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WO2025223534A1 - Méthodes d'expansion de lymphocytes t - Google Patents

Méthodes d'expansion de lymphocytes t

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Publication number
WO2025223534A1
WO2025223534A1 PCT/CN2025/091116 CN2025091116W WO2025223534A1 WO 2025223534 A1 WO2025223534 A1 WO 2025223534A1 CN 2025091116 W CN2025091116 W CN 2025091116W WO 2025223534 A1 WO2025223534 A1 WO 2025223534A1
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WO
WIPO (PCT)
Prior art keywords
cells
less
fold
hla
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/091116
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English (en)
Inventor
Wanbing TANG
Yanliang ZHU
Yafeng Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Legend Biotechnology Co Ltd
Legend Biotech Ireland Ltd
Original Assignee
Nanjing Legend Biotechnology Co Ltd
Legend Biotech Ireland Ltd
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Application filed by Nanjing Legend Biotechnology Co Ltd, Legend Biotech Ireland Ltd filed Critical Nanjing Legend Biotechnology Co Ltd
Publication of WO2025223534A1 publication Critical patent/WO2025223534A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This disclosure relates methods for expanding ⁇ T cells that have an eliminated or reduced expression of endogenous MHC Class I molecules by expressing a single-chain fusion HLA Class I protein.
  • the disclosure also relates to modified ⁇ T cells produced by the methods described herein.
  • Chimeric antigen receptor (CAR) expressing immune cell therapies are promising in cancer immunotherapy. Due to the labor-intensive, time-consuming processing, high cost, and other limitations of autologous CAR expressing T cell therapy, autologous immune cell production approach is limited to small-scale clinical applications. Universal CAR expressing immune cell therapies are promising methods for cancer immunotherapy.
  • ⁇ T Gamma delta T ( ⁇ T) cells from healthy donor have been given to patients with cancer and were shown to expand without inducing significant graft-versus-host diseases.
  • allogeneic ⁇ T cells can be rapidly rejected by the host’s immune system because of mismatching of human leukocyte antigen (HLA) .
  • HLA human leukocyte antigen
  • knockout (KO) of beta-2 microglobulin (B2M) an essential subunit of HLA Class I molecules, has shown to be effective.
  • B2M beta-2 microglobulin
  • some subsets of ⁇ T cells may recognize and lyse HLA Class I-deficient cells (e.g., fratricide) , which makes it difficult to prepare B2M -/- ⁇ T cells.
  • HLA Class I-deficient cells e.g., fratricide
  • the present application provides a platform to overcome fratricide of ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., B2M KO ⁇ T cells) , by expressing a single-chain fusion HLA Class I protein.
  • the single-chain fusion HLA Class I protein includes at least a portion of B2M protein and at least a portion of HLA-E heavy chain.
  • the single-chain fusion HLA Class I protein can effectively increase proliferation and/or reduce fratricide of the ⁇ T cells for therapeutic use.
  • the disclosure also relates to modified cells produced by the methods described herein.
  • the modified cells when grafted to a subject in need thereof, can reduce the risk of eliciting a host-versus-graft (HvG) reaction induced by host cells.
  • HvG host-versus-graft
  • the modified cells can reduce the cytotoxicity induced by host ⁇ T cells and/or NK cells.
  • the disclosure also relates to protein constructs used in the methods described herein, including any of the single-chain fusion HLA Class I proteins, engineered receptors (e.g., any of the CARs described herein) , and exogenous human CD47 or variants thereof.
  • the disclosure relates to nucleic acids or vectors encoding any of the protein constructs described herein.
  • the disclosure relates to methods of using the modified cells or protein constructs in cancer immunotherapy.
  • the disclosure is related to a method of expanding a population of ⁇ T cells, the method comprising: a) providing a starting population of ⁇ T cells; and b) modifying the starting population of ⁇ T cells by eliminating or reducing expression of endogenous MHC Class I molecules; and expressing a single-chain fusion human leukocyte antigen (HLA) Class I protein.
  • HLA human leukocyte antigen
  • the disclosure is related to a method of expanding a population of ⁇ T cells, in some embodiments, the ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules, the method comprising: expressing a single-chain fusion HLA Class I protein.
  • 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.
  • the ⁇ T cells further comprise a peptide antigen that is presented by the single-chain fusion HLA Class I protein on the cell surface.
  • 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.
  • the peptide antigen is a peptide derived from HLA-G or HLA-C, in some embodiments, the peptide antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 13 or 14.
  • the single-chain fusion HLA Class I protein increases proliferation of the ⁇ T cells by 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 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to that of a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, in some embodiments, the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules.
  • the single-chain fusion HLA Class I protein reduces fratricide of the ⁇ T cells to 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 a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, in some embodiments, the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules.
  • the starting population of ⁇ T cells are isolated from one or more healthy donors.
  • a polynucleotide encoding the single-chain fusion HLA Class I protein is introduced to the ⁇ T cells via viral transduction (e.g., lentiviral transduction) .
  • the eliminating or reducing expression of endogenous MHC Class I molecules is achieved by disrupting endogenous beta-2 microglobulin (B2M) gene of the ⁇ T cells.
  • B2M beta-2 microglobulin
  • the disrupting endogenous B2M gene is achieved by using a gene editing system (e.g., clustered regularly interspaced short palindromic repeats/Cas9 protein (CRISPR/Cas9) ) .
  • the gene editing system comprises a guide RNA (gRNA) targeting the endogenous B2M gene of the ⁇ T cells.
  • the ⁇ T cells further express an engineered receptor.
  • 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.
  • the engineered receptor specifically recognizes GPC2, GPRC5D, CD7, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD70, CD123, CD228, CD138, BCMA, AFP, GPC3, CEA, Claudin 18.2, CLL1, folate receptor (FR ⁇ ) , mesothelin (MSLN) , DLL3, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, NKG2D ligand, ULBP and/or HER-2.
  • GPC2 GPRC5D
  • CD7 CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD70, CD123, CD228, CD138, BCMA, AFP, GPC3, CEA, Claudin 18.2, CLL1, folate receptor (FR ⁇ ) , mesothelin (MSLN) , DLL3, CD276,
  • the ⁇ T cells further express an exogenous human CD47 or a variant thereof.
  • the exogenous human CD47 or the variant thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 18, 19 or 20.
  • the exogenous human CD47 or the variant thereof with 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: 5, 6 or 7.
  • the exogenous human CD47 or the variant thereof increases proliferation of the ⁇ T cells by 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 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, in some embodiments, the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules.
  • the exogenous human CD47 or the variant thereof reduces fratricide of the ⁇ T cells to 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 a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, in some embodiments, the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules.
  • the single-chain fusion HLA Class I protein comprises, optionally from N-terminus to C-terminus: a signal peptide, a peptide antigen, optionally a first linker (e.g., a flexible linker) , a human B2M protein, optionally a second linker (e.g., a flexible linker) , and a human HLA-E heavy chain.
  • the human HLA-E heavy chain is HLA-E*01: 01 heavy chain or HLA-E*01: 03 heavy chain.
  • the human B2M protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 11.
  • the human HLA-E heavy chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 12.
  • the first linker and/or the second linker comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 10.
  • 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 SEQ ID NO: 17.
  • the single-chain fusion HLA Class I protein with a signal peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 2.
  • the disclosure is related to a population of modified ⁇ T cells produced using the method described herein.
  • the disclosure is related to a population of modified ⁇ T cells whose endogenous B2M gene is disrupted, in some embodiments, the modified ⁇ T cells express a single-chain fusion HLA Class I protein comprising at least a portion of B2M protein and at least a portion of HLA-E heavy chain.
  • 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.
  • the population of modified ⁇ T cells further express an engineered receptor (e.g., CAR) on the cell surface.
  • the population of modified ⁇ T cells further express an exogenous human CD47 or a variant thereof.
  • the population of modified ⁇ T cells when grafted to a subject in need thereof, can reduce the risk of eliciting a host-versus-graft reaction induced by host cells.
  • the host cells comprise ⁇ T cells and/or NK cells.
  • the disclosure is related to a pharmaceutical composition, comprising the population of modified ⁇ T cells described herein, and a pharmaceutically acceptable carrier.
  • the disclosure is related to a kit comprising the population of modified ⁇ T cells described herein, or the pharmaceutical composition described herein.
  • the kit described herein further comprises written instructions for using the population of modified ⁇ T cells, or the pharmaceutical composition.
  • the disclosure is related to a method of treating a disease or disorder in a subject, the method comprising administering to the subject in need thereof the population of modified ⁇ T cells described herein, or the pharmaceutical composition described herein.
  • the disease or disorder is a cancer, an autoimmune disease, or an injury.
  • FIG. 1A shows the specific cytotoxicity of CAR- ⁇ T cells when co-incubated with target cells, as determined by flow cytometry.
  • FIG. 1B shows a set of flow cytometry results of long-term killing assays.
  • CD33/CLL1CAR-, CLL1CAR-, or 19CAR-expressing ⁇ T or ⁇ T cells were co-incubated with their corresponding target cells for 2-3 days, before the cell mixtures were stained for flow cytometry analysis.
  • R0 means before the first re-challenge.
  • R1, R2, R3, and R4 mean after the first, second, third, and fourth round of re-challenge, respectively.
  • Either B2M KO CAR-expressing ⁇ T cells ( “ ⁇ T” ; left) or B2M KO CAR-expressing ⁇ T cells ( “ ⁇ T” ; right) were used as effector cells.
