WO2022063302A1 - 免疫细胞活性调节 - Google Patents

免疫细胞活性调节 Download PDF

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WO2022063302A1
WO2022063302A1 PCT/CN2021/121016 CN2021121016W WO2022063302A1 WO 2022063302 A1 WO2022063302 A1 WO 2022063302A1 CN 2021121016 W CN2021121016 W CN 2021121016W WO 2022063302 A1 WO2022063302 A1 WO 2022063302A1
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cells
antibody
cell
seq
immune
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PCT/CN2021/121016
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English (en)
French (fr)
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李宗海
王鹏
刘妹
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克莱格医学有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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

Definitions

  • the present invention relates to the killing of NK cells by immune cells expressing chimeric antibodies to CS1 and also relates to methods for developing engineered reactive T cells for immunotherapy, and more particularly to increasing the persistence of allogeneic immune cells and/or method of implantation.
  • the present invention also relates to engineered immune cells and functional derivatives thereof, chimeric antigen receptors (CARs), multi-chain CARs and their use for enhancing the efficiency of immunotherapy.
  • CARs chimeric antigen receptors
  • the second direction is to eliminate the major histocompatibility antigen of allogeneic T cells.
  • the commonly used method is to knock out the B2M of allogeneic T cells.
  • the knockout of B2M makes the diverse HLA-ABC proteins unable to be expressed on the cell membrane.
  • host T cells are prevented from attacking it, but the deletion of HLA-I molecules will lead to the elimination of HLA-I-deficient cells by host NK cells. Therefore, in order to improve the survival of allogeneic T cells in vivo for a longer time and better exert their antitumor effect, it is urgent to develop new strategies to resist the elimination of allogeneic T cells by host T cells or NK cells.
  • the present invention constructs a CAR targeting CS1 or a bispecific antibody construct targeting host NK cells and host and/or donor T cells to kill or eliminate NK cells, and/or resist NK cells in the host against the same species Killing of allogeneic T cells; in addition, in order to further enhance the clearance of strongly active NK cells that may exist in the host by allogeneic T cells, the CS1 CAR T cells or bispecific antibody constructs can simultaneously express exogenous NK cells Ligands or antibody fragments of inhibitory receptors, such as NKG2A.
  • the present invention provides a method for effectively solving the rejection of B2M knockout allogeneic T cells by host NK cells and a related pharmaceutical composition and preparation thereof, so as to increase the immunity of allogeneic immune cells in the presence of host immune cells. Persistence and/or graft survival.
  • the method or related pharmaceutical composition provided by the present invention can significantly improve the anti-tumor or anti-infection curative effect of the combined anti-tumor or anti-infection CAR-T cells.
  • the first aspect of the present invention provides the use of an engineered immune cell expressing CS1-targeting chimeric receptor 1 for preparing a drug for killing or eliminating NK cells.
  • MHC expression, activity and/or signaling in said immune cells is reduced or inhibited.
  • the MHC is MHC class I molecule; more preferably, the MHC I is HLA; more preferably, the HLA is HLA-I; more preferably, the HLA-I is selected from HLA-A, One or more of HLA-B, HLA-C, and B2M; most preferably, the HLA-I includes HLA-A and B2M;
  • said being reduced or inhibited is by using TAL nucleases, meganucleases, zinc finger nucleases, Cas9 and Argonaute;
  • the immune cells comprise inhibitory nucleic acid molecules or gRNA molecules targeting genes encoding MHC;
  • the inhibitory nucleic acid molecule comprises a sequence complementary to the gene encoding the MHC;
  • the inhibitory nucleic acid comprises an RNA interfering agent
  • the inhibitory nucleic acid comprises siRNA, shRNA or miRNA
  • the sgRNA sequence contains the sequences shown in SEQ ID NOs: 17 and 18;
  • said reduction in MHC expression, activity and/or signaling is permanent, transient or inducible
  • the expression, activity and/or signaling of MHC in the engineered immune cell is reduced by greater than or greater than about 50% compared to the expression, activity and/or signaling of MHC in the unengineered immune cell, 60%, 70%, 80%, 90%, 95% or 100%;
  • the expression of MHC expressed in the immune cells is undetectable using immunoblot assays and/or in flow cytometry.
  • the chimeric receptor 1 comprises: chimeric antigen receptor 1 (CAR1), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell Antigen Coupler (TAC), aTCR-T, or a combination thereof.
  • CAR1 chimeric antigen receptor 1
  • TCR modified T cell
  • TCP T cell fusion protein
  • TAC T cell Antigen Coupler
  • the CAR1 comprises:
  • the nucleic acid molecule of the antibody that specifically recognizes CS1 is at least 80%, preferably 90%, and more preferably 95% identical to SEQ ID NO: 11; or the amino acid sequence of the antibody that specifically recognizes CS1 At least 80%, preferably 90%, and more preferably 95% identical to SEQ ID NO: 12.
  • the nucleic acid molecule of the antibody that specifically recognizes CS1 has at least 80%, 90%, or 95% identity with SEQ ID NO: 11; or the amino acid sequence of the antibody that specifically recognizes CS1 is identical to SEQ ID NO: 11 NO: 12 is at least 80%, 90%, or 95% identical.
  • the immune cells further express ligands or antibody fragments of membrane-bound NK cell inhibitory receptors.
  • the immune cells also express membrane-bound NKG2A antibody or antibody fragment;
  • the membrane-bound NKG2A antibody or antibody fragment comprises a sequence that is at least 80%, 90%, or 95% identical to SEQ ID NO: 14 or 38;
  • nucleic acid molecule of the membrane-bound NKG2A antibody or antibody fragment is at least 80%, 90%, or 95% identical to SEQ ID NO:13.
  • the endogenous CS1 gene of the immune cell is knocked out, preferably using CRISPR/Cas9 technology to knock out the endogenous CS1 gene of the immune cell.
  • the gRNA used in CRISPR/Cas9 technology is shown in SEQ ID NO: 19, 20, 40, 41, 42, 43, 44 and/or 45.
  • the immune cells are administered in combination with T cells expressing non-CS1-targeting chimeric antigen receptors or the immune cells also express non-CS1-targeting chimeric antigen receptors,
  • the non-CS1 targeting chimeric antigen receptor targets tumor or pathogen antigens
  • the non-CS1 targeting chimeric antigen receptor targets BCMA, CD19, GPC3, Claudin18.2, EGFR, EGFRvIII or a combination thereof.
  • the immune cells are derived from natural T cells and/or T cells induced by pluripotent stem cells;
  • the T cells are autologous/allogeneic T cells
  • the T cells are primary T cells
  • the T cells are derived from human autologous T cells.
  • the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), and effector memory T cells. cells (Tem), ⁇ T cells, or a combination thereof.
  • the endogenous MHC and endogenous TCR of the immune cells are knocked out, preferably by using CRISPR/Cas9 technology to knock out the endogenous MHC and endogenous TCR.
  • the gRNA used for knocking out B2M using CRISPR/Cas9 technology is shown in SEQ ID NO: 18, and/or the gRNA used for knocking out TRAC is shown in SEQ ID NO: 17.
  • the immune cells can enhance the killing of tumor cells by T cells and/or CAR-T cells that are introduced into the subject before, at the same time, and later, and enhance the killing of tumor cells. Survival and proliferation of T cells and/or CAR-T cells.
  • the immune cells are administered in combination with an agent that enhances their function, preferably, in combination with a chemotherapeutic agent;
  • the immune cells are administered in combination with an agent that ameliorates one or more side effects associated therewith.
  • the non-targeting CS1 chimeric receptors include: chimeric antigen receptor (CAR) 2, modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen Coupler (TAC), aTCR-T, or a combination thereof;
  • CAR chimeric antigen receptor
  • TCR modified T cell
  • TCP T cell fusion protein
  • TAC T cell antigen Coupler
  • the CAR2 comprises:
  • the tumor antigens include Claudin18.2, Claudin18.1, Claudin 6, vascular endothelial growth factor receptor, glypican-3 (GPC3), B cell maturation antigen (BCMA), carbonic anhydrase 9(CAIX), tEGFR, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelin 2 (EPG) -2), Epiglin 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor body, GD2, GD3, HMW-MAA, IL-22R- ⁇ , IL-13R- ⁇ 2, kinase insertion domain receptor (k
  • the infected cell antigens include pathogen antigens and viral antigens;
  • the pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites;
  • the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen;
  • the immune cells when the immune cells are co-cultured with host NK cells, the immune cells are capable of killing host NK cells.
  • the immune cells when the immune cells are co-cultured with host NK cells, the immune cells can resist the killing of the immune cells by host NK cells;
  • the immune cells are resistant to killing of the immune cells by NK cells activated by cytokines in host NK cells;
  • the immune cells are resistant to killing of the immune cells by host NK cells expressing NKG2A;
  • the immune cells can significantly resist the killing of the immune cells by host NK cells that express low NKG2A.
  • the immune cells are administered in combination with an agent that enhances their function, preferably, in combination with a chemotherapeutic drug; or the immune cell is administered in combination with an agent that ameliorates one or more side effects associated therewith .
  • a second aspect of the present invention provides a method for increasing the persistence and/or transplantation survival rate of allogeneic immune cells in the presence of host immune cells, comprising:
  • CRISPR/Cas9 technology is used to knock out endogenous MHC and endogenous TCR.
  • the CRISPR/Cas9 technology is used to knock out the gRNA used by B2M as shown in SEQ ID NO: 18, and/or the gRNA used by TRAC as shown in SEQ ID NO: 17.
  • the chimeric receptor 1 includes: chimeric antigen receptor 1 (CAR1), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen Coupler (TAC), aTCR-T, or a combination thereof.
  • CAR1 chimeric antigen receptor 1
  • TCR modified T cell
  • TCP T cell fusion protein
  • TAC T cell antigen Coupler
  • the CAR1 comprises:
  • the nucleic acid molecule of the antibody that specifically recognizes CS1 has at least 80%, 90%, or 95% identity with SEQ ID NO: 11; or the amino acid sequence of the antibody that specifically recognizes CS1 is identical to SEQ ID NO: 11 NO: 12 is at least 80%, 90%, or 95% identical.
  • step d) modifying the cell with a non-endogenous polynucleotide encoding a ligand or antibody fragment of a membrane-bound NK cell inhibitory receptor.
  • step d) modifying the cell with a non-endogenous polynucleotide encoding the immune cell membrane-bound NKG2A antibody or antibody fragment;
  • the NKG2A antibody or antibody fragment comprises a sequence that is at least 80%, 90%, or 95% identical to SEQ ID NO: 14 or 38;
  • nucleic acid molecule of the membrane-bound NKG2A antibody or antibody fragment is at least 80%, 90%, or 95% identical to SEQ ID NO:13.
  • the gRNA used in CRISPR/Cas9 technology is the sequence shown in SEQ ID NO: 19, 20, 40, 41, 42, 43, 44 and/or 45.
  • step e) encoding a non-endogenous polynucleotide targeting tumor antigens, and/or pathogen antigens, and/or viral antigens, chimeric antigen receptors to modify the cells;
  • the tumor antigens include BCMA, CD19, GPC3, Claudin18.2, EGFR, EGFRvIII or a combination thereof.
  • the immune cells are derived from natural T cells and/or T cells induced by pluripotent stem cells;
  • the T cells are autologous/allogeneic T cells
  • the T cells are primary T cells
  • the T cells are derived from human autologous T cells.
  • the immune cells prepared by the method when the immune cells prepared by the method are co-cultured with host NK cells, the immune cells can kill host NK cells.
  • the immune cells prepared by the method are administered in combination with an agent that enhances their function, preferably, in combination with a chemotherapeutic drug;
  • the immune cells produced by the method are administered in combination with an agent that ameliorates one or more side effects associated therewith.
  • a method of increasing the persistence and/or engraftment survival of allogeneic immune cells in the presence of host immune cells comprising:
  • the polypeptide in step b) is selected from MHC, and the MHC is an MHC class I molecule; more preferably, the MHC I is HLA; more preferably, the HLA is HLA- I; more preferably, the HLA-I is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M; most preferably, the HLA-I includes HLA-A and B2M .
  • the tumor antigens include Claudin18.2, Claudin18.1, Claudin 6, vascular endothelial growth factor receptor, glypican-3 (GPC3), B cell maturation antigen (BCMA), carbonic anhydrase 9(CAIX), tEGFR, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelin 2 (EPG) -2), Epiglin 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor body, GD2, GD3, HMW-MAA, IL-22R- ⁇ , IL-13R- ⁇ 2, kinase insertion domain receptor (k
  • the infected cell antigens include pathogen antigens and viral antigens;
  • the pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites;
  • the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.
