 51-62, wherein the subject is a human subject.

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