  • FIG. 1C shows the percentage of B2M -/- CAR-expressing ⁇ T cells in CAR + ⁇ T cells during the long-term killing assays (corresponding to left panel of FIG. 1B) .
  • FIG. 1D shows the percentage of B2M -/- CAR-expressing ⁇ T cells in CAR + ⁇ T cells during the long-term killing assays (corresponding to right panel of FIG. 1B) .
  • FIG. 2A shows a set of flow cytometry results that indicate the surface expression of CAR and B2M of B2M knockout ⁇ T cells.
  • the expression was detected on Day 7, Day 11 and Day 14 after the lentiviral transduction.
  • Either the 19CAR- ⁇ T/B2M KO group or the 19CAR-HLAE- ⁇ T/B2M KO group was subjected to flow cytometry analysis.
  • the 19CAR-HLAE- ⁇ T/B2M KO group the cells on Day 7 and Day 11 (within a dashed-line rectangle) were further subjected to detect HLA-E expression.
  • the CAR + B2M - and CAR + HLA-E + cell populations are circled.
  • FIG. 2B shows the percentage of B2M -/- CAR + ⁇ T cells in the 19CAR- ⁇ T/B2M KO group or the 19CAR-HLAE- ⁇ T/B2M KO group. Untransduced ⁇ T/B2M KO cells ( “Un- ⁇ T/B2M KO” ) were used as controls.
  • FIG. 3B shows residual CAR- ⁇ T cell number after co-incubation with un-alloreactive- ⁇ T or allogenic- ⁇ T cells.
  • FIG. 3C shows the lysis percentage of CAR- ⁇ T cells after co-incubation with allogenic- ⁇ T cells.
  • the lysis percentage induced by allogenic ⁇ T cells was calculated by using 100%to minus the percentage value which calculated by dividing the residual CAR- ⁇ T cell number of the co-incubation groups by the residual CAR- ⁇ T cell number of a ⁇ T cells-only group.
  • FIG. 4A shows a set of flow cytometry results when 19CAR-HLAE- ⁇ T/B2M KO cells or un- ⁇ T/B2M KO cells were co-incubated with pre-activated NK cells at different NK: ⁇ T ratios.
  • the cell surface expression of B2M and CAR was detected for 19CAR-HLAE- ⁇ T/B2M KO cells that were incubated with NK cells for 20 hours.
  • the cell surface expression of B2M and HLA-E was detected for un- ⁇ T/B2M KO cells that were incubated with NK cells for 20 hours.
  • FIG. 4B shows the NK cell-mediated lysis percentage of 19CAR-HLAE- ⁇ T/B2M KO or un- ⁇ T/B2M KO cells after co-incubation with pre-activated NK cells at different NK: ⁇ T ratios.
  • the negative value of lysis % was likely caused by non-specific stimulation-induced ⁇ T proliferation.
  • FIG. 5A shows a set of flow cytometry results that indicate the surface expression of CAR and B2M of B2M -/- CAR- ⁇ T cells.
  • the expression was detected on Day 11 after the lentiviral transduction.
  • the B2M -/- CAR- ⁇ T cells 11.16%of 19CAR- ⁇ T/B2M KO cells were retained; 24.20%of 19CAR+HLAE- ⁇ T/B2M KO cells were retained; 27.28%of 19CAR-wt47- ⁇ T/B2M KO cells were retained; 41.64%of 19CAR-47m1- ⁇ T/B2M KO cells were retained; 64.42%of 19CAR-47m1-HLAE- ⁇ T/B2M KO cells were retained; 42.40%of 19CAR-47m2- ⁇ T/B2M KO cells were retained; and 61.56%of 19CAR-47m2-HLAE- ⁇ T/B2M KO cells were retained.
  • Untransduced ⁇ T cells “
  • FIG. 5B shows the relative expansion folds of B2M -/- CAR- ⁇ T cells (or B2M -/- CAR + - ⁇ T cells) on Day 11 after the lentiviral transduction.
  • FIG. 6 lists sequences discussed in the disclosure.
  • GvHD graft-versus-host disease
  • HvG host-versus-graft
  • TCR T cell receptor
  • KO knockout
  • ⁇ and/or ⁇ constant domain of one of its chains
  • ⁇ T Gamma delta T
  • B2M KO is an effective way to preclude HvG response induced by mismatch of HLA Class I molecules.
  • some subsets of ⁇ T cells may recognize and lyse HLA Class I-deficient cells, which impedes B2M KO ⁇ T production.
  • the present disclosure endeavors, in part, to solve this problem and provides methods of expanding ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., B2M KO ⁇ T cells) .
  • a single-chain fusion HLA Class I protein e.g., a HLA-E trimeric construct described herein
  • the methods can effectively increase proliferation and/or reduce fratricide of these cells before they are subjected for treating patients in need.
  • ⁇ T cells that have an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., B2M KO ⁇ T cells) by expressing a single-chain fusion HLA Class I protein.
  • endogenous MHC Class I molecules e.g., B2M KO ⁇ T cells
  • a higher yield of ⁇ T cells can be obtained for subsequent therapeutic use (e.g., administration to patients in need thereof) .
  • the ⁇ T cells are expanded (e.g., cultured) for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 15 days) before harvested, tested, and/or further modified for subsequent therapeutic use.
  • the ⁇ T cells either before, during, or after the expansion, can be modified to express an engineered receptor (e.g., a CAR) .
  • an engineered receptor e.g., a CAR
  • the ⁇ T cells can be modified to further express an exogenous human CD47 or a variant thereof, which may exhibit a synergistic effect for ⁇ T cell proliferation.
  • expression of the single-chain fusion HLA Class I protein described herein can protect the obtained ⁇ T cells from lysis induced by host cells (e.g., ⁇ T cells and/or NK cells) .
  • compositions and kits using the methods, compositions, and/or cells described herein.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • engineered receptor refers to an exogenous receptor (such as CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR (antibody-coupled T cell receptor) ) , engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , T cell antigen coupler (TAC) , or TAC-like chimeric receptor) that retains its biological activity after being introduced into the T cells or modified cells described herein.
  • CAR e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR (antibody-coupled T cell receptor)
  • engineered TCR e.g., traditional engineered TCR, chimeric TCR (cTCR)
  • TAC T cell antigen coupler
  • TAC-like chimeric receptor TAC-like chimeric receptor
  • the biological activity includes but are not limited to the ability of the engineered receptor in specifically binding to a molecule (e.g., cancer antigen) , properly transducing downstream signals, such as inducing cellular proliferation, cytokine production and/or performance of regulatory or cytolytic effector functions.
  • a molecule e.g., cancer antigen
  • chimeric antigen receptor 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.
  • CLL1CAR refers to a CAR that specifically recognizes CLL1.
  • 19CAR refers to a CAR that specifically recognizes CD19.
  • GvHD Growth-versus-host disease
  • HvG Health-versus-graft response or rejection refers to a pathological condition where the immune system of a host generates an immune response against transplanted cells.
  • 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.
  • malignancies of the various organ systems such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, endocrine systems and neuroendocrine tumor (NET) ; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, small cell lung cancer (SCLC) , large cell neuroendocrine cancer (LCNC) , neuroendocrine prostate cancer (NEPC) , pancreatic neuroendocrine tumor (PNET) , gastrointestinal neuroendocrine cancers, and cancer of the small intestine.
  • SCLC small cell lung cancer
  • LCNC large cell neuroendocrine cancer
  • NEPC neuroendocrine prostate cancer
  • PNET pancreatic neuroendocrine tumor
  • gastrointestinal neuroendocrine cancers 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.
  • 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.
  • 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.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art.
  • autologous is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic refers to a graft derived from a different individual of the same species.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • a term “no less than” refers to the “equal to” and/or “more than”
  • a term “no more than” refers to the “equal to” and/or “less than” .
  • references to "about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se.
  • description referring to "about X” includes description of "X” .
  • the term “about X-Y” used herein has the same meaning as “about X to about Y. ”
  • T cells Gamma delta ( ⁇ ) T cells (or ⁇ T cells) are a subgroup of T cells with distinct T cell receptors (TCRs) ⁇ and ⁇ 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 ( ⁇ chain) in 1984. In contrast, the most T cells in normal human body are ⁇ T cells (65-70%) with TCR composed of two glycoprotein chains called ⁇ and ⁇ TCR chains. These cells are generally simply referred to as “T cells” .
  • ⁇ T cells are much less common than ⁇ T cells, they are at their highest abundance in the gut mucosa, within a population of lymphocytes known as intraepithelial lymphocytes.
  • MHC major histocompatibility complex
  • ⁇ T cells can recognize generic determinants expressed by cells that have become dysregulated as a result of either malignant transformation or viral infection. Consequently, ⁇ 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.
  • ⁇ T cells have shown to possess the ability to bridge innate and adaptive immunity.
  • the majority of ⁇ T cells in adult human blood exhibit V ⁇ 9V ⁇ 2 T cell receptors and respond to small phosphorylated nonpeptide antigens, called phosphoantigens (pAgs) , which are commonly produced by malignant cells.
  • pAgs phosphoantigens
  • ⁇ 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.
  • MHC major histocompatibility complex
  • GvHD graft-versus-host disease
  • ⁇ T cells have several other unique features that make them ideal cellular carriers for developing off-the-shelf cellular therapy for cancer.