  • step e) modifying the cell with a non-endogenous polynucleotide encoding a ligand or antibody fragment of an NK cell inhibitory receptor;
  • step e) is further included to modify the cell by a non-endogenous polynucleotide encoding the immune cell NKG2A binding molecule;
  • the NKG2A binding molecule is a cell membrane-bound protein or a secreted protein
  • the NKG2A binding molecule comprises only an extracellular domain, a transmembrane domain; or an extracellular domain, a transmembrane domain and an intracellular domain;
  • the NKG2A binding molecule is an NKG2A antibody or antibody fragment bound to the cell membrane;
  • the nucleic acid molecule of the NKG2A binding molecule is at least 80%, preferably 90%, and more preferably 95% identical to SEQ ID NO:13; or the NKG2A amino acid sequence is at least 80% identical to SEQ ID NO:14 %, preferably 90%, and more preferably 95% identity.
  • the immune cells prepared by the method when the immune cells prepared by the method are co-cultured with host NK cells, the immune cells can kill host NK cells.
  • the immune cells prepared by the method when the immune cells prepared by the method are co-cultured with host NK cells, the immune cells can resist the killing of the immune cells by host NK cells;
  • the immune cells are resistant to killing of the immune cells by NK cells activated by cytokines in host NK cells;
  • the immune cells are resistant to killing of the immune cells by host NK cells expressing NKG2A;
  • the immune cells can significantly resist the killing of the immune cells by host NK cells that express low NKG2A.
  • the immune cells prepared by the method are administered in combination with an agent that enhances their function, preferably, in combination with a chemotherapeutic drug; or the immune cells prepared by the method are administered in combination with improving one or more of the related drugs. co-administration of drugs with side effects.
  • a third aspect of the present invention provides a bispecific antibody construct, characterized in that the antibody construct comprises a first binding domain of CS1 bound to the surface of target cells and a first binding domain of CD3 bound to the surface of T cells second binding domain.
  • the first binding domain binds to human or rhesus monkey CS1; and/or the second binding domain binds to human CD3 ⁇ , common marmoset, cotton top marmoset or squirrel monkey CD3 ⁇ .
  • the antibody construct is selected from the following formats: scFv, (scFv) 2 , scFv-single domain mAbs, diabodies and oligomers thereof.
  • the VH region contained in the first binding domain contains CDR1 shown in SEQ ID NO: 21, CDR2 shown in SEQ ID NO: 22 and CDR3 shown in SEQ ID NO: 23,
  • the VL region contained in a binding domain contains CDR1 shown in SEQ ID NO: 24, CDR2 shown in SEQ ID NO: 25 and CDR3 shown in SEQ ID NO: 26;
  • the VH region of the first binding domain is shown in SEQ ID NO:27 and the VL region is shown in SEQ ID NO:28.
  • the antibody construct comprises, in order from N-terminus to C-terminus:
  • the antibody construct comprises the amino acid sequence shown in SEQ ID NO:31.
  • a fourth aspect of the present invention provides a polynucleotide encoding an antibody construct as defined in the third aspect of the present invention.
  • a fifth aspect of the present invention provides a vector comprising the polynucleotide as defined in the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides an immune cell transformed or transfected with the polynucleotide as defined in the fourth aspect of the present invention or with the vector as defined in the fifth aspect of the present invention;
  • the immune cells are capable of secreting the construct according to the third aspect of the present invention.
  • MHC expression, activity and/or signaling in said immune cells is reduced or inhibited, and the construct according to the third aspect of the invention is secreted;
  • the immune cells also express chimeric receptors targeting tumor antigens, and/or pathogen antigens, and/or viral antigens;
  • the CAR includes:
  • the immune cells also express ligands or antibody fragments of NK cell inhibitory receptors;
  • the immune cells also express NKG2A binding molecules;
  • the NKG2A binding molecule is a cell membrane-bound protein or a secreted protein
  • the NKG2A binding molecule comprises only an extracellular domain and a transmembrane domain; or comprises an extracellular domain, a transmembrane domain and an intracellular domain;
  • the NKG2A binding molecule is an NKG2A antibody or antibody fragment bound to the cell membrane;
  • the nucleic acid molecule of the NKG2A binding molecule shares at least 80%, preferably 90%, and more preferably 95% identity with SEQ ID NO: 13; or the NKG2A amino acid sequence shares at least 80% with SEQ ID NO: 14 %, preferably 90%, and more preferably 95% identity.
  • a seventh aspect of the invention provides a method for producing an antibody construct according to the third aspect of the invention, said method comprising culturing under conditions allowing expression of said antibody construct as defined in the third aspect of the invention The host cell as defined in the sixth aspect of the invention and the recovery of the antibody construct produced from said culture.
  • the eighth aspect of the present invention provides a pharmaceutical composition comprising the antibody construct according to the third aspect of the present invention or the antibody construct produced according to the method of the seventh aspect of the present invention.
  • An eighth aspect of the present invention provides a method of increasing the persistence and/or transplantation survival rate of allogeneic immune cells in the presence of host immune cells, comprising administering to a subject in need thereof according to the third aspect of the present invention The steps of the antibody construct or the antibody construct produced according to the method of the seventh aspect of the invention.
  • the ninth aspect of the present invention provides a kit, which comprises the antibody construct according to the third aspect of the present invention, the antibody construct produced according to the method of the seventh aspect of the present invention, as described in the fourth aspect of the present invention.
  • Figure 1 Schematic diagram of PRRLsin-CS1-BBZ, PRRLSIN-NKG2A, PRRLsin-CS1-BBZ-F2A-NK G2A, PRRLsin-BCMA-BBZ, PRRLsin-BCMA-BBZ-F2A-NKG2A plasmids;
  • FIG. 2A Detection of knockout efficiency of TCR/B2M knockout and TCR/B2M/CS1 knockout T cells
  • Figure 2B The proportion of TCR/B2M knockout and TCR/B2M/CS1 knockout T cells after negative enrichment
  • FIG. 3A and 3B Detection of CAR positive rate in TCR/B2M knockout and TCR/B2M/CS1 knockout T cells;
  • FIG. 6A and Figure 6B Detection of the killing ability of CS1 CAR-T cells to human primary NK cells
  • FIG. 7A and Figure 7B Detection of the killing ability of CS1 CAR-T cells to human primary NK cells after co-culture for 24 hours and 48 hours;
  • FIG. 1 Schematic diagram of PRRLsin-CS1-CD3 BITE plasmid
  • Figure 14 Killing assay of co-cultured NK cells by T-BITE cell culture supernatant.
  • autologous or allogeneic T cells can express CS1-CAR, express/secrete CS1-CD3 bispecific antibody constructs, or utilize a stand-alone CS1-CD3 bispecific antibody.
  • the constructs can significantly enhance killing of host NK cells, clear host quiescent or activated NK cells, thereby increasing the persistence and/or engraftment survival of allogeneic T cells in the presence of host immune cells.
  • any concentration range, percentage range, ratio range, or integer range described herein should be understood to include any integer within the stated range, as well as, where appropriate, fractions thereof (eg, one tenth of an integer and one percent).
  • CS1 (also known as SLAMF7, CD319 or CRACC-NCBI reference sequence: NP_067004.3) is a member of the lymphocyte activation molecule family 7, involved in cell adhesion and NK cell activation, mainly expressed in plasma cells, NK cells, CD8+ In T cells, activated B cells and mononuclear dendritic cells, it is basically not expressed in hematopoietic lineage progenitor cells and other human tissues.
  • the CS1 molecule is also highly expressed in multiple myeloma (MM) cells, and its monoclonal antibody, Elotuzumab (huLuc63), is used in combination with immunomodulators and proteasome inhibitors in the treatment of relapsed or refractory MM patients.
  • Persistence refers to the ability of a cell to resist rejection and to maintain and/or increase a number (eg, days, weeks, months, years) in the body over time.
  • the engineered immune cells of the invention are found in the patient's blood for at least 10 days, preferably at least 20 days, more preferably at least 25 days, even more preferably at least 30 days after infusion into said patient .
  • Increasing persistence and/or engraftment means that during the course of treatment, allogeneic immune cells engineered so that they persist, compared to the situation in which non-engineered immune cells are administered to the patient (ie, non-persistent) The number remains high.
  • This enhanced persistence and/or engraftment of allogeneic immune cells (eg, T cells) to be injected into the patient is part of immune tolerance (or “tolerisation”), which An unresponsive state of the host immune system relative to the immune cells is described, while the immune cells retain the ability to elicit an immune response.
  • cell refers to cells of human or non-human animal origin.
  • the engineered cells or engineered cells refer to T cells expressing CS1-CAR.
  • the term "host” or “subject” refers to the recipient of the transplant, which, in some embodiments, may be an individual, such as a human, who has received the engraftment of exogenous cells.
  • a "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, and the like.
  • the subject may be suspected of having a disease characterized by cellular proliferation or having a disease characterized by cellular proliferation, be diagnosed with a disease characterized by cellular proliferation, or be confirmed not to have a disease characterized by cellular proliferation. Control subjects with disease characterized by proliferation.
  • the subject is or may be suffering from an immune disease, such as an autoimmune disease, or following treatment with a transplant.
  • engineing refers to the application of the principles and methods of cell biology and molecular biology, through some engineering means, at the overall level of cells or at the level of organelles, to change the genetic material in cells or obtain cells according to people's wishes.
  • the terms “genetic modification”, “genetic modification”, “genetically engineered” or “modified” refer to methods of modifying cells, including but not limited to, by means of gene editing, in the coding or non-coding regions of genes or their expression regulatory regions. ; Or through endonuclease and/or antisense RNA technology; or increase the introduction of exogenous proteins and/or complexes, small molecule inhibitors to change the protein expression level of the gene to cause gene defects.
  • modified cells refers to a change in the state or structure of a protein or polypeptide of the invention. Modifications can be chemical, structural and functional.
  • the modified cells are stem cells (eg, hematopoietic stem cells (HSC) or progenitor cells, embryonic stem cells (ES), induced pluripotent stem (iPS) cells), lymphocytes (eg, T cells), which can is obtained from the subject or donor.
  • Cells can be modified to express foreign constructs, such as pre-TCR ⁇ protein, chimeric antigen receptor (CAR) or T cell receptor (TCR), which can be integrated into the cell genome.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • pluripotent stem cell has the potential to differentiate into any of the three germ layers: endoderm (eg, gastric junction, gastrointestinal tract, lung, etc.), mesoderm (eg, muscle, bone, blood, urogenital tissue, etc.) ) or ectoderm (eg epidermal tissue and nervous system tissue).
  • endoderm eg, gastric junction, gastrointestinal tract, lung, etc.
  • mesoderm eg, muscle, bone, blood, urogenital tissue, etc.
  • ectoderm eg epidermal tissue and nervous system tissue.
  • the term “pluripotent stem cell” also encompasses "induced pluripotent stem cell” or "iPSC,” a type of pluripotent stem cell derived from a non-pluripotent cell.
  • the pluripotent stem cells are derived from cells that have the characteristics of pluripotent stem cells by reprogramming somatic cells. Such "iPS” or “iPSC” cells can be generated by induc
  • pluripotent stem cell characteristics refers to cell characteristics that distinguish pluripotent stem cells from other cells.
  • human pluripotent stem cells express at least several of the following markers: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin Protein, UTF-1, Oct4, Rex1 and Nanog. Having a cell morphology associated with pluripotent stem cells is also characteristic of pluripotent stem cells.
  • non-endogenous polypeptide refers to a polypeptide not normally expressed by a donor immune cell, preferably a polypeptide expressed from an exogenous polynucleotide that has been introduced into the immune cell's genome.
  • exemplary, non-endogenous polypeptides are bispecific antibody constructs targeting CS1 and CD3.
  • immune cell refers to a cell involved in an immune response that produces an immune effect, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, etc.
  • the immune cells are T cells, NK cells, NKT cells.
  • the T cells can be autologous T cells, xenogeneic T cells, allogeneic T cells.
  • the NK cells are allogeneic NK cells.
  • artificially engineered cells with immune effector cell function refers to a cell or cell line without immune effector that has acquired immune effector cell function after being artificially engineered or stimulated by a stimulus.
  • 293T cells are artificially modified to have the function of immune effector cells; for example, stem cells are induced in vitro to differentiate into immune effector cells.
  • T cells can be PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue and natural T cells obtained from infection sites, ascites, pleural effusion, spleen tissue, tumor tissue, and can also be A population of cells with specific phenotypic characteristics obtained by sorting, etc., or a mixed population of cells with different phenotypic characteristics, such as "T cells” can be cells comprising at least one subset of T cells: memory stem-like T cells (stem). cell-like memory T cells, Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells (Tem).
  • Tstem memory stem-like T cells
  • Tscm cells central memory T cells
  • effector T cells Tef, Teff
  • Tregs regulatory T cells
  • Tregs effector memory T cells
  • a "T cell” may be a particular subtype of T cell, such as ⁇ T cells.
  • T cells can be obtained from blood collected from an individual using any number of techniques known to those of skill in the art, such as FicollTM separation and/or apheresis.
  • the T cells are derived from induced pluripotent stem cells.
  • cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps.
  • the T cells can be derived from a healthy donor, or from an individual diagnosed with cancer.
  • T cells can be autologous T cells or allogeneic T cells.
  • the T cells can be primary T cells.
  • T cells can also be T cells that carry exogenous proteins that recognize target antigens, such as CAR T cells, TCR-T cells, T cell antigen couplers (TACs), T cell fusion proteins, and the like.
  • target antigens such as CAR T cells, TCR-T cells, T cell antigen couplers (TACs), T cell fusion proteins, and the like.
  • MHC histocompatibility complex
  • HLA antigens in human cells. Rejection is mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.
  • MHC proteins play a crucial role in T cell stimulation, antigen-presenting cells (usually dendritic cells) display peptides that are degradation products of foreign proteins on the cell surface on the MHC, in the presence of a costimulatory signal , T cells are activated and act on target cells that also display the same peptide/MHC complex.
  • stimulated T helper cells will target macrophages that display antigens bound to their MHC, or cytotoxic T cells (CTLs) will act on virus-infected cells displaying exogenous viral peptides.
  • MHC antigens are divided into NHC class I antigens and MHC class II antigens.
  • NKG2A (OMIM 161555, the entire disclosure of which is hereby incorporated by reference) is a member of the NKG2 transcriptome, which together with CD94 forms in NK cells, alpha/beta T cells, gamma/delta T cells, and subsets of NKT cells The heterodimeric inhibitory receptor CD94/NKG2A found on the surface.
  • NKG2A refers to the NKG2A gene or encoded protein and any variant, derivative or isoform.
  • NKG2A/CD94 is a heterodimer and is the receptor of non-classical HLA-I molecule HLA-E, which is distributed on the surface of most NK cells and plays an inhibitory role. After binding to HLA-E, NKG2A transmits inhibitory signals to suppress the cytotoxic activity of these immune cells, thereby attenuating T cell clearance of viruses such as polyoma or human cytomegalovirus, or suppressing immune cells against tumors killing of cells.
  • HLA human leukocyte antigen
  • HLA Human leukocyte antigen
  • HLA human leukocyte antigen
  • HLA is the gene encoding the major histocompatibility complex in humans, located on chromosome 6 (6p21.31), and is closely related to the function of the human immune system.
  • HLA includes class I, class II and class III gene portions.
  • the antigens expressed by HLA class I and class II genes are located on the cell membrane and are MHC-I (encoded by HLA-A, HLA-B, HLA-C sites) and MHC-II (encoded by HLA-D region),
  • HLA I Class II is distributed on the surface of almost all cells in the body and is a heterodimer composed of heavy chain ( ⁇ chain) and ⁇ 2 microglobulin (B2M).
  • Class II is mainly a glycoprotein located on the surface of macrophages and B lymphocytes .
  • B2M beta-2 microglobulin, also known as B2M, the light chain of an MHC class I molecule.
  • B2M is encoded by the b2m gene located on chromosome 15, as opposed to other MHC genes located as gene clusters on chromosome 6.
  • Studies have shown that when the B2M gene is mutated, hematopoietic grafts from mice lacking normal cell surface MHC I expression are rejected by NK cells in normal mice, suggesting that defective expression of MHC I molecules predisposes cells to the host immune system. exclusion (Bix et al. 1991).
  • chimeric receptor refers to a fusion molecule formed by linking DNA fragments or protein corresponding cDNAs from different sources by gene recombination technology, including extracellular domain, transmembrane domain and intracellular domain.
  • Chimeric receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor (TCR), T Cell Antigen Coupler (TAC).
  • chimeric antigen receptor includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and/or a costimulatory molecule, in one aspect, the stimulatory molecule is a zeta chain bound to a T cell receptor complex; in one aspect, a cytoplasmic signaling Conductive domains further include functional signaling domains of one or more costimulatory molecules, eg, 4-1BB (ie, CD137), CD27, and/or CD28.
  • T cell receptor mediates T cell recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens, including classical TCR receptors and optimized TCR receptors. body.
  • the classic TCR receptor is composed of two peptide chains, ⁇ and ⁇ . Each peptide chain can be divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region. The specificity exists in the V region, and the V region (V ⁇ , V ⁇ ) has three hypervariable regions, CDR1, CDR2, and CDR3.
  • T cells expressing the classic TCR can be stimulated by antigens. Induce specificity of T-cell TCRs for target antigens.
  • chimeric T cell receptor includes recombinant polypeptides derived from the various polypeptides that make up the TCR, which are capable of binding to surface antigens on target cells and interacting with other polypeptides of the intact TCR complex, usually co-localized at T cell surface.
  • the chimeric T cell receptor is composed of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, the TCR cell
  • the stimulation domain of the intracellular signaling domain of the intradomain; the TCR subunit is effectively linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3 ⁇ or CD3 ⁇ , CD3z , the alpha chain of TCR, or the beta chain of TCR, and the chimeric T cell receptor integrates into the TCR/CD3 complex expressed on T cells.
  • T cell antigen coupler includes three functional domains: 1. Antigen binding domain, including single-chain antibody, designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) or other targeting groups; 2. the extracellular domain, a single-chain antibody that binds to CD3, thereby making the TAC receptor close to the TCR receptor; 3. the transmembrane region and the intracellular region of the CD4 co-receptor, wherein , the intracellular domain-linked protein kinase LCK, catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • signaling domain also known as “cytoplasmic signaling domain” refers to the functional portion of a protein that acts by transmitting information within a cell, either by producing a second messenger or by responding to such a messenger. Effector action modulates the activity of cells via defined signaling pathways.
  • the intracellular signaling domain may include the entire intracellular portion of the protein, or the entire native intracellular signaling domain, or functional fragments or derivatives thereof.
  • the signaling domain of a CS1-targeting, BCMA-targeting CAR includes CD3 ⁇ .
  • CD3 ⁇ is a human CD3 ⁇ molecule comprising the sequence shown in SEQ ID NO:5 or SEQ ID NO:6.
  • costimulatory molecule is a cell surface molecule and its ligands that provide a costimulatory signal for full activation of T (or B) cells, and when combined with a cell stimulatory signal molecule, such as TCR/CD3, the combination results in T cell proliferation and/or Signals of up- or down-regulation of key molecules, thereby mediating the co-stimulatory response of T cells.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137) .
  • CD137 is a human CD137 molecule comprising the sequence shown in SEQ ID NO:7 or SEQ ID NO:8.
  • activation and “activation” are used interchangeably and can refer to the process by which cells change from a resting state to an active state. This process may include responses to phenotypic or genetic changes in antigenic, migratory and/or functionally active states.
  • activation can refer to the process of stepwise activation of T cells. The activation process is co-regulated by the first stimulatory signal and the co-stimulatory signal. Activation of T cells is a dynamic process, and its duration and degree of activation are affected by external stimulation.
  • T cell activation or “T cell activation” refers to the state of T cells that are stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • the engineered T cells are co-incubated with tumor cells containing a specific target antigen or activated after viral infection.
  • Gene editing refers to genetic engineering techniques that utilize site-specific nucleases to insert, knock out, modify or replace DNA at specific locations in the genome of an organism, which will change the DNA sequence. This technique is sometimes called “gene editing” or “genome engineering. Gene editing can be used to achieve precise, efficient gene knockout or gene knock-in.
  • Nuclease-guided genome targeted modification technology usually consists of a DNA recognition domain and a non-specific endonuclease domain.
  • the DNA recognition domain recognizes the target site and locates the nuclease to the genomic region that needs to be edited. Then, the DNA double-strand is cut by the non-specific endonuclease, causing the DNA breakage self-repair mechanism, thereby triggering the mutation of the gene sequence and promoting the occurrence of homologous recombination.
  • the endonuclease may be a Meganuclease, a zinc finger nuclease, a CRISPR/Cas9 nuclease, a MBBBD-nuclease or a TALEN-nuclease.
  • the endonuclease is CRISPR/Cas9 nuclease, TALEN-nuclease.
  • Gene knockout techniques using nucleases include CRISPR/Cas9 technology, ZFN technology, TALE technology and TALE-CRISPR/Cas9 technology.
  • the gene editing technology is selected from single base editing (Base Editor) technology, Prime Editor technology, homing endonuclease (Meganuclease) technology.
  • ZFN artificial zinc finger nuclease
  • ZFN zinc finger Nucleases
  • TALE transcription activator-like effector
  • the term "transcription activator-like effector (TALE)" has DNA binding specificity and a module that can specifically recognize bases, and the operation is simple and convenient.
  • the TALE-DNA binding domain is composed of tandem repeat units, most of which contain 34 amino acids.
  • the 12th and 13th amino acids of the unit are designed as variable regions (repeat variable residues, RVD).
  • RVD variat variable residues
  • the RVD of TALE recognizes the 4 bases of DNA sequence with high specificity, and the 13th amino acid directly binds specifically to the base of DNA.
  • a specific TALEDN recognition and binding domain can be constructed at any site, which can be widely used in gene sequence mutation modification and gene targeting.
  • TALE-DNA binding domain sets the DNA target sequence, assemble the TALE-DNA binding domain, fuse the non-specific DNA cleavage domain of Fok I endonuclease, and assemble into TALE nucleases (tanscription activator-like effector nucleases, TALENs).
  • TALENs target and bind to DNA, resulting in DNA double-strand breaks (DNA double-srand breaks, DSBs).
  • CRISPR/Cas9 is the third generation of gene editing technology.
  • the "CRISPR system” collectively refers to transcripts and other elements involved in the expression or directing activity of CRISPR-associated (“Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (eg, tracrRNA or active moieties) tracrRNA), tracr mate sequence (covering "direct repeats” and partial direct repeats of tracrRNA processing in the context of endogenous CRISPR systems), guide sequences (also known as “spacers” in the context of endogenous CRISPR systems) ”), or other sequences and transcripts from CRISPR loci.
  • CRISPR systems are characterized by elements that facilitate the formation of a CRISPR complex (also referred to as a protospacer in the context of an endogenous CRISPR system) at the site of a target sequence.
  • a "target sequence” refers to a sequence to which a guide sequence is designed to be complementary, wherein hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Perfect complementarity is not required, provided that sufficient complementarity is present to cause hybridization and facilitate the formation of a CRISPR complex.
  • a target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • gene silencing refers to the phenomenon that a gene is not expressed or is underexpressed due to various reasons. Gene silencing can be gene silencing at the transcriptional level due to DNA methylation, heterochromatinization, and position effects, or post-transcriptional gene silencing, that is, specific inhibition of target RNA at the post-transcriptional level. Inactivation of genes, including antisense RNA, RNA interference and microRNA-mediated translation inhibition.
  • TCR silencing refers to no or low expression of endogenous TCR.
  • MHC silencing refers to no or low expression of endogenous MHC.
  • CRISPR Clustered regularly interspaced short palindromicrepeats
  • Cas9 CRISPR associated nuclease
  • Cas9 CRISPR associated nuclease
  • RNA-guided technology that uses Cas9 nuclease to edit targeted genes.
  • a guide sequence is any polynucleic acid sequence that is sufficiently complementary to a target polynucleic acid sequence to hybridize to the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
  • gRNAs are used to guide, bind or recognize Cas enzymes.
  • the degree of complementarity between the guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80% when optimally aligned using a suitable alignment algorithm , 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Algorithms based on Burrows-Wheeler Transform (e.g. Burrows-Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND Corporation (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Aligner e.g. Burrows-Wheeler Aligner
  • ClustalW Clustal X
  • BLAT Novoalign
  • ELAND Corporation Illumina, San Diego, CA
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net.
  • the CRISPR enzyme comprises one or more heterologous protein domains (eg, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains) part of a fusion protein.
  • the CRISPR enzyme fusion protein can comprise any other protein, and optionally a linker sequence between any two domains.
  • protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity , transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags .
  • Cas9 enzyme can be wild-type Cas9 or engineered Cas9.
  • sgRNA refers to short gRNAs.