  • ⁇ T cells have roles in cancer immunosurveillance; 2) ⁇ T cells have the remarkable capacity to target tumors independent of tumor antigen-and major histocompatibility complex (MHC) -restrictions; 3) ⁇ T cells can employ multiple mechanisms to attack tumor cells through direct killing and adjuvant effects; and 4) ⁇ T cells express a surface receptor, Fc ⁇ RIII (CD16) , that is involved in antibody-dependent cellular cytotoxicity (ADCC) and can be potentially combined with monoclonal antibody for cancer therapy.
  • Fc ⁇ RIII CD16
  • ⁇ T cells Details of ⁇ 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 (2016) : 393121; Zou, C., et al. " ⁇ T cells in cancer immunotherapy. " Oncotarget 8.5 (2017) : 8900; and Zhao, Y., et al. "Gamma-delta ( ⁇ ) T cells: friend or foe in cancer development? . " Journal of Translational Medicine 16 (2016) : 1-13; each of which is incorporated herein by reference in its entirety.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • Mismatched HLA proteins that are presented to T cells as foreign antigens activate this allo-immune response.
  • 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) ⁇ 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 ⁇ chain) and ⁇ -2 microglobulin (B2M) to form a functional heterodimer.
  • 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.
  • MHC Class II e.g., HLA-DR, -DQ, -DP
  • APCs professional antigen presenting cells
  • CD4 T cells help to drive a B-cell mediated antibody response to host antigens.
  • the present disclosure provides ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules.
  • the ⁇ T cells comprise a genetically engineered disruption in a beta-2 microglobulin (B2M) gene.
  • B2M beta-2 microglobulin
  • the ⁇ T cells comprise genetically engineered disruptions of all copies of the B2M gene.
  • 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 is shown in SEQ ID NO: 11. 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.
  • SNPs single nucleotide polymorphisms
  • B2M deficient cells encompass cells that comprise a B2M -/- genetic background (referred to as B2M -/- cells) .
  • B2M -/- cells refers 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.
  • a recipient or patient refers to a primate, and optionally a human.
  • the cell is a human cell and the patient is a human.
  • 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.
  • the disruption results in expression of non-functional B2M proteins, including but not limited to truncations, deletions, point mutations and insertions.
  • the disruption results in no protein expression from the B2M gene.
  • HLA Class I-deficiency provides further benefits; for example, cells without HLA Class I expression cannot present auto-antigens that would otherwise prevent successful cell therapies for autoimmune diseases such as diabetes and rheumatoid arthritis.
  • 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. 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. These techniques may optionally include steps to remove non-human DNA sequences from the cells after B2M gene disruption.
  • An exemplary embodiment of this method is as disclosed throughout the application, using an adeno-associated virus gene targeting vector, optionally including 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 entirety. Exemplary targeting vectors and exemplary vector diagrams are also disclosed herein. It is within the level of those of skill in the art, based on the teachings herein and known in the art, to utilize a variety of techniques for making the B2M -/- cells, optionally human cells.
  • 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.
  • expression of the endogenous MHC Class I molecules is eliminated or reduced by disrupting endogenous B2M gene (s) .
  • the disruption is achieved by using a gene editing system (e.g., clustered regularly interspaced short palindromic repeats/Cas9 protein (CRISPR/Cas9) ) .
  • the gene editing system comprises a guide RNA (gRNA) targeting the endogenous B2M gene (s) .
  • the B2M gRNA sequence is shown in SEQ ID NO: 21.
  • the disclosure provides a method of producing a modified cell (e.g., a modified ⁇ 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 interference (RNAi) technology, transcription activator-like (TAL) effector nucleases (TALENs) and Zinc finger nucleases (ZFNs) .
  • the methods may involve knocking out the ⁇ 2 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 modified cells.
  • the disclosure provides a method of producing a modified cell (e.g., a modified ⁇ 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.
  • CIITA major histocompatibility complex transactivator
  • 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.
  • 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) .
  • 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.
  • the modified 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 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.
  • 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) .
  • Additional gRNAs may be used to knock out the CIITA gene.
  • the method described herein may further comprise disrupting the expression of endogenous TCR.
  • 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%.
  • endogenous MHC Class I molecules e.g., endogenous B2M gene
  • 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%.
  • 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.
  • insertion or deletion e.g., NHEJ-mediated insertion or deletion
  • deletion e.g., NHEJ-mediated deletion
  • a genomic sequence including at least a portion of the gene
  • 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 thereof) .
  • a locus e.g., an endogenous MHC Class I locus or the coding region thereof
  • 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.
  • 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 MHC Class I locus or the coding region thereof) .
  • 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)
  • a locus e.g., an endogenous MHC Class I locus or the coding region thereof
  • the method may comprise the introduction of two double stand breaks-one 5’a nd 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 polyadenylation signal) ) of an endogenous MHC Class I locus or the coding region thereof.
  • 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.
  • 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.
  • 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.
  • 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
  • the break can be positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
  • 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
  • the break can be positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
  • 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 alleles of a gene.
  • 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.
  • a targeted knockdown 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.
  • 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 modified 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) .
  • NK cells natural killer cells
  • overexpressing HLA-E can protect the modified cells from NK cell mediated cell killing.
  • the methods may further involve disrupting the expression of an endogenous TCR.
  • 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.
  • HLA-E-CD94/NKG2A engagement has been shown to regulate NK cell-mediated lysis and represents an important component of immune homeostasis.
  • HLA-E overexpression on B2M knockout allogenic graft cells may protect the cells against allogeneic NK cell-mediated lysis, thereby overcoming the host-versus-graft response.
  • 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 HLA-E*01: 07
  • HLA-E*01: 09 HLA-E*01: 10.
  • HLA-E HLA-E complexed with HLA class I signal sequence–derived peptides by CD94/NKG2 confers protection from natural killer cell–mediated lysis.
  • European Journal of Immunology 51.10 (2021) 2513-2521; and U.S. Patent Application Publication No. 2023/0014010 A1; each of which is incorporated herein by reference in its entirety.
  • the cells instead can be engineered to recombinantly express a single-chain fusion HLA class I protein in a B2M -/- genetic background.
  • the B2M -/- cells as used herein also encompass ⁇ T cells that express one or more single-chain fusion HLA Class I proteins in a B2M -/- genetic background.
  • the B2M -/- cells recombinantly expressing a single-chain fusion HLA Class I protein are nevertheless deficient in normal B2M function in that the cells do not express wildtype B2M protein that form a non-covalently associated heterodimer with any HLA Class I ⁇ chain on the cell surface.
  • single-chain fusion HLA Class I protein 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 ⁇ chain.
  • 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 ⁇ chain expressed on the surface of a wildtype cell.
  • HLA Class I ⁇ chain or “HLA-I heavy chain” refers to the ⁇ chain of the HLA Class I heterodimer.
  • HLA Class I heavy chain includes without limitation HLA Class I ⁇ chains HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.
  • the HLA Class I ⁇ chain described herein is a HLA-E heavy chain.
  • 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) .
  • the HLA ⁇ 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 ⁇ chain (leaderless HLA ⁇ chain) .
  • 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 ⁇ chain to ensure proper folding of the single-chain fusion HLA Class I protein on the cell surface.
  • a leader sequence or signal peptide
  • the leader sequence can be the leader sequence of the B2M protein, the leader sequence of an HLA ⁇ chain protein or the leader sequence of other secretary proteins.
  • the single-chain fusion HLA Class I protein comprises a B2M protein with its leader sequence removed.
  • the single-chain fusion HLA Class I protein comprises an HLA ⁇ chain protein with its leader sequence removed.
  • Certain HLA Class I ⁇ chains are highly polymorphic. As will be understood by those of skill in the art, the human cells and methods of the disclosure are applicable to any such HLA ⁇ chains and polymorphism thereof.
  • Single-chain fusion HLA Class I proteins comprising sequence variants and fragments of B2M and/or HLA ⁇ 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 ⁇ T cells.
  • 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: 11 or HLA-E heavy chain as shown in SEQ ID NO: 12.
  • Natural killer (NK) cells are part of the innate immune response.
  • 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 ⁇ chain alleles thereby preventing NK-medicated apoptosis in uninfected normal cells.
  • 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.
  • HLA-E is a ligand for the CD94/NKG2 receptor of NK cells that inhibits NK cell-mediated apoptosis.
  • the B2M -/- 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.
  • 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.
  • the B2M -/- 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.
  • 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.
  • 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) .
  • SCT single-chain trimer
  • 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: 13) or a HLA-C peptide antigen (SEQ ID NO: 14) ) designed to occupy the peptide binding cleft of the single-chain fusion HLA Class I protein.
  • a peptide antigen such as, but not limited to, a HLA-G peptide antigen (SEQ ID NO: 13) or a HLA-C peptide antigen (SEQ ID NO: 14)
  • 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: 17.
  • the single-chain fusion HLA Class I protein with a 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: 2.
  • 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.
  • 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.
  • the peptide antigen comprises a peptide of a tumor antigen.
  • the peptide antigen comprises a peptide of a protein from a pathogen including without limitation a bacterium, a virus, a fungus and a parasite.
  • the peptide antigen comprises a peptide of a tumor antigen.
  • the B2M -/- 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 B2M -/- 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.
  • 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 a B2M -/- 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.
  • the viral vector is derived from human foamy virus, a type of retrovirus.
  • suitable viral vectors include without limitation vectors derived from retrovirus, adenoviral virus, adeno-associated virus, lentivirus, herpes simplex virus, vaccinia virus, and pox virus.
  • the polynucleotide capable of encoding a single-chain fusion HLA Class I protein is integrated into the chromosome of the cells, optionally into the B2M or the HLA loci, for stable expression.
  • 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.
  • the polynucleotide capable of expressing a single-chain 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.
  • any immune effects of the single-chain fusion HLA class I protein expressing B2M -/- cells can be studied by various means.
  • the cytotoxicity of B2M -/- cells expressing a single-chain fusion HLA Class I protein can be determined by flow cytometry.
  • the allogenic ⁇ T cell killing and NK cell-mediated lysis of the B2M -/- ⁇ T cells can also be determined by flow cytometry.
  • the disclosure provides an HLA Class I-typed B2M -/- cell bank, wherein the cells of the cell bank comprise a B2M -/- genetic background and are engineered to express one or more types of single-chain fusion HLA Class I proteins in which the HLA ⁇ chain is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G heavy chains.
  • the cell bank comprises a population of cells that expresses a single-chain fusion HLA Class I protein in which the HLA ⁇ chain comprises HLA-E heavy chain.
  • 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) .
  • 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
  • the signal peptide described herein is a human B2M signal peptide.
  • 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 ID NO: 15.
  • the human B2M protein described herein is a wildtype human B2M protein (e.g., NCBI Reference No. NP_004039.1) .
  • 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: 11.
  • the human B2M protein with 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: 15.
  • 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: 16.
  • the human HLA-E heavy chain described herein is a wildtype human HLA-E heavy chain (e.g., NCBI Reference No. NP_005507.3) .
  • 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: 12.
  • the human HLA-E heavy chain with 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: 16.
  • the first linker and/or the second linker described herein are flexible linkers.
  • the first linker and/or the second linker described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 10.
  • the flexible linker is a GS linker. Details of flexible linkers can be found, e.g., in Chen, X., et al. "Fusion protein linkers: property, design and functionality. " Advanced Drug Delivery Reviews 65.10 (2013) : 1357-1369, which is incorporated herein by reference in its entirety.
  • the peptide antigen described herein is a human HLA-G peptide antigen or a human HLA-C peptide antigen.
  • 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: 13.
  • 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: 14.
  • 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: 17.
  • the single-chain fusion HLA Class I protein with a signal peptide described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 2.
  • the engineered receptor can comprise an extracellular ligand binding domain or an extracellular antigen binding domain that specifically binds to an antigen (e.g., a tumor antigen) , a transmembrane domain, and an intracellular signaling domain.
  • the engineered receptor may comprise a signal peptide located at the N-terminus of the extracellular ligand binding domain.
  • the engineered receptor can be encoded by a heterologous polynucleotide operably linked to a promoter (such as a constitutive promoter or an inducible promoter) .
  • Exemplary engineered receptors include, but are not limited to, chimeric antigen receptor (CAR) , engineered T-cell receptor (TCR) , and T-cell antigen coupler (TAC) receptor.
  • CAR chimeric antigen receptor
  • TCR engineered T-cell receptor
  • TAC T-cell antigen coupler
  • the engineered receptors described herein are expressed from a vector having a truncated EF1 ⁇ promoter (SEQ ID NO: 8) .
  • the engineered receptor can be monovalent and monospecific.
  • the engineered receptor can be multivalent and monospecific.
  • the engineered receptor can be multivalent and multispecific.
  • the engineered receptor can comprise: (a) an extracellular ligand binding domain, (b) a transmembrane domain (e.g., derived from CD8 ⁇ ) , and (c) an intracellular signaling domain (ISD) comprising a chimeric signaling domain (CMSD) .
  • the CMSD may contain one or a plurality signaling motifs known as Immunoreceptor Tyrosine-based Activation Motifs, or ITAMs.
  • ITAMs Immunoreceptor Tyrosine-based Activation Motifs
  • the plurality of ITAMs may be connected by one or more linkers.
  • the plurality of ITAMs may be directly connected to each other.
  • the ITAMs may be derived from 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, Fcgamma R IIa, DAP10, and DAP 12.
  • the engineered receptor may have an intracellular signaling domain derived from CD3 ⁇ .
  • the engineered receptor may not have any sequence derived from CD3 ⁇ (e.g., human CD3 ⁇ ) .
  • the antigen of the engineered receptor may be a tumor antigen selected from the group consisting of BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor- ⁇ , GD2, GD3, hTERT, IL-13R ⁇ 2, ⁇ -light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, proteinase-3 (PR3) , tyrosinase, survivin, hTERT, EphA2, NY-ES
  • the antigen may be CD20.
  • the tumor antigen can be derived from an intracellular protein of tumor cells.
  • the tumor antigen can be expressed on the surface of tumor cells.
  • Many TCRs specific for tumor 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) .
  • the engineered receptor described herein can be a chimeric antigen receptor (CAR) .
  • CAR chimeric antigen receptor
  • Many chimeric antigen receptors are known in the art and can be suitable for the modified cells 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.
  • CARs of the present disclosure may 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.
  • 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.
  • immune effector function e.g., in a CAR-T cell, include cytolytic activity (such as antibody-dependent cellular toxicity, or ADCC) and helper activity (such as the secretion of cytokines) .
  • the intracellular signaling domain may generate a signal that promotes proliferation and/or survival of the CAR containing cell.
  • 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.
  • the intracellular signaling domain of a CAR can 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.
  • co-stimulatory molecule refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival.
  • Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response.
  • a co-stimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins) , and activating NK cell receptors.
  • Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) , ICOS (CD278) , and 4-1BB (CD137) .
  • co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile)
  • the CAR can comprise a single co-stimulatory signaling domain.
  • the CAR can comprise two or more co-stimulatory signaling domains.
  • the intracellular signaling domain can comprise a functional primary intracellular signaling domain (e.g., a chimeric signaling domain (CMSD) set forth herein) and one or more co-stimulatory signaling domains.
  • the CAR may not comprise a functional primary intracellular signaling domain (such as CD3 ⁇ ) .
  • the CAR can comprise an intracellular signaling domain consisting of or consisting essentially of one or more co-stimulatory signaling domains.
  • the CAR can comprise an intracellular signaling domain consisting of or consisting essentially of a nonfunctional or attenuated primary intracellular signaling domain (such as a mutant CD3 ⁇ ) and one or more co-stimulatory signaling domains.
  • 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 can be derived from one or more molecules selected from the group consisting of CD27, CD28, 4-1BB (i.e., CD137) , OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specially bind to CD83.
  • CD27, CD28, 4-1BB i.e., CD137
  • OX40 i.e., CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specially bind to CD83.
  • LFA-1 lymphocyte function-associated antigen-1
  • the antigen binding domain of a CAR may comprise one or more (such as any one of 1, 2, 3, 4, 5, 6 or more) antibodies or antibody fragments, which can be selected from an scFv, a Fv, a Fab, a (Fab’) 2 , a minibody, a diabody, a single domain antibody (sdAb) , or a V H H domain.
  • the antigen binding domain of a CAR can comprise a ligand or an extracellular portion of a receptor that specifically binds to a tumor antigen.
  • the CAR can be a monospecific, bispecific or multispecific CAR.
  • the antigen binding domain of a CAR can specifically bind to a single tumor antigen.
  • the antigen binding domain of a CAR can bind to two or more tumor antigens.
  • the engineered receptor e.g., CAR
  • CAR chimeric antigen receptor
  • TCR chimeric antigen receptor
  • the transmembrane region of a CAR comprises a transmembrane region can be selected from the transmembrane region of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d,
  • the extracellular domain can be connected to the transmembrane domain by a hinge domain.
  • the hinge domain can be a hinge domain of CD8 ⁇ .
  • the CAR can also comprise a signal peptide (SP) , such as a CD8 ⁇ signal peptide.
  • SP signal peptide
  • CD19 CARs or BCMA CARs 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.
  • the CAR described herein can specifically recognize CLL1 or CD19.
  • the CAR described herein is a CLL1CAR, which includes from N-terminus to C-terminus, a CLL1-targeting sdAb, an intracellular co-stimulatory region of 4-1BB, and an intracellular domain of CD3z.
  • the CLL1CAR described herein comprises an amin acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3.
  • the CAR described herein is a 19CAR, which includes from N-terminus to C-terminus, a CD19-binding scFv, an intracellular co-stimulatory region of 4-1BB, and an intracellular domain of CD3z.
  • the 19CAR described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 1.
  • cells described herein can express a CD33-targeting engineered receptor that includes from N-terminus to C-terminus, a CD33-targeting sdAb, and an intracellular domain of TLR4.
  • the CD33-targeting engineered receptor is co-expressed with the CLL1CAR described herein together.
  • cells described herein can express the CD33-targeting engineered receptor fused with the CLL1CAR via a P2A linker (SEQ ID NO: 9) , and the fusion protein ( “CD33/CLL1CAR” ) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 4.
  • the engineered receptor can be a modified T-cell receptor or engineered T-cell receptor.
  • the engineered TCR can be specific for any tumor antigen set forth herein. Any of the TCRs known in the art can be used.
  • the TCR can 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.