  • the administered gRNA, tracr pairing sequence, and tracr sequence can be administered alone, or a complete RNA sequence can be administered.
  • the combination of Cas9 protein and gRNA can cut DNA at a specific site.
  • the recognition sequence of the CRISPR/Cas system derived from Streptococcus pyogenes is 23bp and can target 20bp.
  • the last 3 NGG sequence of its recognition site is called PAM ( protospacer adjacent motif) sequence.
  • CRISPR/Cas9 transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.
  • vectors eg, AAV, adenovirus, lentivirus
  • particles and/or nanoparticles e.g., electroporation.
  • the exons of the corresponding coding genes in the constant regions of one or both of the ⁇ and ⁇ chains of CS1, B2M, and TCR are knocked out by CRISPR/Cas technology, so that the endogenous CS1, B2M or TCR have no activity; preferably, the first exon of the endogenous TCR ⁇ chain constant region is targeted to knock out, and the gRNA used is selected from the sequence shown in SEQ ID NO: 17.
  • the endogenous CS1 gene of the engineered T cell is knocked out using CRISPR/Cas9 technology, and the gRNA used is selected from the group consisting of SEQ ID NOs: 19, 20, 40, 41, 42, 43, 44 and/or 45 shows the sequence.
  • CRISPR/Cas9 technology was used to knock out the endogenous B2M gene of the engineered T cell, and the gRNA used was selected from the sequence shown in SEQ ID NO: 18.
  • “Suppressing” or “suppressing” the expression of B2M or TCR or CS1 means reducing the expression of B2M or TCR or CS1 in a cell by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • inhibiting means reducing the level of B2M in a cell by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% , at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • Protein expression or content in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using antibodies specific for B2M, TCR, CS1.
  • RNA interfering agent as used herein is defined as any agent that interferes with or inhibits the expression of a target gene by RNA interference (RNAi).
  • RNA interfering agents include, but are not limited to, nucleic acid molecules, short interfering RNAs (siRNAs), shRNAs, or miRNAs that are RNA molecules homologous to the target gene or fragments thereof, and small molecules that interfere or inhibit the expression of the target gene by RNA interference (RNAi). molecular.
  • an “antibody” as used herein can be a full-length antibody or a fragment of an antibody that retains antigen-binding ability.
  • An antibody fragment refers to any molecule comprising the antigen-binding portion (eg, CDRs) of an antibody from which the molecule is derived. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies and multispecific antibodies formed from antigen binding molecules.
  • an antibody refers to a fragment of an antibody that specifically binds an antigen, including one or more complementarity determining regions (CDRs) thereof, in further embodiments, an antibody may be a single chain antibody (scFv).
  • the antibody comprises or consists of a high affinity multimer (avimer).
  • an antibody or functional fragment thereof that recognizes CS1 contains HCDR1 as set forth in SEQ ID NO:21, and/or HCDR2 as set forth in SEQ ID NO:22, and/or HCDR3 as set forth in SEQ ID NO:23, and /or LCDR1 shown in SEQ ID NO:24, and/or LCDR2 shown in SEQ ID NO:25, and/or LCDR3 shown in SEQ ID NO:26.
  • an antibody or functional fragment thereof that recognizes CS1 contains the heavy chain variable region set forth in SEQ ID NO:27 and/or the light chain variable region set forth in SEQ ID NO:28.
  • an antibody or functional fragment thereof that recognizes CS1 contains the scFV sequence set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • an antibody or functional fragment thereof that recognizes BCMA contains the scFV sequence set forth in SEQ ID NO: 15 or 16.
  • an antibody or functional fragment thereof that recognizes NKG2A contains the scFV sequence set forth in SEQ ID NO: 13 or 14.
  • antibody construct refers to a molecule whose structure and/or function is based on that of an antibody (eg, a full-length or intact immunoglobulin molecule).
  • the antibody construct is capable of binding to its specific target or antigen.
  • the antibody constructs according to the present invention comprise the minimum structural requirements of the antibody to allow binding of the target antigen. This minimal requirement can be met, for example, by the presence of at least three light chain CDRs (ie CDR1, CDR2 and CDR3 of the VL region) and/or three heavy chain CDRs (ie CDR1, CDR2 and CDR3 of the VH region), preferably all six CDRs definition.
  • Antibodies on which the constructs according to the invention are based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies and human antibodies.
  • full-length or intact antibodies are also included within the definition of "antibody constructs" according to the present invention, including camelid antibodies and other immunoglobulin antibodies produced by biotechnology or protein engineering methods or processes. These full-length antibodies can be, for example, monoclonal, recombinant, chimeric, deimmunized, humanized, and human antibodies. Fragments of full-length antibodies (such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or "r IgG"("half-antibody”)) are also described in "Antibody Construction"body” definition.
  • Antibody constructs according to the invention may also be modified fragments of antibodies, also known as antibody variants, such as scFv; di-scFv or bi(s)-scFv; scFv-Fc; scFv-zipper; scFab Fab2; Fab3; diabody; single chain diabody; tandem diabody (Tandab); tandem di-scFv; tandem tri-scFv; VL-CH3) 2 , (scFv-CH3) 2 , ((scFv) 2 -CH3+CH3), ((scFv) 2 -CH3) or (scFv-CH3-scFv) 2 ; multifunctional antibodies, such as trifunctional antibodies or tetrafunctional antibodies; and single domain antibodies, such as Nanobodies, or containing only one variable domain (which may be VHH, VH or VL) that specifically binds antigens or epitopes independently of other V regions or domains of single variable domain antibodies
  • a binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it is not necessary to include both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • Fd fragments have two VH regions and generally retain some antigen-binding function of the intact antigen-binding domain.
  • Additional examples of forms of antibody fragments, antibody variants or binding domains include (1) Fab fragments, which are monovalent fragments having VL, VH, CL and CH1 domains; (2) F(ab') 2 fragments, which are is a bivalent fragment with two Fab fragments linked at the hinge region by a disulfide bridge; (3) an Fd fragment, which has two VH and CH1 domains; (4) an Fv fragment, which has the VL of a single arm of the antibody and VH domains; (5) dAb fragments (Ward et al.
  • antibody construct includes monovalent, bivalent and multivalent constructs, and thus includes monospecific constructs that specifically bind to only one antigenic structure as well as that specifically bind to more than one antigen via different binding domains Bispecific and multispecific constructs of structures (eg, two, three, or more).
  • antibody construct includes molecules consisting of only one polypeptide chain as well as molecules consisting of more than one polypeptide chain, which chains may be the same (homodimer, homotrimer or homooligomer) or different (heterodimer, heterotrimer or heterooligomer).
  • the antibody construct of the present invention is preferably an "in vitro produced antibody construct".
  • the term refers to an antibody construct according to the above definition, wherein all or a portion of the variable region (eg at least one CDR) is selected on a non-immune cell (eg in vitro phage display) protein chip or any other testable candidate sequence for the ability to bind to an antigen method produced.
  • the term preferably excludes sequences that are produced solely by genome rearrangement in immune cells in animals.
  • a "recombinant antibody” is an antibody prepared by using recombinant DNA technology or genetic engineering.
  • the antibody construct is a bispecific T cell engagement antibody (BITE) targeting CS1 and CD3.
  • BITE bispecific T cell engagement antibody
  • T cells expressing BITE in the present invention are also referred to as T-BITE cells.
  • mAb monoclonal antibody
  • monoclonal antibody construct refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population except for possible naturally occurring ones that may occur in small amounts Identical except for mutation and/or post-translational modification (eg, isomerization, amidation).
  • Monoclonal antibodies are highly specific for a single antigenic site or determinant on an antigen, as compared to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (or epitopes).
  • monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are therefore not contaminated with other immunoglobulins.
  • the modifier "monoclonal” indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring that the antibody be produced by any particular method.
  • transfection refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • nucleic acid molecule encoding refers to the sequence or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The sequence of these deoxyribonucleotides determines the sequence of amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.
  • Subjects of the present invention include, but are not limited to, humans, non-human primates (eg, rhesus monkeys or other types of rhesus monkeys), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • non-human primates eg, rhesus monkeys or other types of rhesus monkeys
  • mice pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • peripheral blood mononuclear cells refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, and the like.
  • sequence when used in reference to nucleic acid sequences may include DNA or RNA, and may be single-stranded or double-stranded.
  • the term "effective amount” refers to an amount that provides a therapeutic or prophylactic benefit.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleic acid sequence to be expressed.
  • Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be provided by host cells or in vitro expression systems.
  • Expression vectors include all those known in the art, such as plasmids, viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • vector is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • Non-plasmid and non-viral compounds that facilitate nucleic acid transfer into cells may also be included, such as polylysine compounds, liposomes, and the like.
  • sequence identity determines percent identity by comparing two best matched sequences over a comparison window (eg, at least 20 positions), wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise Additions or deletions (ie, gaps), eg, 20% or less gaps (eg, 5 to 15%, or 10 to 12%, for the two sequences that best match) compared to the reference sequence (which does not contain additions or deletions) %). Percentages are usually calculated by determining the number of positions in the two sequences at which identical nucleic acid bases or amino acid residues occur to yield the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence ( i.e. window size) and multiply the result by 100 to yield the percent sequence identity.
  • exogenous refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or the expression level is insufficient to achieve the function that it has when overexpressed.
  • endogenous refers to a nucleic acid molecule or polypeptide or the like that is derived from the organism itself.
  • chimeric receptor refers to a fusion molecule formed by linking DNA fragments or protein corresponding cDNAs from different sources by gene recombination technology, including extracellular domain, transmembrane domain and intracellular domain.
  • Chimeric receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor (TCR), T Cell Antigen Coupler (TAC).
  • CAR Chimeric Antigen Receptor
  • TCR Chimeric T Cell Receptor
  • TAC T Cell Antigen Coupler
  • the chimeric receptors of the invention are chimeric antigen receptors.
  • chimeric antigen receptor refers to two or more groups of polypeptides; when immune effector cells express the CAR, the immune cells can specifically target target cells (the present invention includes tumor cells, NK cells) , and generate intracellular signals.
  • a CAR includes at least one extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to as a "cytoplasmic signaling domain").
  • the intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and/or a costimulatory molecule, in one aspect, the stimulatory molecule is a zeta chain bound to a T cell receptor complex; in one aspect, a cytoplasmic signaling Conductive domains further include functional signaling domains of one or more costimulatory molecules, eg, 4-1BB (ie, CD137), CD27, and/or CD28. In certain embodiments, groups of polypeptides are contiguous with each other.
  • a CAR targeting CS1 comprises the sequence of SEQ ID NO:37.
  • Cells expressing a CAR targeting CS1 and expressing a membrane-bound NKG2A antibody include the amino acid sequences shown in SEQ ID NOs: 37 and 38.
  • Cells expressing a CAR targeting BCMA and expressing a membrane-bound NKG2A antibody include the amino acid sequences shown in SEQ ID NOs: 38 and 39.
  • Chimeric antigen receptors typically contain an extracellular antigen binding region.
  • the extracellular antigen binding region can be fully human, humanized, murine, or a chimera in the extracellular antigen binding region consists of amino acid sequences from at least two different animals.
  • extracellular antigen binding regions may be scFv, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain fragments, or natural ligands that engage their cognate receptors, and their any derivative of .
  • the extracellular antigen binding region can comprise light chain CDRs specific for the antigen.
  • a light chain CDR may comprise two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, etc.
  • the light chain CDRs may comprise three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively.
  • a set of CDRs present on a common light chain may be collectively referred to as light chain CDRs.
  • the extracellular antigen binding region can comprise heavy chain CDRs specific for the antigen.
  • the heavy chain CDRs may be the heavy chain complementarity determining regions of an antigen binding unit such as an scFv.
  • a heavy chain CDR may comprise two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, etc.
  • the heavy chain CDRs may comprise three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively.
  • a set of CDRs present on a common heavy chain may be collectively referred to as heavy chain CDRs.
  • the extracellular antigen binding region can be modified in various ways.
  • the extracellular antigen-binding region can be mutated so that the extracellular antigen-binding region can be selected to have a higher affinity for its target antigen.
  • the affinity of the extracellular antigen binding region for its target antigen can be optimized for a target antigen that can be expressed at low levels on normal tissues.
  • clones of extracellular antigen-binding regions with higher affinity for the membrane-bound form of the target antigen may be preferred over their soluble counterparts.
  • the extracellular antigen binding region also includes a hinge or spacer region, the terms hinge and spacer region being used interchangeably.
  • the hinge can be thought of as part of the CAR used to provide flexibility to the extracellular antigen-binding region.