  • the TCR receptor complex is an octomeric complex formed by variable TCR receptor ⁇ and ⁇ chains (or ⁇ and ⁇ chains on case of ⁇ T cells) with three dimeric signaling modules CD3 ⁇ / ⁇ , CD3 ⁇ / ⁇ and CD247 (T-cell surface glycoprotein CD3 zeta chain) ⁇ / ⁇ or ⁇ / ⁇ . 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.
  • the engineered receptor can be an engineered TCR comprising one or more T-cell receptor (TCR) fusion proteins (TFPs) .
  • TCR T-cell receptor
  • TFPs T-cell receptor fusion proteins
  • Exemplary TFPs have been described, for example, in US20170166622A1, which is incorporated herein by reference in its entirety.
  • the TFP can 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 can 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 can comprise a transmembrane region that comprises a transmembrane region of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • a transmembrane region that comprises a transmembrane region of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta T
  • the TFP can comprise a TCR subunit comprising 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.
  • the engineered receptor can be a T-cell antigen coupler (TAC) receptor.
  • TAC T-cell antigen coupler
  • Exemplary TAC receptors have been described, for example, in US20160368964A1, which is incorporated herein by reference.
  • the TAC can 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 can be an antibody fragment, such as scFv or V H H, which specifically binds to a tumor antigen.
  • the antigen binding domain can be a designed Ankyrin repeat (DARPin) polypeptide.
  • the tumor antigen can be any of the antigens set forth herein.
  • the protein associated with the TCR complex can be CD3, such as CD3E.
  • the TCR-binding domain can be a single-chain antibody, such as scFv, or a V H H.
  • the TCR-binding domain can be derived from UCHT1.
  • the TAC receptor can comprise a cytosolic domain and a transmembrane region.
  • the T-cell receptor signaling domain can 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 CD 154.
  • the TAC receptor can comprise a transmembrane region and a cytosolic domain derived from CD4.
  • the TAC receptor can comprise a transmembrane region and a cytosolic domain derived from CD8 (such as CD8 ⁇ ) .
  • T cell co-receptors are expressed as membrane proteins on T cells. They can provide stabilization of the TCR: peptide: MEC complex and facilitate signal transduction.
  • the CD4 co-receptor can only stabilize TCR: MEC II complexes while the CD8 co-receptor can only stabilize the TCR: MEC 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.
  • the engineered receptor (such as CAR, engineered TCR, or TAC) can 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) , ⁇ -human chorionic gonadotropin, alphafetoprotein (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
  • the tumor antigen can comprise one or more antigenic cancer epitopes associated with a malignant tumor.
  • Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2/Neu/ErbB-2.
  • Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA) .
  • CEA carcinoembryonic antigen
  • 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.
  • the tumor antigen can be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA) .
  • TSA tumor-specific antigen
  • TAA tumor-associated antigen
  • 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.
  • TSA or TAA antigens include the following: Differentiation antigens such as MART-1/MelanA (MART-I) , gp 100 (Pmel 17) , tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • Differentiation antigens such as MART-1/MelanA (
  • 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 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.
  • CD47 was first identified as a membrane protein involved in ⁇ 3 integrin-mediated signaling on leukocytes, it is now known to also interact with thrombospondin-1, signal regulatory protein-alpha (SIRP ⁇ , also known as SIRPA, Sirp ⁇ , Sirpa, or CD172A) , and others to regulate various cellular functions including cell migration, axon extension, cytokine production, and T cell activation.
  • SIRP ⁇ signal regulatory protein-alpha
  • SIRP ⁇ 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-1/BIT/CD172a)
  • SHPS-1/BIT/CD172a 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
  • SIRP ⁇ cytoplasmic immunoreceptor tyrosine-based inhibition
  • ITIM cytoplasmic immunoreceptor tyrosine-based inhibition
  • One resulting downstream effect is the prevention of myosin-IIA accumulation at the phagocytic synapse and consequently inhibition of phagocytosis.
  • CD47-SIRP ⁇ 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.
  • 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 SIRP ⁇ 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.
  • CSCs cancer stem cells
  • 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.
  • RBCs red blood cells
  • CD47 antagonists when used as therapies, it is also very important to assess its toxicities.
  • 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.
  • the present disclosure provides an modified cell (e.g., ⁇ T cell) expressing an exogenous human CD47 or variants thereof.
  • 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: 18.
  • 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: 19 or 20.
  • the exogenous human CD47 is a wildtype human CD47 with a 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.
  • the exogenous human CD47 variant is a mutated human CD47 with a 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.
  • the present disclosure provides an modified cell (e.g., ⁇ T cell) having an eliminated or reduced expression of endogenous MHC Class I molecules.
  • the modified cell may comprise disrupted B2M genes.
  • the modified cell may comprise reduced surface expression of B2M genes.
  • the cell surface expression of B2M genes may be down-regulated by at least about any of 50%, 60%, 70%, 80%, 90%, 95%or 97%.
  • the expression of one or more endogenous MHC Class I molecules e.g., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and/or HLA-G
  • HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and/or HLA-G may be not down-regulated.
  • the modified cells may be modified to knock out the B2M gene.
  • the modified cells may be further modified to knock out the CIITA gene.
  • the modified cell may be further modified to overexpress HLA-E.
  • HLA-E can be overexpressed in a form of a single-chain trimer (SCT) , comprising a signal peptide, a peptide antigen, a first linker, a human B2M protein, a second linker, and a HLA-E heavy chain.
  • SCT single-chain trimer
  • the modified cells can express any of the single-chain fusion HLA Class I proteins described herein.
  • the modified cells can express any of the engineered receptors described herein. In some embodiments, the modified cells can express any of the exogenous human CD47 or variants thereof described herein.
  • the starting cell that is to be engineered can be obtained from e.g., humans or non-human animals.
  • the starting cell can be obtained from humans, rats or mice.
  • the starting cell may be a blood cell.
  • the starting cell may be a leukocyte (e.g., a T cell) , lymphocyte or any other suitable blood cell type.
  • the starting cell may be a peripheral blood cell.
  • the starting cell may be a tumor-infiltrating lymphocyte (TIL) .
  • TIL tumor-infiltrating lymphocyte
  • the starting cell may be a T cell, a B cell, an NKT cell or an NK cell.
  • the starting cells may be human peripheral blood mononuclear cells (PBMCs) .
  • the human PBMCs may be CD3+ cells.
  • the human PBMCs may be CD8+ cells or CD4+ cells.
  • the starting cell may be a T cell.
  • the starting T cells may express one or more engineered receptors that recognize a specific antigen on the surface of a target cell.
  • T cells can be obtained by various methods known in the art, e.g., in vitro culture of T cells (e.g., tumor infiltrating lymphocytes) isolated from subjects.
  • the T cells may be CD4+ T cells, CD8+ T cells, or regulatory T cells.
  • the T cells may be T helper type 1 T cells and/or T helper type 2 T cells.
  • the T cell may be an ⁇ T cell.
  • the T cell may be a ⁇ T cell.
  • the T cells may be central memory T cells.
  • the T cells may be effector memory T cells.
  • the T cells may be T cells.
  • the starting cells may be stem cells, such as hematopoietic stem cell, embryonic stem cell and pluripotent stem cell, including induced pluripotent stem cells (iPSCs) .
  • the starting cells can be primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the stem cells may be cultured with additional differentiation factors to obtain desired cell types (e.g., T cells) .
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. Any known method for separation based on such markers can be used.
  • the separation may be affinity-or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. Both fractions may be retained for further use. Negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • the modified cells may have reduced expressions of endogenous MHC Class I molecules (e.g., endogenous B2M) . Comparing to the starting cells, the modified cells described herein may elicit reduced HvG response in a histoincompatible individual. The modified cells may have reduced expressions of Class II major histocompatibility complex transactivator (CIITA) . The modified cells may have reduced expressions of endogenous TCR.
  • endogenous MHC Class I molecules e.g., endogenous B2M
  • CIITA major histocompatibility complex transactivator
  • the modified cells can be autologous cells, syngeneic cells, allogeneic cells, or xenogeneic cells with respect to the individual receiving them.
  • the modified cells can 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.
  • MHC major histocompatibility complex
  • the cytotoxicity of the modified cells can be determined by a cytotoxicity assay.
  • the modified cells may be co-cultured with corresponding target cells at an E: T ratio of 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 for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.
  • the target cells can be stained by CFSE. Residual cells can be stained by DAPI and analyzed by FACS. The cells may be also be stained for LIVE/DEAD to check the viability of cells via flow cytometry analysis. More specifically, the CLL1CAR ⁇ T cells (as effector cells) can be co-cultured with U-937 cells (as target cells) ; the CD33/CLL1CAR ⁇ T cells (as effector cells) can be co-cultured with U-937 cells (as target cells) ; and the 19CAR ⁇ T cells (as effector cells) can be co-cultured with Raji cells (as target cells) .
  • eliminating or reducing expression of endogenous MHC Class I molecules does not reduce the cytotoxicity of the modified cells (e.g., any of the modified ⁇ T cells described herein) .
  • the cytotoxicity of the modified cells having an eliminated or reduced expression of endogenous MHC Class I molecules is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%as compared to that of a reference population of cells expressing endogenous MHC Class I molecules (e.g., ⁇ T cells whose endogenous B2M gene is not disrupted) .
  • the anti-HvG efficacy of the modified cells can be determined by co-culture with alloreactive T cells derived from PBMCs.