  • the hinge can be the native hinge region of the CD8 ⁇ molecule.
  • transmembrane domain can anchor a chimeric protein to the plasma membrane of a cell.
  • CD28 CD8[alpha] (also known as the CD8 transmembrane domain)
  • CD8 transmembrane domain can be employed.
  • module refers to positive or negative changes. Adjustment examples include 1%, 2%, 10%, 25%, 50%, 75%, or 100% variation. In a specific embodiment, it refers to a negative change.
  • treatment refers to interventions that attempt to modify the disease process, either prophylactically or clinically.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the rate of disease progression, improving or relieving the condition, relieving or improving the prognosis, etc.
  • prevention refers to interventions that are attempted prior to the development of a disease such as rejection of a cell transplant.
  • transplant immune rejection refers to the fact that after the host has transplanted an allogeneic tissue, organ, or cell transplant, the foreign graft is recognized by the host's immune system as an "alien component" and initiates targeting of the graft. immune response to attack, destruction, and clearance.
  • the present invention provides an anti-transplant immune rejection cell and a method for resisting and inhibiting rejection.
  • the engineered T cells provided by the present invention can be used to treat, prevent or ameliorate autoimmune diseases or inflammatory diseases, especially inflammatory diseases associated with autoimmune diseases, such as arthritis (eg, rheumatoid arthritis, chronic progressive arthritis (arthritis chronicica progrediente and osteoarthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthropathies (including ankylosing spondylitis), Reiter Syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic and enteropathic arthritis, enthesitis, hypersensitivity reactions (including airway hypersensitivity and skin hypersensitivity) and allergies .
  • arthritis eg, rheumatoid arthritis, chronic progressive arthritis (arthritis chronicica progrediente and osteoarthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthropathies (including an
  • the engineered T cells provided by the present invention are used for the treatment and prevention of autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, pure red cell anemia and idiopathic thrombocytopenia), systemic lupus erythematosus (SLE).
  • autoimmune hematological disorders including, for example, hemolytic anemia, aplastic anemia, pure red cell anemia and idiopathic thrombocytopenia
  • SLE systemic lupus erythematosus
  • lupus nephritis inflammatory muscle disease (dermatomyositis), periodontitis, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis , Stephen Johnson syndrome, spontaneous sprue, autoimmune inflammatory bowel disease (including, for example, ulcerative colitis, Crohn's disease, and irritable bowel syndrome), endocrine eye disease, Graves disease, Sarcoidosis, multiple sclerosis, systemic sclerosis, fibrotic diseases, primary biliary cirrhosis, juvenile diabetes (type I diabetes), uveitis, keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial Pulmonary fibrosis, periprosthetic osteolysis, glomerulonephritis (with and without nephrotic syndrome, including
  • the engineered T cells provided by the present invention can be used to treat, prevent or improve asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema and other obstructive or inflammatory diseases of the airway.
  • the engineered T cells of the invention can be used as the sole active ingredient or in combination with other drugs such as immunosuppressive or immunomodulatory agents or other anti-inflammatory or other cytotoxic or anti-cancer agents (eg as adjuvants thereof or in combination with them). ), for example, to treat or prevent diseases associated with immune disorders.
  • drugs such as immunosuppressive or immunomodulatory agents or other anti-inflammatory or other cytotoxic or anti-cancer agents (eg as adjuvants thereof or in combination with them). ), for example, to treat or prevent diseases associated with immune disorders.
  • the antibodies of the invention can be used in combination with DMARDs such as gold salts, sulfasalazine, antimalarial drugs, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, other Crolimus, sirolimus, minocycline, leflunomide, glucocorticoids; calcineurin inhibitors such as cyclosporine A or FK 506; modulators of lymphocyte recycling such as FTY720 and FTY720 analogs; mTOR inhibitors such as rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; Ascomycetes with immunosuppressive properties corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxyspergualin or its immunosuppressive homologs , analogs or
  • Tumor antigen refers to an antigen that is new or overexpressed during the development, progression of a hyperproliferative disease.
  • the hyperproliferative disorder of the present invention refers to cancer.
  • the tumor antigens of the present invention can be solid tumor antigens or hematological tumor antigens.
  • the tumor antigens of the present invention include, but are not limited to: Thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2); Lewis (Y) Antigen; CD24; Platelet-
  • the pathogen antigens are selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; the viral antigens are selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.
  • the uses provided by the present invention may be combined with treatment with one or more anti-cancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy and radiation therapy group within groups.
  • the use of the engineered immune cells provided by the present invention can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, blood transfusion, implantation or transplantation.
  • the compositions described herein can be administered to a patient by intravenous or intralymphatic injection or intraperitoneal subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular.
  • the cellular composition of the invention is preferably administered by intravenous injection.
  • the cells or cell populations useful in the present invention may be administered in one or more doses.
  • the effective amount of cells is administered in a single dose.
  • the effective amount of cells is administered in more than one dose over a period of time.
  • the timing of administration is within the judgment of the managing physician and depends on the clinical condition of the patient.
  • Cells or cell populations can be obtained from any source, such as a blood bank or a donor. Although individual needs vary, it is within the skill of the art to determine the optimal range of effective amounts for a given cell type for a particular disease or condition.
  • An effective amount refers to an amount that provides a therapeutic or prophylactic benefit.
  • the dose administered will depend on the age, health and weight of the recipient, the type of concurrent treatment (if any), the frequency of treatment and the nature of the desired effect.
  • the cells may be administered to a patient in conjunction with any number of relevant therapeutic modalities (eg, before, concurrently, or after) including, but not limited to, treatments such as anti-tumor therapy, anti-viral therapy, cidol Treatment with fovir and interleukin-2, cytarabine (also known as ARA-C) or nataliziimab for MS patients or efaliztimab for psoriasis ) treatment or other treatments for PML patients.
  • relevant therapeutic modalities eg, before, concurrently, or after
  • treatments such as anti-tumor therapy, anti-viral therapy, cidol Treatment with fovir and interleukin-2, cytarabine (also known as ARA-C) or nataliziimab for MS patients or efaliztimab for psoriasis ) treatment or other treatments for PML patients.
  • chemotherapy radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506 antibodies or other immunoablative agents such as CAM PATH, anti- - CD3 antibody or other antibody therapy, cytotoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and irradiation using T cells of the invention.
  • immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506 antibodies or other immunoablative agents such as CAM PATH, anti- - CD3 antibody or other antibody therapy, cytotoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and irradiation using T cells of the invention.
  • chemotherapeutic agents such as fludarabine, external radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or alemtuzumab (CAMPATH ) T cell depletion therapy
  • XRT external radiation therapy
  • CAMPATH alemtuzumab
  • the cell composition of the present invention is administered to a patient.
  • a cellular composition of the invention eg, a CD20 reactive agent such as rituximab, is administered following B cell depletion therapy.
  • the subject may receive standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • following transplantation the subject receives an infusion of the expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • Said modified cells obtained by any of the methods described herein can be used in certain aspects of the invention for the treatment of host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in a patient in need thereof;
  • a method of treating host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in a patient in need thereof comprising by administering to said patient an effective amount of a modified cells to treat the patient, the modified cells comprising inactivated TCR ⁇ and/or TCR ⁇ genes.
  • This article provides a T cell genetically engineered to express CS1-CAR or CS1-CD3 BITE (bispecific T-cell engager) for killing NK cells; a method for preparing the gene functionalized T cells for killing NK cells is provided. and methods for increasing the persistence and/or transplantation survival of allogeneic immune cells in the presence of host immune cells.
  • the engineered T cells are genetically engineered to express CS1-CAR or CS1-CD3 BITE.
  • the engineered T cells are genetically engineered to express CS1-CAR or CS1-CD3 BITE, which also reduces or eliminates endogenous CS1 expression or activity.
  • the engineered T cells are genetically engineered to express CS1-CAR or CS1-CD3 BITE, also reduce or eliminate endogenous CS1 expression or activity, and are also genetically engineered to express an NKG2A binding protein, preferably NKG2A membrane-bound antibody.
  • the engineered T cells are genetically engineered to express CS1-CAR or CS1-CD3 BITE, also reduce or eliminate endogenous CS1 expression or activity, and are also genetically engineered to express an NKG2A binding protein, preferably NKG2A membrane-bound antibody; the cells are also genetically engineered to express at least a chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR, or a combination thereof) that does not target CS1 that specifically binds Tumor antigens and/or viral antigens.
  • CAR chimeric receptor
  • the engineered T cells are genetically engineered to express CS1-CAR or CS1-CD3 BITE, and also reduce or eliminate the expression or activity of B2M and TCR, the cells are also genetically engineered to express at least one non-targeted Chimeric receptors for CS1 (CAR, modified TCR, TFP, TAC, aTCR, or a combination thereof).
  • the engineered T cells are genetically engineered to express CS1-CAR or CS1-CD3 BITE, and also reduce or eliminate the expression or activity of B2M and TCR, the cells are also genetically engineered to express at least non-targeted CS1 A chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR, or a combination thereof) that specifically binds tumor antigens and/or viral antigens.
  • CAR non-targeted CS1 A chimeric receptor
  • the engineered T cells are genetically engineered to express CS1-CAR or CS1-CD3 BITE, and also reduce or eliminate the expression or activity of B2M and TCR, and the cells are also genetically engineered to express non-CS1 targeting A chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR, or a combination thereof), wherein at least one of said chimeric receptors specifically binds a tumor antigen or a viral antigen, and is also genetically engineered to express an NKG2A binding protein, Preferably it is an NKG2A membrane-bound antibody.
  • the engineered T cells are genetically engineered to express CS1-CAR or CS1-CD3 BITE, and also reduce or eliminate the expression or activity of B2M, TCR and endogenous CS1, the cells are also genetically engineered to express A chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR, or a combination thereof) that does not target CS1, wherein at least one of said chimeric receptors specifically binds to a tumor antigen or viral antigen, further genetically engineered An NKG2A binding protein is expressed, preferably an NKG2A membrane bound antibody.
  • the amino acid sequence of CS1-CD3 BITE is shown in SEQ ID NO.31
  • the nucleic acid sequence is shown in SEQ ID NO.36.
  • the genetically engineered T cells provided by the present invention can enhance the killing of tumor cells by the T cells and/or the CAR-T cells carrying the targeted target antigens introduced into the subject before, at the same time, and later, and enhance the T cells. Survival and proliferation of cells and/or CAR-T cells.
  • the drug combination scheme is provided herein: 1.
  • the genetically engineered T cells provided by the present invention are administered in combination with T cells that do not target CS1, preferably, the chimeric antigen receptor 2 targets To tumor or pathogen antigens, more preferably, the chimeric antigen receptor 2 targets CD19, GPC3, Claudin18.2, EGFR, EGFRvIII, or a combination thereof.
  • the genetically engineered T cells provided by the present invention are administered in combination with an agent that enhances their function, preferably, a chemotherapeutic drug; or an agent that improves one or more side effects associated therewith.
  • This patent includes, for example, Chinese Patent Application Publication No. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN104140974A, CN 108884459 A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN109503715A, CN 109908176 A, CN109880803A, CN 110055275 A, CN110123837A, CN 110438082 A, CN 110468105 A international Patent application Publication No.
  • the engineered T cells provided by the invention have a killing effect on NK cells, and provide a new treatment method for anti-NK cell tumors. Methods of increasing the persistence and/or transplantation survival of allogeneic immune cells in the presence of host immune cells
  • the expression vector was constructed using conventional molecular biology methods in the art (Fig. 1). Will consist of CD8 ⁇ signal peptide (SEQ ID NO: 1), CS1-scfv (SEQ ID NO: 11), CD8 ⁇ hinge and transmembrane domain (SEQ ID NO: 32), CD137 intracellular signaling domain (SEQ ID NO: 7 ) and the CS1-BBZ fragment composed of CD3 ⁇ (SEQ ID NO:5) is inserted into the PRRLsin vector to construct the vector PRRLsin-CS1-BBZ; it will contain CD8 ⁇ signal peptide (SEQ ID NO:1), NKG2A-scfv (SEQ ID NO:13) , CD8 ⁇ hinge and transmembrane domain (SEQ ID NO: 32) fragments are inserted into PRRLsin vector to construct vector PRRLSIN-NKG2A; CS1-BBZ fragment, F2A (SEQ ID NO: 9), NKG2A-scfv (SEQ ID NO: 13) , CD
  • the above vectors PRRLsin-CS1-BBZ, PRRLSIN-NKG2A, PRRLsin-CS1-BBZ-F2A-NKG2A, PRRLsin-BCMA-BBZ, PRRLsin-BCMA-BBZ-F2A-NKG2A were respectively transfected into 293T cells, and the lentiviruses were packaged to obtain the corresponding vectors.
  • the infection method is a conventional infection method in the preparation of T cells expressing the chimeric antigen receptor in the art.
  • PBMCs Mononuclear cells
  • PBMCs Mononuclear cells
  • CS1-BBZ, NKG2A, CS1-BBZ-F2A-NKG2A, BCMA-BBZ and BCMA-BBZ-F2A-NKG2A were respectively infected with activated T cells and cultured and expanded to the required number to obtain CS1 CAR-T cells, NKG2A-T cells, CS1 CAR-NKG2A-T cells, BCMA CAR-T and BCMA CAR-NKG2A-T cells.
  • CS1 CAR-T cells, NKG2A-T cells, CS1 CAR-NKG2A-T cells, BCMA CAR-T cells and BCMA CAR-NKG2A-T cells were screened out.
  • reagent instructions GeneArt TM Precision gRNA Synthesis Kit, Thermo Tisher, the gRNA sequences targeting TCR, B2M, and CS1 were synthesized in vitro as shown in SEQ ID NOs: 17, 18, and 19, respectively.
  • Cas9 enzyme and gRNA in a 1:4 ratio After incubation at room temperature, the cells were mixed with Cas9 enzyme and gRNA complex (RNP), and the RNP complex was introduced into CAR-T cells using a maxcyte electroporator.
  • RNP Cas9 enzyme and gRNA complex
  • TCR/B2M /CS1 knockout T cells In vitro expansion of TCR/B2M knockout T cells, TCR/B2M /CS1 knockout T cells, double knockout cells were labeled with B2M antibody, CD3 antibody, or triple knockout cells were labeled with B2M antibody, CD3 antibody, CS1 antibody, and then conjugated with phycoerythrin (PE) After labeling with the secondary antibody, the labeled cells were sorted with anti-PE magnetic beads through a sorting column, and CD3, B2M double-negative cells, or CD3, B2M, CS1 triple-negative cells were collected (the sorting kit was purchased from Miltenyi), that is, to obtain TCR, B2M-depleted double knockout T cells (also known as DKO-T cells): CS1 CAR-DKO-T cells, NKG2A-DKO-T cells, CS1 CAR-NKG2A-DKO-T cells, BCMA-CAR-DKO-T cells,
  • the knockout efficiency is shown in Figure 2A.
  • CS1 CAR-T cells for double knockout or triple knockout the knockout efficiency can reach more than 90%; and the proportion of CD3-negative or B2M-negative T cells after magnetic bead-negative enrichment is shown in Figure 2B, which can reach 97% or more.
  • CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used to detect cytotoxicity. For details, please refer to the instructions of CytoTox 96 non-radioactive cytotoxicity detection kit.
  • Target cells inoculated with multiple myeloma cells RPMI-8226 (Shanghai Chinese Academy of Sciences Cell Bank), NCI-H929 (Shanghai Chinese Academy of Sciences Cell Bank), MM.1R (Shanghai Chinese Academy of Sciences Cell Bank) and MM.1S cells (Shanghai Chinese Academy of Sciences Cell Bank)
  • RPMI-8226 and NCI-H929 cells are low-expressing CS1 cells
  • MM.1R and MM.1S are high-expressing CS1 cells;
  • Effector cells 3:1, 1:1, 0.3:1 plus UTD (virus-uninfected T cells), CS1 CAR-T cells, CS1 CAR-DKO-T cells and CS1 CAR-TKO-T cells at effector-target ratios of 3:1, 1:1, 0.3:1 incubate;
  • CS1 is specifically expressed on immune cells, including NK cells, T cells, plasma cells and activated monocytes.
  • NK cells On resting NK cells (rNK), the proportion of CS1 expression exceeded 95%, and the proportion of CS1 expression on NK cells (aNK) activated by cytokines IL2 and IL15 in vitro reached 98% ( Figure 5) ( Figure 5 NK-1, NK-2, NK-3, and NK-4 were obtained from 4 donors, respectively).
  • Cytotoxicity was detected using CytoTox 96 non-radioactive cytotoxicity assay kit (Promega).
  • Target cells seeded rNK or aNK cells in 96-well plates;
  • Effector cells Incubate with UTD and CS1 CAR-T cells for 6 hours according to the effector-target ratio of 3:1, 1:1, 0.3:1;
  • CytoTox 96 non-radioactive cytotoxicity assay kit (Promega) was used to detect NK cytotoxicity.
  • Target cells inoculate UTD and DKO-T cells in 96-well plates respectively;
  • Effector cells Incubate for 18 hours with rNK cells and in vitro cytokine-activated aNK cells at an effector-target ratio of 2:1, 0.4:1;
  • Effector cells respectively seeded aNK-1 cells (high expression of NKG2A) and aNK-2 cells (low expression of NKG2A) ( Figure 9), in 96-well plates;
  • Target cells add TKO-T cells (untransfected UTD cells), BCMA CAR-TKO-T cells, CS1 CAR-TKO-T cells, NKG2A-TKO-T cells, BCMA CAR-T cells according to the effect-target ratio of 1:1 NKG2A-TKO-T cells and CS1 CAR-NKG2A-TKO-T cells were co-cultured for 24 and 48 hours. The proportion and number of T cells in the co-culture system of T cells and NK cells were detected by flow cytometry.
  • aNK-1 cells or aNK-2 cells have obvious killing activity on TCR/B2M/CS1-deficient T cells such as TKO-T cells and BCMA CAR-TKO-T cells; when TKO-T cells Cells expressing CS1CAR, such as CS1 CAR-TKO-T cells, can significantly inhibit the killing of TKO-T cells by aNK-2 cells with low NKG2A expression, but have poor resistance to aNK-1 cells with high NKG2A expression; Cells expressing NKG2A, such as NKG2A-TKO-T cells, BCMA CAR-NKG2A-TKO-T cells, can inhibit the killing of TKO-T cells by aNK-1 cells with high NKG2A expression, but on aNK-2 cells with low NKG2A expression Poor resistance; when TKO-T expresses both CS1 CAR and NKG2A, such as CS1 CAR-NKG2A-TKO-T cells can be very effective against the killing of
  • the expression vector CS1-CD3 BITE was constructed using conventional molecular biology methods in the art. Will consist of CD8 ⁇ signal peptide (SEQ ID NO: 1), CS1-scfv (SEQ ID NO: 11), BITE linker nucleic acid (SEQ ID NO: 34), CD3 scFv (SEQ ID NO: 35), F2A (SEQ ID NO: 35), F2A (SEQ ID NO: 11) 9) The fragment consisting of EGFP (SEQ ID NO: 3) was inserted into the PRRLsin vector to construct the vector PRRLsin-CS1-CD3 BITE ( Figure 11).
  • the vector PRRLsin-CS1-CD3 BITE was transfected into 293T cells, and the lentivirus was packaged to obtain the corresponding lentivirus CS1BITE.
  • Ficoll-Paque (GE bioscience) was used for density gradient centrifugation to separate PBMC from human peripheral blood, and anti-CD3/CD28 magnetic beads were added to activate and expand in vitro to obtain T cells.
  • lentivirus CS1BITE infected and activated T cells were cultured and expanded to the required number to obtain T-BITE cells. Since BITE is a secreted protein, the expression of EGFP was used to detect the expression of BITE in this experiment. After flow cytometry, the expression of EGFP was shown in Figure 12.
  • Example 7 Killing detection of co-cultured NK cells by T-BITE cells
  • Activated NK cells from the same donor were co-cultured with T-BITE or UTD cells at a ratio of 1:1, and the ratio of CD56 + NK cells in the culture system was detected by flow cytometry at 24 hours.
  • the results are shown in Figure 13, the addition of T-BITE eliminated or killed the NK cells in the culture system. It shows that the constructed BITE has biological activity.
  • Example 8 Killing detection of T-BITE cell culture supernatant on co-cultured NK cells
  • T-BITE cells and uninfected T cells were cultured overnight, respectively, and the supernatant was collected by centrifugation.