  • the alloreactive T cells are also referred to as alloreactive ⁇ T cells because ⁇ T cells account for the majority (e.g., at least 80%, 85%, 90%, 95%, 96%, 96%, 98%, or 99%) of total cells.
  • the modified ⁇ T cells may be co-cultured with the alloreactive ⁇ T cells at an ⁇ T: ⁇ T ratio of 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 for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.
  • eliminating or reducing expression of endogenous MHC Class I molecules can increase the residual number of the modified cells (e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein) by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold as compared to that of a reference population of cells expressing endogenous MHC Class I molecules (e.g., ⁇ T cells whose endogenous B2M gene is not disrupted) .
  • the modified cells e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein
  • eliminating or reducing expression of endogenous MHC Class I molecules can decrease the lysis of the modified cells (e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein) caused by the alloreactive ⁇ T cells to less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%as compared to that of a reference population of cells expressing endogenous MHC Class I molecules (e.g., ⁇ T cells whose endogenous B2M gene is not disrupted) .
  • modified cells e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein
  • the anti-HvG efficacy of the modified cells can be determined by co-culture with allogenic NK cells derived from PBMCs.
  • the modified ⁇ T cells may be co-cultured with the allogenic NK cells at an NK: ⁇ T ratio of 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 for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.
  • expression of the single-chain fusion HLA Class I protein described herein can decrease the lysis of the modified cells (e.g., any of the ⁇ T cells having a disrupted endogenous B2M gene and expressing a HLA-E trimeric construct described herein) caused by the allogenic NK cells to less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%as compared to that of a reference population of cells not expressing the single-chain fusion HLA Class I protein (e.g., any of the ⁇ T cells having a disrupted endogenous B2M gene and not expressing a HLA-E trimeric construct described herein) .
  • the modified cells e.g., any of the ⁇ T cells having a disrupted endogenous B2M gene and expressing a HLA-E trimeric construct described herein
  • the modified cells may be labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with CD8+ T cells derived from another donor.
  • CFSE carboxyfluorescein succinimidyl ester
  • the modified cells may be labeled with CFSE and co-cultured with NK cells derived from another donor.
  • the modified cell described herein can have reduced cell killing by allogeneic CD8+ T cells.
  • the viability of the modified cells may be at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%after incubation with allogeneic CD8+ T cells.
  • the viability of the starting cells may be 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 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%or less than 96%after incubation with allogeneic CD8+ T cells.
  • the viability of the modified cells may be at least 10%, at least 20%, at least 30%, least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least 50 times, or at least 100 times higher after incubation with allogeneic CD8+ T cells.
  • the methods described herein can decrease the killing efficiency by allogeneic CD8+ T cells against the modified cells.
  • the methods described herein can decrease the killing efficiency by allogeneic CD8+ T cells against the modified cells by at least 10%, at least 20%, at least 30%, least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • the modified cell described herein can have reduced cell killing by allogeneic NK cells.
  • the viability of the modified cells may be at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%after incubation with allogeneic NK cells.
  • the viability of the starting cells may be 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 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%or less than 96%after incubation with allogeneic NK cells.
  • the viability of the modified cells may be at least 10%, at least 20%, at least 30%, least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least 50 times, or at least 100 times higher after incubation with allogeneic NK cells.
  • the methods described herein can decrease the killing efficiency by allogeneic NK cells against the modified cells.
  • the methods described herein can decrease the killing efficiency by allogeneic NK cells against the modified cells by at least 10%, at least 20%, at least 30%, least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • HLA-E overexpression can also decrease the killing efficiency by allogeneic CD8+ T cells.
  • HLA-E overexpression can also decrease the killing efficiency by allogeneic CD8+ T cells by at least 10%, at least 20%, at least 30%, least 40%, or at least 50%.
  • HLA-E overexpression can also decrease the killing efficiency by allogeneic NK cells.
  • HLA-E overexpression can also decrease the killing efficiency by allogeneic NK cells by at least 10%, at least 20%, at least 30%, least 40%, or at least 50%.
  • Knocking out the B2M gene can also decrease the killing efficiency by allogeneic CD8+T cells.
  • Knocking out the B2M gene can increase the killing efficiency by allogeneic NK cells by at least 10%, at least 20%, at least 30%, least 40%, or at least 50%.
  • This increased killing efficiency by allogeneic NK cells can be mitigated by HLA-E overexpression.
  • This increased killing efficiency by allogeneic NK cells can be mitigated by HLA-E overexpression by at least 10%, at least 20%, at least 30%, least 40%, or at least 50%.
  • the present disclosure also provides nucleic acids and/or vectors (e.g., lentiviral vectors) encoding any of the protein constructs described herein.
  • nucleic acids and/or vectors e.g., lentiviral vectors
  • the vectors described herein include a truncated EF1 ⁇ promoter (SEQ ID NO: 8) .
  • nucleic acids and/or vectors encoding any of the amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7.
  • the nucleic acids and/or vectors described herein comprises a sequence that encodes, optionally from N-terminus to C-terminus, an engineered receptor (e.g., any of the engineered receptors described herein) , a linker (e.g., a P2A linker) , and a single-chain fusion HLA Class I protein (e.g., any of the single-chain fusion HLA Class I proteins described herein) .
  • the nucleic acids and vectors described herein can encode a fusion protein comprising, optionally from N-terminus to C-terminus, SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 2.
  • the nucleic acids and vectors described herein comprises a sequence that encodes, optionally from N-terminus to C-terminus, an engineered receptor (e.g., any of the engineered receptors described herein) , a linker (e.g., a P2A linker) , and an exogenous human CD47 or a variant thereof (e.g., any of the exogenous human CD47 or variants thereof described herein) .
  • the nucleic acids and vectors described herein can encode a fusion protein comprising, optionally from N-terminus to C-terminus, SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 5.
  • the nucleic acids and vectors described herein comprises a sequence that encodes, optionally from N-terminus to C-terminus, an engineered receptor (e.g., any of the engineered receptors described herein) , a first linker (e.g., a P2A linker) , an exogenous human CD47 or a variant thereof (e.g., any of the exogenous human CD47 or variants thereof described herein) , a second linker (e.g., a P2A linker) , and a single-chain fusion HLA Class I protein (e.g., any of the single-chain fusion HLA Class I proteins described herein) .
  • an engineered receptor e.g., any of the engineered receptors described herein
  • a first linker e.g., a P2A linker
  • an exogenous human CD47 or a variant thereof e.g., any of the exogenous human CD47 or variants thereof described herein
  • nucleic acids and vectors described herein can encode a fusion protein comprising, optionally from N-terminus to C-terminus, SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 2.
  • methods of expanding a population of ⁇ T cells comprising: a) providing a starting population of ⁇ T cells; and b) modifying the starting population of ⁇ T cells by eliminating or reducing expression of endogenous MHC Class I molecules (e.g., using any of the methods described herein to eliminate or reduce expression of endogenous MHC Class I molecules) , and expressing a single-chain fusion HLA Class I protein (e.g., any of the single-chain fusion HLA Class I proteins described herein) .
  • endogenous MHC Class I molecules e.g., using any of the methods described herein to eliminate or reduce expression of endogenous MHC Class I molecules
  • a single-chain fusion HLA Class I protein e.g., any of the single-chain fusion HLA Class I proteins described herein
  • the proliferation of ⁇ T cells can be determined by flow cytometry after co-culturing the ⁇ T cells (as effector cells) with corresponding target cells at an E: T ratio of 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 for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.
  • the residual cell mixtures can be rechallenged for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times by adding fresh target cells to ensure there are more than 20%of target cells in the cell mixture.
  • the percentage of ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein) after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rechallenges is less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%or less than 30%in all ⁇ T cells.
  • the percentage of ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, or less than 50%as compared to that of ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules.
  • the proliferation of ⁇ T cells can be determined by cell counting and/or flow cytometry after culturing the ⁇ T cells for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, or at least 25 days, e.g., after viral transduction.
  • the ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., those with a disrupted endogenous B2M gene described herein) .
  • the ⁇ T cells also express a single-chain fusion HLA Class I protein (e.g., any of the single-chain fusion HLA Class I proteins described herein) .
  • expression of the single-chain fusion HLA Class I protein can increase the proliferation of ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein) by 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 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to that of a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, wherein the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules, and do not express the single-chain
  • expression of the single-chain fusion HLA Class I protein can reduce fratricide of ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein) to 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 of a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, wherein the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules, and do not express the single-chain fusion HLA Class I protein.
  • endogenous MHC Class I molecules e.g., any of the ⁇ T cells with a disrupted endogenous B
  • the ⁇ T cells described herein may also express an exogenous human CD47 or a variant thereof (e.g., any of the exogenous human CD47 or variants thereof described herein) .
  • expression of exogenous human CD47 or the variant thereof can increase the proliferation of ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein) by 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 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to that of a reference population of ⁇ T cells that are expanded (
  • expression of the exogenous human CD47 or the variant thereof can reduce fratricide of ⁇ T cells having an eliminated or reduced expression of endogenous MHC Class I molecules (e.g., any of the ⁇ T cells with a disrupted endogenous B2M gene described herein) to 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 of a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, wherein the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules, and do not express the exogenous human CD47 or the variant thereof.
  • endogenous MHC Class I molecules e.g., any of the ⁇ T cells with a disrupted endogenous B
  • expression of both the single-chain fusion HLA Class I protein and the exogenous human CD47 or variants thereof described herein can synergistically increase proliferation of the ⁇ T cells (any of the modified ⁇ T cells described herein) by 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 15-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to that of a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, wherein the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules, and express either one of the single-chain fusion HLA Class I protein or the exogen
  • expression of both the single-chain fusion HLA Class I protein and the exogenous human CD47 or variants thereof described herein can synergistically reduce fratricide of the ⁇ T cells (any of the modified ⁇ T cells described herein) to 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 of a reference population of ⁇ T cells that are expanded (e.g., cultured) for the same period of time, wherein the reference population of ⁇ T cells have an eliminated or reduced expression of endogenous MHC Class I molecules, and express either one of the single-chain fusion HLA Class I protein or the exogenous human CD47 or the variant thereof.
  • modified cells e.g., modified ⁇ T cells
  • modified ⁇ T cells can be used in a variety of experimental, therapeutic and commercial applications.
  • the disclosure provides a method of treating a disease or disorder in a subject (e.g., human subject) , the method comprising administering to the subject, an effective amount of the modified cell described herein, or the pharmaceutical composition described herein.
  • the disease or disorder may be a cancer, an autoimmune disease, or an infection.
  • Solid tumor refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer) , or malignant (cancer) . Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors.
  • the disclosure provides a method of modulating an immune response comprising administering an effective amount of modified cells described herein to a subject in need thereof.
  • the present disclosure provides a method for treating cancer comprising administering an effective amount of modified cells described herein to a subject in need thereof.
  • cancer that can be treated include, but are not limited to, small cell lung cancer (SCLC) , large cell neuroendocrine cancer (LCNC) , neuroendocrine prostate cancer (NEPC) , pancreatic neuroendocrine tumor (PNET) , gastrointestinal neuroendocrine cancers, leukemias including chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, and T cell and B cell leukemias, lymphomas (Hodgkin’s and non-Hodgkins) , lymphoproliferative disorders, plasmacytomas, histiocytomas, melanomas, adenomas, sarcomas, carcinomas of solid tissues, hypoxic tumors, squamous cell carcinomas, genitourinary cancers such as cervical and bladder cancer
  • the disclosure further includes the use of the modified cells described herein in the manufacture of a medicament or pharmaceutical composition to modulate an immune response, to treat an infection or to treat cancer as described herein above.
  • the modified cells can also be used in experimental models, for example, to further study and elucidate the function of the cells.
  • One or more of the modified cells described herein can be administered to a subject in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions or injections, or subcutaneous injections.
  • the modified cells can expand within a subject’s body, in vivo, after administration to a subject.
  • the modified cells can be frozen to provide cells for multiple treatments with the same cell preparation.
  • the modified cells of the disclosure, and pharmaceutical compositions comprising the same can be packaged as a kit.
  • a kit can include instructions (e.g., written instructions) on the use of the modified cells and compositions comprising the same.
  • a method of treatment can comprise administering to a subject a therapeutically effective amount of the modified cells.
  • the therapeutically effective amount of the modified cells may be administered for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year.
  • the therapeutically effective amount of the modified cells may be administered for at least one week.
  • the therapeutically effective amount of the modified cells may be administered for at least two weeks.
  • the modified cells described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the modified cells can vary.
  • the modified cells can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition.
  • the modified cells can be administered to a subject during or as soon as possible after the onset of the symptoms.
  • the administration of the modified cells can be initiated immediately within the onset of symptoms, within the first 3 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within 48 hours of the onset of the symptoms, or within any period of time from the onset of symptoms.
  • the initial administration can be via any route practical (e.g., intravenous infusions or injections) , such as by any route described herein using any formulation described herein.
  • the administration of the modified cells of the disclosure is an intravenous administration.
  • One or multiple dosages of the modified cells can be administered as soon as is practicable after the onset of a cancer or an infectious disease, and for a length of time necessary for the treatment of the disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months.
  • one or multiple dosages of the modified cells can be administered years after onset of the cancer and before or after other treatments.
  • the modified cells can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years at least 3 years, at least 4 years, or at least 5 years.
  • the length of treatment can vary for each subject.
  • the cell therapy e.g., adoptive T cell therapy can 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.
  • 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.
  • the cell therapy (e.g., adoptive T cell therapy) can 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 then are 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 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.
  • the subject may be 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., allogenic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the administration can effectively treat the subject despite the subject having become resistant to another therapy.
  • the subject may be responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden.
  • the subject may be initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time.
  • the subject may have not 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.
  • the subject may has not received prior treatment with another 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., allogenic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the administration may effectively treat the subject despite the subject having become resistant to another therapy.
  • the modified cells described herein can be administered to an animal, such as a mammal, even more a human, to treat a cancer.
  • the modified cells can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell (s) , where it is desirable to treat or alleviate the disease.
  • the modified cells e.g., modified ⁇ T cells
  • the composition can include a pharmaceutical composition and further include a pharmaceutically acceptable carrier.
  • the disclosure provides a pharmaceutical composition comprising the modified cell described herein and a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified cells can be administered.
  • the modified cells can be immediately used in the above therapeutic, experimental or commercial applications or the cells can be cryopreserved for use at a later date.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the modified cells disclosed herein can be formulated in unit dosage forms suitable for single administration of precise dosages.
  • the unit dosage forms may comprise additional lymphocytes.
  • 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.
  • the pharmaceutical composition may 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.
  • the disclosure provides a method of transplantation in a patient in need thereof comprising the step of administering to the patient an effective amount of the cells of the disclosure for transplantation.
  • the B2M -/- cells do not express wildtype HLA Class I protein on the cell surface, the cells when administered to a patient elicit minimal or no immune responses in the patient.
  • transplantation using the B2M -/- cells limits the need for taking immune suppressant therapies.
  • the patient is immune competent.
  • the cell is an isogeneic cell.
  • the cell is an allogeneic cell.
  • the lentivirus packaging plasmid mixture including pCMV- ⁇ R-8.47 and pMD2. G (Addgene, Cat#: 12259) was mixed with the appropriate construct-encoding plasmid at a pre-optimized ratio with polyethyleneimine.
  • HEK293 cells were transfected with the mixture and were cultured overnight. The culture supernatant was collected and centrifuged to remove cell debris. The supernatant was filtered through a 0.45 ⁇ m PES filter. The virus particles were pelleted and rinsed with pre-chilled DPBS. The virus was aliquoted and stored at -80°C immediately. The virus titer was determined by measuring SUP-T1 cell line transduction efficiency using the FACSCelesta TM Cell Analyzer (BD) .
  • PBMCs Peripheral blood mononuclear cells
  • Ficoll-Paque TM PLUS Media (Cytiva, Cat#: 17144002)
  • PBMCs were subsequently pre-activated for 48 hours in 24 Well Plates (Wilson Wolf, P/N 80192M) with 3 ⁇ 10 6 T cells/well.
  • the pre-activated PBMCs were transduced with the lentivirus stock described above. The cells were cultured for approximately 72 hours at 37°C.
  • the transduced cells were then electroporated with ribonucleoprotein (RNP) complex (180 pmol of sgRNA (GenScript) targeting B2M (SEQ ID NO: 21) and 60 pmol of Cas9 Nuclease (KACTUS, KACTUS-CAS9) ) and Human T Cell Kit (Lonza, Cat#: VPA-1002) using the 2B Nucleofector TM system (Lonza) and the program T-020, 3 ⁇ 10 6 cells for each electroporation response (see Ren, J., et al. "Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. " Clinical Cancer Research 23.9 (2017) : 2255-2266) .
  • RNP ribonucleoprotein
  • Electroporated cells were transferred into new 24 Well Plates for another 6-7 days of culture, with half of the culture medium changed every 2-3 days.
  • Brilliant Violet 785 TM anti-human CD3 BioLegend, Cat#: 344842
  • Brilliant Violet 421 TM anti-human TCR V ⁇ 2 Antibody BioLegend, Cat#: 3314278
  • mouse anti-camel sdAb mouse anti-camel sdAb
  • APC anti-human ⁇ 2-microglobulin Antibody BioLegend, Cat#: 316312
  • Brilliant Violet 421 TM anti-human HLA-E Antibody BioLegend, Cat#: 342612
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs Peripheral blood mononuclear cells
  • Pan T cells were isolated from PBMCs using Pan T Cell Isolation Kit (Miltenyi, Cat#: 130-096-535) .
  • Isolated pan T cells were then activated using T Cell Activation/Expansion Kit (Miltenyi, Cat#: 130-091-441) . After 24 hours of activation, T cells were transduced with the lentivirus stock described above. The cells were cultured for approximately 72 hours at 37°C.
  • the transduced cells were then electroporated with ribonucleoprotein (RNP) complex (180 pmol of sgRNA (GenScript) targeting B2M (SEQ ID NO: 21) and 60 pmol of Cas9 Nuclease (KACTUS, KACTUS-CAS9) ) and Human T Cell Kit (Lonza, Cat#: VPA-1002) using the 2B Nucleofector TM system (Lonza) and the program T-020, 3 ⁇ 10 6 cells for each electroporation response (see Ren, J., et al. "Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. " Clinical Cancer Research 23.9 (2017) : 2255-2266) .
  • RNP ribonucleoprotein
  • Electroporated cells were transferred into new 24 Well Plates for another 6-7 days of culture, with half of the culture medium changed every 2-3 days.