  • NK cells and T cells from the same donor were co-cultured at a ratio of 1:1, and 50% of the above UTD or T-BITE culture supernatant was added. After 24 hours, NK cells in the culture system were detected by flow cytometry proportion.
  • NK cells CD56 +
  • NK cells CD56 +
  • soluble CS1-CD3 BITE can effectively eliminate NK cells in the presence of T cells.

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Abstract

提供了一种表达靶向CS1的嵌合受体1的工程化免疫细胞用于制备杀伤NK细胞的药物的用途，一种包含结合到靶细胞表面的CS1的第一结构域和结合到T细胞表面上的CD3第二结合结构域的双特异抗体构建体，编码所述嵌合受体1、膜结合型NK细胞抑制性受体的配体或抗体片段、抗体构建体的多核苷酸，包含所述多核苷酸的载体，能够分泌所述抗体构建体的免疫细胞，含有上述抗体构建体、免疫细胞的药物组合物和试剂盒。

Description

免疫细胞活性调节 技术领域
本发明涉及表达CS1的嵌合抗体的免疫细胞对NK细胞的杀伤还涉及用于开发工程化反应性T细胞进行免疫治疗的方法，且更具体地涉及用于增加同种异体免疫细胞的持久性和/或植入的方法。本发明还涉及工程化免疫细胞及其功能衍生物、嵌合抗原受体(CAR)、多链CAR及其用于增强免疫治疗效率的用途。
背景技术
传统的免疫细胞疗法是利用患者体内自体的免疫细胞，在体外进行激活、扩增或基因修饰，再输注到患者体内发挥作用。这种自体免疫细胞疗法因其特殊的技术特点，存在昂贵费用、不能现货供应、难以规模化以及因患者本身免疫细胞质量问题而不能或制备的免疫细胞质量不佳导致治疗效果不理想等问题。因此，仍然需要开发能够大规模制备、质量稳定、随时供货的通用型免疫细胞的制备方法。
利用基因编辑技术对健康人T细胞进行基因编辑制备同种异体的通用型T细胞，有望克服以上问题。同种异体T细胞的制备首先要克服的就是异体T细胞对宿主细胞的攻击，目前已有相对比较成熟的方法，即通过对异体T细胞的TCR受体进行敲除从而避免移植物抗宿主反应(GvHD)。此外影响异体T细胞在宿主体内持久性进而影响疗效的最主要的是宿主T对异体细胞排斥问题(HvDR)，针对此问题，目前有两类策略：第一个方向是清除宿主体内可能会排斥异体细胞的T细胞，此类策略针对的宿主的T细胞，但宿主T细胞或活化型T细胞的长期缺失会严重影响宿主自身免疫系统。第二类方向是消除异体T细胞的主要组织相容性抗原，常用的方法是对异体T细胞的B2M进行敲除，B2M的敲除使多样性丰富的HLA-ABC蛋白无法在细胞膜上表达，从而避免宿主T细胞对其攻击，但是HLA-I类分子缺失会导致宿主NK细胞对HLA-I类分子缺失细胞的清除。因此，为了提高同种异体的T细胞能够在体内存活更久从而更好的发挥其抗肿瘤效果，急需开发出新的策略用于抵抗宿主T细胞或NK细胞对同种异体T细胞的清除。
发明内容
本发明构建了靶向CS1的CAR或靶向宿主NK细胞和宿主和/或供体T细胞的双特异性抗体构建体用以杀伤或清除NK细胞、和/或抵抗宿主体内NK细胞对同种异体T细胞的杀伤；此外为了进一步增强同种异体T细胞对宿主体内可能存在的强活性NK细胞的清除，所述的CS1 CAR T细胞或双特异性抗体构建体可同时表达外源性NK细胞抑制性受体的配体或抗体片段，如NKG2A。本发明提供了通过有效的解决宿主NK细胞对B2M敲除的同种异体T细胞的排斥的方法以及相关药物组合物及其制备，来增加了同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率。本发明所提供的方法或相关药物组合物，能显著提高联用的抗肿瘤或抗感染的CAR-T细胞在提供抗肿瘤或抗感染的疗效。
本发明第一方面提供了一种表达靶向CS1的嵌合受体1的工程化免疫细胞用于制备杀伤或清除NK细胞的药物的用途。
优选地，所述免疫细胞中的MHC表达、活性和/或信号传导被降低或抑制。
优选地，所述MHC为MHC I类分子；更优选地，所述MHC I为HLA；更优选地，所述HLA为HLA-I；更优选地，所述HLA-I选自HLA-A、HLA-B、HLA-C、B2M中的一种或两种以上；最优选的，所述HLA-I包括HLA-A和B2M；
优选地，所述被降低或抑制是通过使用TAL核酸酶、巨核酸酶、锌指核酸酶、Cas9和Argonaute来进行；
优选地，所述免疫细胞包含靶向编码MHC的基因的抑制性核酸分子或gRNA分子；
优选地，所述抑制性核酸分子包含与所述编码MHC的基因互补的序列；
优选地，所述抑制性核酸包含RNA干扰剂；
优选地，所述抑制性核酸包含siRNA、shRNA或miRNA；
优选地，所述sgRNA序列含有SEQ ID NO：17和18所示序列；
优选地，所述MHC的表达、活性和/或信号传导减少是永久的、短暂的或可诱导的；
优选地，与未基因工程化的免疫细胞中MHC的表达、活性和/或信号传导相比，所述工程化免疫细胞中MHC的表达、活性和/或信号传导减少大于或者大于约50％、60％、70％、80％、90％、95％或100％；
优选地，利用免疫印迹测定和/或在流式检测，检测不到所述免疫细胞中表达的MHC的表达。
在一优选的实施例中，所述嵌合受体1包括：嵌合抗原受体1(CAR1)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)、aTCR-T或其组合。
在一优选的实施例中，所述CAR1包括：
(i)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；和/或
(ii)特异性识CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或
(iii)特异性识CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ；
(iv)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区和CD3ζ；
优选的，所述特异性识别CS1的抗体的核酸分子与SEQ ID NO：11具有至少80％、优选90％、并且更优选95％的同一性；或所述特异性识别CS1的抗体的氨基酸序列与SEQ ID NO：12具有至少80％、优选90％、并且更优选95％的同一性。
优选的，所述特异性识别CS1的抗体的核酸分子与SEQ ID NO：11具有至少80％、90％、或95％的同一性；或所述特异性识别CS1的抗体的氨基酸序列与SEQ ID NO：12具有至少80％、90％、或95％的同一性。
在一优选的方式中，所述免疫细胞还表达膜结合型NK细胞抑制性受体的配体或抗体片段。
在一优选的方式中，所述免疫细胞还表达膜结合型NKG2A抗体或抗体片段；
优选的，所述膜结合型NKG2A抗体或抗体片段包括与SEQ ID NO：14或38具有至少80％、90％、或95％的同一性序列；
或者所述膜结合型NKG2A抗体或抗体片段的核酸分子与SEQ ID NO：13具有至少80％、90％、或95％的同一性。
在一优选的方式中，所述免疫细胞的内源性CS1基因被敲除，优选采用CRISPR/Cas9技术敲除所述免疫细胞的内源性CS1基因。
在一优选的方式中，CRISPR/Cas9技术使用的gRNA如SEQ ID NO:19、20、40、41、42、43、44和/或45所示。
在一优选的方式中，所述免疫细胞和表达非靶向CS1的嵌合抗原受体的T细胞联合施用或者所述免疫细胞还表达非靶向CS1的嵌合抗原受体，
优选地，所述非靶向CS1的嵌合抗原受体靶向肿瘤或病原体抗原，
更优选地，所述非靶向CS1的嵌合抗原受体靶向BCMA、CD19、GPC3、Claudin18.2、EGFR、EGFRvIII或它们的组合。
在一优选的方式中，所述免疫细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；
优选地，所述T细胞为自体/同种异体T细胞；
优选地，所述T细胞为原代T细胞；
优选地，所述T细胞来源于人的自体T细胞。
在一优选的方式中，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。
在一优选的方式中，所述免疫细胞的内源性MHC和内源性TCR被敲除，优选采用CRISPR/Cas9技术敲除内源性MHC和内源性TCR。
在一优选的方式中，采用CRISPR/Cas9技术敲除B2M使用的gRNA如SEQ ID NO:18所示，和/或敲除TRAC使用的gRNA如SEQ ID NO:17所示。
在一优选的方式中，所述免疫细胞能增强在先、同时、在后导入受试者的T细胞和/或携带靶向靶抗原的CAR-T细胞对肿瘤细胞的杀伤、以及增强所述T细胞和/或CAR-T细胞的存活、增殖。
在一优选的方式中，所述免疫细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；
或所述免疫细胞与改善其相关的一种或多种副作用的药剂联合施用。
优选地，所述非靶向CS1的嵌合受体包括：嵌合抗原受体(CAR)2、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)、aTCR-T或其组合；
优选地，所述CAR2包括：
(i)特异性识别肿瘤抗原、和/或感染细胞抗原的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；和/或
(ii)特异性识别肿瘤抗原、和/或感染细胞抗原的抗体或其功能片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或
(iii)特异性识别肿瘤抗原、和/或感染细胞抗原的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ；
(iv)特异性识别肿瘤抗原、和/或感染细胞抗原的抗体或其功能片段、CD28或CD8的跨膜区和CD3ζ；
优选的，所述肿瘤抗原包括Claudin18.2、Claudin18.1、Claudin 6、血管内皮生长因子受体、磷脂酰肌醇蛋白聚糖-3(GPC3)、B细胞成熟抗原(BCMA)、碳酸酐酶9(CAIX)、tEGFR、CD19、CD20、CD22、间皮素、CEA和乙型肝炎表面抗原、抗叶酸受体、CD23、CD24、CD30、CD33、CD38、CD44、EGFR、上皮糖蛋白2(EPG-2)、上皮糖蛋白40(EPG-40)、 EPHa2、erb-B2、erb-B3、erb-B4、erbB二聚体、EGFR vIII、叶酸结合蛋白(FBP)、FCRL5、FCRH5、胎儿乙酰胆碱受体、GD2、GD3、HMW-MAA、IL-22R-α、IL-13R-α2、激酶插入结构域受体(kdr)、κ轻链、路易斯Y、L1细胞粘附分子(L1-CAM)、黑色素瘤相关抗原(MAGE)-A1、MAGE-A3、MAGE-A6、优先表达的黑色素瘤抗原(PRAME)、存活蛋白、TAG72、B7-H6、IL-13受体α2(IL-13Ra2)、CA9、GD3、HMW-MAA、CD171、G250/CAIX、HLA-AI MAGEA1、HLA-A2、PSCA、叶酸受体-a、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、NKG2D配体、CD44v6、双重抗原、癌症-睾丸抗原、间皮素、鼠CMV、粘蛋白1(MUC1)、MUC16、PSCA、NKG2D、NY-ESO-1、MART-1、gp100、G蛋白偶联受体5D(GPCR5D)、ROR1、TAG72、VEGF-R2、癌胚抗原(CEA)、前列腺特异性抗原、PSMA、Her2/neu、雌激素受体、孕酮受体、肝配蛋白B2、CD123、c-Met、GD-2、O-乙酰化GD2(OGD2)、CE7、Wilms肿瘤1(WT-1)、细胞周期蛋白、细胞周期蛋白A2、CCL-1、CD138、病原体特异性抗原和与通用标签相关的抗原；
优选地，所述感染细胞抗原包括病原体抗原和病毒抗原；
优选地，所述病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；
优选地，所述病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原；
在一优选的实施例中，当所述免疫细胞与宿主NK细胞共培养时，所述免疫细胞能杀伤宿主NK细胞。
在一优选的实施例中，当所述免疫细胞与宿主NK细胞共培养时，所述免疫细胞能抵抗宿主NK细胞对所述免疫细胞的杀伤；
优选地，所述免疫细胞能抵抗宿主NK细胞中被细胞因子活化的NK细胞对所述免疫细胞的杀伤；
优选地，所述免疫细胞能抵抗表达NKG2A的宿主NK细胞对所述免疫细胞的杀伤；
优选地，所述免疫细胞能显著地抵抗低表达NKG2A的宿主NK细胞对所述免疫细胞的杀伤。
在一优选的实施例中，所述免疫细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；或所述免疫细胞与改善其相关的一种或多种副作用的药剂联合施用。
本发明第二方面提供了一种增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率的方法，包括：
a)提供同种异体免疫细胞；
b)任选通过编码参与针对自体和非自体抗原识别的响应的多肽的至少一种内源基因 表达、活性和/或信号传导被降低或抑制来修饰所述细胞；以及
c)利用编码靶向CS1的嵌合受体1多核苷酸来修饰所述细胞。
在一优选的方式中，采用CRISPR/Cas9技术敲除内源性MHC和内源性TCR。
在一优选的方式中，采用CRISPR/Cas9技术敲除B2M使用的gRNA如SEQ ID NO:18示序列，和/或敲除TRAC使用的gRNA如SEQ ID NO:17示序列。
在一优选的方式中，所述嵌核受体1包括：嵌合抗原受体1(CAR1)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)、aTCR-T或其组合。
优选地，所述CAR1包括：
(i)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；和/或
(ii)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或
(iii)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ；
(iv)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区和CD3ζ；
优选的，所述特异性识别CS1的抗体的核酸分子与SEQ ID NO：11具有至少80％、90％、或95％的同一性；或所述特异性识别CS1的抗体的氨基酸序列与SEQ ID NO：12具有至少80％、90％、或95％的同一性。
在一优选的方式中，还包括步骤d)编码膜结合型NK细胞抑制性受体的配体或抗体片段的非内源性多核苷酸来修饰所述细胞。
在一优选的方式中，还包括步骤d)编码所述免疫细胞膜结合型NKG2A抗体或抗体片段的非内源性多核苷酸来修饰所述细胞；
优选的，所述NKG2A抗体或抗体片段包括与SEQ ID NO：14或38具有至少80％、90％、或95％的同一性序列；
或者所述膜结合型NKG2A抗体或抗体片段的核酸分子与SEQ ID NO：13具有至少80％、90％、或95％的同一性。
在一优选的方式中，还包括步骤b)采用CRISPR/Cas9技术敲除所述工程化T细胞内源性CS1基因。
在一优选的方式中，CRISPR/Cas9技术使用的gRNA如SEQ ID NO:19、20、40、41、42、43、44和/或45所示序列。