  • Brilliant Violet 785 TM anti-human CD3 and mouse anti-camel sdAb (GenScript, Piscataway, NJ) were added to detect the cell surface CD3 and sdAb, respectively.
  • APC anti-human ⁇ 2-microglobulin Antibody and Brilliant Violet 421 TM anti-human HLA-E Antibody were added to detect the cell surface B2M and HLA-E, respectively.
  • B2M KO CAR ⁇ T and CAR ⁇ T were produced. All constructions described herein were linked to a truncated EF1 ⁇ promoter (SEQ ID NO: 8) , which was described in Chandler, R. J., et al. "Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1. " Human Molecular Genetics 26.1 (2017) : 52-64.
  • Exemplary CLL1CAR (SEQ ID NO: 3) was constructed by cloning a sequence encoding CLL1-targeting sdAb into a lentiviral expression vector with the sequences encoding the intracellular co-stimulatory region of 4-1BB and the intracellular domain of CD3z.
  • Exemplary CD33/CLL1CAR (SEQ ID NO: 4) was constructed by fusing a CD33-targeting sdAb with Toll-like receptor 4 (TLR4) intracellular domain and the Exemplary CLL1CAR described herein via a P2A linker (SEQ ID NO: 9) .
  • Exemplary 19CAR (SEQ ID NO: 1) was constructed by cloning a sequence encoding FMC63 (see Zola, H., et al. "Preparation and characterization of a chimeric CD19 monoclonal antibody. " Immunology and Cell Biology 69.6 (1991) : 411-422) , which binds to CD19 protein, into a lentiviral expression vector with the sequences encoding the intracellular co-stimulatory region of 4-1BB and the intracellular domain of CD3z.
  • CD33/CLL1CAR ⁇ T cells In vitro cytotoxicity was compared between CD33/CLL1CAR ⁇ T cells and B2M KO CD33/CLL1CAR ⁇ T cells. Specifically, CD33/CLL1CAR ⁇ T cells or B2M KO CD33/CLL1CAR ⁇ T cells were co-incubated with CellTrace TM CFSE (Thermo Fisher, Cat#: C34554) labeled U-937 cells (ATCC, CRL-1593.2) at an E/T ratio (effector cells : target cells) of 3: 1, 1: 1, or 1: 3 for 20 hours.
  • CellTrace TM CFSE Thermo Fisher, Cat#: C34554
  • Residual cell mixtures were applied to the FACSCelesta TM Cell Analyzer (BD) after 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI, Thermo Fisher, Cat#: D1306) staining.
  • Specific cytotoxicity was calculated by subtracting the percentage of DPAI + cells in CFSE + population of the target cells-only group from that of the co-incubated groups.
  • FIG. 1A after co-incubation with target cells, both wildtype CD33/CLL1CAR ⁇ T cells and B2M knockout CD33/CLL1CAR ⁇ T cells killed target cells effectively. No significant difference in cytotoxicity was observed between groups, which indicated that B2M KO didn’ t affect the cytotoxicity of CAR- ⁇ T cells.
  • FIG. 1B B2M KO CAR-expressing ⁇ T and ⁇ T cells were applied to long-term killing assays (FIG. 1B) , in which CD33/CLL1CAR-and CLL1CAR-expressing T cells were co-incubated with U-937 cells at an E/T ratio of 1: 2, whereas 19CAR-expressing T cells were co-incubated with Raji (ATCC, CCL-86) cells at an E/T ratio of 1: 1.
  • Example 3 B2M knockout protected ⁇ T from allogenic ⁇ T cell killing and expression of HLA-E trimeric construct rescued survival of B2M knockout ⁇ T from NK cell lysis
  • Example 1 The process of CAR- ⁇ T preparation is described in Example 1.
  • Exemplary 19CAR-HLAE was constructed by fusing a HLA-E trimeric construct (SEQ ID NO: 2) and 19CAR (SEQ ID NO: 1) via a P2A linker (SEQ ID NO: 9) .
  • expression levels of CAR, B2M and HLA-E were detected by flow cytometry on Day 7, Day 11, and Day 14 after the lentiviral transduction.
  • Cell counting was also performed using the K2 Cell Counter (Nexcelom) on the same days when the transduction efficiency was determined. As shown in FIGS.
  • the percentage of B2M -/- CAR + ⁇ T cells decreased from about 10%to about 1%in the 19CAR- ⁇ T/B2M KO group during the in vitro culture, whereas the percentage of B2M -/- CAR + ⁇ T cells increased from about 10%to about 39%in the 19CAR-HLAE- ⁇ T/B2M KO group.
  • Example 1 The process of CAR- ⁇ T preparation is described in Example 1.
  • allogenic Pan T cells were isolated from healthy donor-derived PBMCs using the Pan T Isolation Kit (Miltenyi, Cat#: 130-096-535) .
  • the isolated Pan T cells were stimulated using un-transduced ⁇ T cells at a Pan T/ ⁇ T ratio of 100: 1 in the presence of Recombinant Human Interleukin-2 (IL-2; Jiangsu Kingsley Pharmaceutical, Jiangsu, China) .
  • IL-2 Human Interleukin-2
  • the stimulated Pan T cells were re-challenged with un-transduced ⁇ T cells every week for another two weeks, and the harvested T cells were named alloreactive T cells.
  • the isolated Pan T cells were stimulated using the T Cell Activation/Expansion Kit (Miltenyi, Cat#: 130-091-441) for 9 days to obtain un-alloreactive T cells. Because the obtained alloreactive T and un-alloreactive T cells mainly contained ⁇ T cells, the alloreactive T cells are also referred to as “alloreactive- ⁇ T” and the un-alloreactive T cells are also referred to as “un-alloreactive- ⁇ T” below.
  • CAR- ⁇ T and CAR-HLAE ⁇ T/B2M KO cells were co-incubated with alloreactive- ⁇ T or un-alloreactive- ⁇ T cells at an ⁇ T: ⁇ T ratio of 1: 1. After 20 hours of co-incubation, the residual percentage of CAR + ⁇ T cells and B2M -/- CAR + HLAE + ⁇ T cells were determined using the FACSCelesta TM Cell Analyzer, and cell counting were performed using the K2 Cell Counter.
  • FIG. 3A compared to un-alloreactive T cells, alloreactive T cells lysed CAR + and CAR - ⁇ T populations with no preference (see upper two panels in FIG. 3A) .
  • FIG. 3B summarizes the cytotoxicity of alloreactive T cells against 19CAR- ⁇ T and 19CAR-HLAE- ⁇ T/B2M KO cells, as compared to that when un-alloreactive T cells were used. As shown in FIG.
  • NK cells were isolated from healthy donor-derived PBMCs using a NK Cell Isolation Kit (Miltenyi, Cat#: 130092657) . Isolated NK cells were stimulated with K562 feeder cells in the presence of IL-2 for 2 weeks. Un- ⁇ T/B2M KO cells and 19CAR-HLAE- ⁇ T/B2M KO cells were co-incubated with pre-activated NK cells at an NK: ⁇ T ratio of 1: 3, 1: 1, or 3: 1 for 20 hours. As shown in FIG.
  • 19CAR-HLAE- ⁇ T/B2M KO cells were partially resistant to NK cell lysis, which indicated that expression of HLA-E trimeric construct could partially protect B2M -/- ⁇ T cells from NK cell-induced cytotoxicity.
  • Example 1 The process of CAR- ⁇ T preparation is described in Example 1.
  • Exemplary 19CAR-wt47 was constructed by fusing full-length wildtype CD47 (SEQ ID NO: 5) and 19CAR (SEQ ID NO: 1) via a P2A linker (SEQ ID NO: 9) .
  • Exemplary 19CAR-47m1-HLAE and 19CAR-47m2-HLAE were constructed by fusing CD47 mutant 1 (SEQ ID NO: 6) or CD47 mutant 2 (SEQ ID NO: 7) and 19CAR-HLAE as described in Example 3 via a P2A linker (SEQ ID NO: 9) , respectively.
  • the percentage of B2M -/- CAR- ⁇ T cells within the 19CAR-47m1-HLAE and 19CAR-47m2-HLAE groups were above 60%, whereas the percentage was under 30%in the 19CAR, 19CAR-HLAE and 19CAR-wt47 groups.
  • the percentage in 19CAR-47m1 and 19CAR-47m2 groups were not comparable to that of the 19CAR-47m1-HLAE or 19CAR-47m2-HLAE group, they were much more than that of the 19CAR or 19CAR-HLAE group.
  • the expansion results (FIG. 5B) showed that 19CAR-47m1-HLAE and 19CAR-47m2-HLAE ⁇ T cells expanded much more than other groups.

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Abstract

La présente divulgation concerne des méthodes d'expansion de lymphocytes T γδ. Plus spécifiquement, la divulgation concerne des procédés d'expansion de lymphocytes T γδ qui présentent une expression éliminée ou réduite des molécules endogènes du CMH de classe I, par expression d'une protéine de fusion HLA de classe I à chaîne unique. Dans certains modes de réalisation, le gène B2M endogène des lymphocytes T γδ est interrompu. Dans certains modes de réalisation, la protéine de fusion HLA de classe I à chaîne unique comprend au moins une partie de la protéine B2M et au moins une partie de la chaîne lourde HLA-E.
PCT/CN2025/091116 2024-04-26 2025-04-25 Méthodes d'expansion de lymphocytes t Pending WO2025223534A1 (fr)

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