在一优选的方式中，还包括步骤e)编码靶向肿瘤抗原、和/或病原体抗原、和/或病 毒抗原的嵌合抗原受体非内源性的多核苷酸来修饰所述细胞；
优选的，所述肿瘤抗原包括BCMA、CD19、GPC3、Claudin18.2、EGFR、EGFRvIII或其组合。
在一优选的方式中，所述免疫细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；
优选地，所述T细胞为自体/同种异体T细胞；
优选地，所述T细胞为原代T细胞；
优选地，所述T细胞来源于人的自体T细胞。
在一优选的方式中，当所述方法制备的免疫细胞与宿主NK细胞共培养时，所述免疫细胞能杀伤宿主NK细胞。
在一优选的方式中，所述方法制备的免疫细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；
或所述方法制备的免疫细胞与改善其相关的一种或多种副作用的药剂联合施用。
一种增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率的方法，包括：
a)提供同种异体细胞；
b)通过编码参与针对自体和非自体抗原识别的响应的多肽的至少一种内源基因表达、活性和/或信号传导被降低或抑制来修饰所述细胞；
c)编码靶向CS1的嵌合受体1多核苷酸来修饰所述细胞。
在一优选的实施例中，步骤b)中的所述多肽选自MHC,所述MHC为MHC I类分子；更优选地，所述MHC I为HLA；更优选地，所述HLA为HLA-I；更优选地，所述HLA-I选自HLA-A、HLA-B、HLA-C、B2M中的一种或两种以上；最优选的，所述HLA-I包括HLA-A和B2M。
优选的，所述肿瘤抗原包括Claudin18.2、Claudin18.1、Claudin 6、血管内皮生长因子受体、磷脂酰肌醇蛋白聚糖-3(GPC3)、B细胞成熟抗原(BCMA)、碳酸酐酶9(CAIX)、tEGFR、CD19、CD20、CD22、间皮素、CEA和乙型肝炎表面抗原、抗叶酸受体、CD23、CD24、CD30、CD33、CD38、CD44、EGFR、上皮糖蛋白2(EPG-2)、上皮糖蛋白40(EPG-40)、EPHa2、erb-B2、erb-B3、erb-B4、erbB二聚体、EGFR vIII、叶酸结合蛋白(FBP)、FCRL5、FCRH5、胎儿乙酰胆碱受体、GD2、GD3、HMW-MAA、IL-22R-α、IL-13R-α2、激酶插入结构域受体(kdr)、κ轻链、路易斯Y、L1细胞粘附分子(L1-CAM)、黑色素瘤相关抗原 (MAGE)-A1、MAGE-A3、MAGE-A6、优先表达的黑色素瘤抗原(PRAME)、存活蛋白、TAG72、B7-H6、IL-13受体α2(IL-13Ra2)、CA9、GD3、HMW-MAA、CD171、G250/CAIX、HLA-AI MAGEA1、HLA-A2、PSCA、叶酸受体-a、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、NKG2D配体、CD44v6、双重抗原、癌症-睾丸抗原、间皮素、鼠CMV、粘蛋白1(MUC1)、MUC16、PSCA、NKG2D、NY-ESO-1、MART-1、gp100、G蛋白偶联受体5D(GPCR5D)、ROR1、TAG72、VEGF-R2、癌胚抗原(CEA)、前列腺特异性抗原、PSMA、Her2/neu、雌激素受体、孕酮受体、肝配蛋白B2、CD123、c-Met、GD-2、O-乙酰化GD2(OGD2)、CE7、Wilms肿瘤1(WT-1)、细胞周期蛋白、细胞周期蛋白A2、CCL-1、CD138、病原体特异性抗原和与通用标签相关的抗原；
优选地，所述感染细胞抗原包括病原体抗原和病毒抗原；
优选地，所述病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；
优选地，所述病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。
在一优选的实施例中，还包括步骤e)编码NK细胞抑制性受体的配体或抗体片段的非内源性多核苷酸来修饰所述细胞；
优选地，还包括步骤e)编码所述免疫细胞NKG2A结合分子的非内源性多核苷酸来修饰所述细胞；
优选地，所述NKG2A结合分子为细胞膜结合蛋白、分泌型蛋白；
优选地，所述NKG2A结合分子仅包含胞外结构域、跨膜域；或包含胞外结构域、跨膜域和胞内区；
优选地，所述NKG2A结合分子为结合在细胞膜上的NKG2A抗体或抗体片段；
优选的，所述NKG2A结合分子的核酸分子与SEQ ID NO：13具有至少80％、优选90％、并且更优选95％的同一性；或所述NKG2A氨基酸序列与SEQ ID NO：14具有至少80％、优选90％、并且更优选95％的同一性。
在一优选的实施例中，当所述方法制备的免疫细胞与宿主NK细胞共培养时，所述免疫细胞能杀伤宿主NK细胞。
在一优选的实施例中，当所述方法制备的免疫细胞与宿主NK细胞共培养时，所述免疫细胞能抵抗宿主NK细胞对所述免疫细胞的杀伤；
优选地，所述免疫细胞能抵抗宿主NK细胞中被细胞因子活化的NK细胞对所述免疫细胞的杀伤；
优选地，所述免疫细胞能抵抗表达NKG2A的宿主NK细胞对所述免疫细胞的杀伤；
优选地，所述免疫细胞能显著地抵抗低表达NKG2A的宿主NK细胞对所述免疫细胞的杀伤。
在一优选的实施例中，所述方法制备的免疫细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；或所述方法制备的免疫细胞与改善其相关的一种或多种副作用的药剂联合施用。
本发明第三方面提供了一种双特异性抗体构建体，其特征在于，所述抗体构建体包含结合到靶细胞表面上的CS1的第一结合结构域和结合到T细胞表面上的CD3的第二结合结构域。
在一优选的方式中，所述第一结合结构域结合到人或猕猴CS1；和/或所述第二结合结构域结合到人CD3ε、普通狨、棉顶狨或松鼠猴CD3ε。
在一优选的方式中，所述抗体构建体选自以下的形式：scFv、(scFv) 2、scFv-单结构域mAb、双功能抗体和它们的寡聚物。
在一优选的方式中，所述第一结合结构域包含的VH区含有SEQ ID NO：21所示的CDR1、SEQ ID NO：22所示的CDR2和SEQ ID NO：23所示的CDR3，第一结合结构域包含的VL区含有SEQ ID NO：24所示的CDR1、SEQ ID NO：25所示的CDR2和SEQ ID NO：26所示的CDR3；
优选所述第一结合结构域的VH区如SEQ ID NO：27所示和VL区如SEQ ID NO：28所示。
在一优选的方式中，所述抗体构建体以从N端到C端的次序包含：
如SEQ ID NO.12所示的氨基酸序列的第一结合结构域：
如SEQ ID NO.29所示的氨基酸序列的肽接头；
如SEQ ID NO.30所示的氨基酸序列的第二结合结构域。
在一优选的方式中，所述抗体构建提包含如SEQ ID NO：31所示的氨基酸序列。
本发明第四方面提供了一种多核苷酸，其编码如本发明第三方面所定义的抗体构建体。
本发明第五方面提供了一种载体，其包含本发明第四方面定义的多核苷酸。
本发明第六方面提供了一种免疫细胞，其用如本发明第四方面所定义的所述多核苷酸或用如本发明第五方面所定义的所述载体转化或转染；
优选地，所述免疫细胞能分泌本发明第三方面所述的构建体；
优选地，所述免疫细胞中的MHC表达、活性和/或信号传导被降低或抑制，且分泌本发明第三方面所述的构建体；
优选地，所述免疫细胞还表达靶向肿瘤抗原、和/或病原体抗原、和/或病毒抗原的嵌合受体；
优选地，所述CAR包括：
(i)特异性识别肿瘤抗原、和/或感染细胞抗原的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；和/或
(ii)特异性识别肿瘤抗原、和/或感染细胞抗原的抗体或其功能片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或
(iii)特异性识别肿瘤抗原、和/或感染细胞抗原的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ；
(iv)特异性识别肿瘤抗原、和/或感染细胞抗原的抗体或其功能片段、CD28或CD8的跨膜区和CD3ζ；
优选地，所述免疫细胞还表达NK细胞抑制性受体的配体或抗体片段；
优选地，所述免疫细胞还表达NKG2A结合分子；
优选地，所述NKG2A结合分子为细胞膜结合蛋白、分泌型蛋白；
优选地，所述NKG2A结合分子仅包含胞外结构域和跨膜域；或包含胞外结构域、跨膜域和胞内区；
优选地，所述NKG2A结合分子为结合在细胞膜上的NKG2A抗体或抗体片段；
优选的，所述NKG2A结合分子的核酸分子与SEQ ID NO：13共有至少80％、优选90％、并且更优选95％的同一性；或所述NKG2A氨基酸序列与SEQ ID NO：14共有至少80％、优选90％、并且更优选95％的同一性。
本发明第七方面提供了一种用于产生根据本发明第三方面的抗体构建体的方法，所述方法包括在允许如本发明第三方面所定义的所述抗体构建体表达的条件下培养如本发明第六方面所定义的宿主细胞和从所述培养物回收所产生的抗体构建体。
本发明第八方面提供了一种药物组合物，其包含根据本发明第三方面所述的抗体构建体或根据本发明第七方面的方法产生的抗体构建体。
根据本发明第三方面所述的抗体构建体或根据本发明第七方面的方法产生的抗体构建体，其用于增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率。
本发明第八方面提供了一种增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率的方法，其包括向有需要的受试者施用根据本发明第三方面的抗体构建体或根据本发明第七方面的方法产生的抗体构建体的步骤。
本发明第九方面提供了一种试剂盒，其包含根据本发明第三方面所述的抗体构建体、根据本发明第七方面所述的方法产生的抗体构建体、如本发明第四方面的多核苷酸、如本发明第五方面的载体和/或如本发明第六方面的宿主细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1.PRRLsin-CS1-BBZ、PRRLSIN-NKG2A、PRRLsin-CS1-BBZ-F2A-NK G2A、PRRLsin-BCMA-BBZ、PRRLsin-BCMA-BBZ-F2A-NKG2A质粒示意图；
图2A.TCR/B2M敲除、TCR/B2M/CS1敲除的T细胞的敲除效率检测；图2B.阴性富集后TCR/B2M敲除、TCR/B2M/CS1敲除的T细胞比例；
图3A和3B.TCR/B2M敲除、TCR/B2M/CS1敲除的T细胞中CAR阳性率检测；
图4.体外肿瘤细胞杀伤功能检测；
图5.CS1在各免疫细胞上的表达情况检测；
图6A和图6B.CS1 CAR-T细胞对人原代NK细胞杀伤能力的检测；
图7A和图7B.共培养24小时、48小时，CS1 CAR-T细胞对人原代NK细胞杀伤能力的检测；
图8.NK细胞对TCR/B2M缺失的T细胞的杀伤检测；
图9.NK细胞中NKG2A表达水平检测；
图10.CS1 CAR-TKO-T细胞抵抗NK细胞杀伤能力的检测；
图11.PRRLsin-CS1-CD3 BITE质粒示意图；
图12.T-BITE细胞分泌BITE水平检测；
图13.T-BITE细胞对共培养的NK细胞的杀伤检测；
图14.T-BITE细胞培养上清对共培养的NK细胞的杀伤检测。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现自体或同种异体T细胞通过表达CS1-CAR、表达/分泌CS1-CD3双特异性抗体构建体或利用独立的CS1-CD3双特异性抗体构建体能显著提升对宿主NK细胞的杀伤，清除宿主静止状态或活化NK细胞，从而增加同种异体T细胞在有宿主免疫细胞存在时的持久性和/或移植成活率。
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗，生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的所有方法和材料都可以在本发明的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，材料、方法和实施例仅是说明性的，而并非旨在进行限制。
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
本发明中请求保护的主题以范围形式呈现的，应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供范围的情况下，应当理解，在该范围的上限和下限之间的较小的范围内可独立地包含这些较小范围的上下限，它们也属于请求保护的主题的范围。明确地排除所述范围的上下限的除外。设定范围包含一个或两个限值时，请求保护的主题也包括排除所述限值之一个或两 个的范围。这适用而无关范围的宽度。
本文使用的术语约是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。在本文中，“约”可以是在所述技术领域内可以接受的误差范围；。例如，可以是指“约”值或参数的±10％范围内的值或参数，例如，约5uM可包括在4.5uM与5.5uM之间的任何数目。
除非另外指出，本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值。
术语
CS1(也称为SLAMF7，CD319或CRACC-NCBI参考序列：NP_067004.3)是淋巴细胞激活分子家族成员7，参与细胞的黏附及NK细胞活化功能，主要表达在浆细胞、NK细胞、CD 8+T细胞、活化的B细胞及单核树突状细胞中，在造血谱系祖细胞及人体其他组织中基本不表达。CS1分子在多发性骨髓瘤(multiple myeloma，MM)细胞中也同样高表达，其单克隆抗体Elotuzumab(huLuc63)，在联合免疫调节剂和蛋白酶体抑制剂在治疗复发或难治的MM患者临床结果中药效良好，已经被FDA批准用于治疗MM。而单抗作用的机制是依赖于其与CD16的结合以及NK细胞的存在，由此我们可以知道，以CS1为靶点的单抗能激活NK细胞的杀伤功能但不能清除NK细胞。本发明意外的发现靶向CS1的CAR的免疫细胞或靶向宿主NK细胞和宿主和/或供体T细胞的双特异性抗体构建体，能抵抗宿主体内NK细胞对同种异体T细胞的杀伤，从而增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率。
“持久性”是指细胞抵抗排斥的能力，并且在体内随时间保持和/或增加数量(例如，天、周、月、年)的数量。通常，在输注到所述患者体内之后，本发明的工程化免疫细胞可在患者的血液中发现至少10天，优选为至少20天，更优选为至少25天，甚至更优选为至少30天。
“增加持久性和/或植入”是指在治疗过程期间，与给予患者非工程化免疫细胞(即，非持久性)的情况相比，工程化以使它们持续的同种异体免疫细胞的数量保持较高。这种提高的持久性和/或待注射到患者中的同种异体免疫细胞的植入(例如，T细胞)是免疫耐受性的一部分(或“免疫耐受作用(tolerisation)”)，其描述了宿主免疫系统相对于所述免疫细胞的无应答性状态，而所述免疫细胞保留引发免疫应答的能力。
“自体和非自体抗原识别”旨在由细胞免疫系统进行筛选，借此由主要组织相容性复合物(MHC)分子上的宿主细胞呈递肽，以评估细胞是否被外源生物体感染。该筛选涉及其他跨膜结构，例如像TCR或TAP1/TAP2或蛋白酶2。
术语“细胞”指人或非人动物来源的细胞。在一个实施例中，所述工程细胞或工程化细胞指表达CS1-CAR的T细胞。
术语“宿主”或“受试者”是指接受移植物移植的受体，在一些实施方式中，可以是接受外源细胞植入的个体，如人。在一些实施方式中，“受试者”可以是临床患者、临床试验志愿者、实验动物等等。所述受试者可能被怀疑患有以细胞增殖为特征的疾病或者具有患有以细胞增殖为特征的疾病、被诊断为患有以细胞增殖为特征的疾病、或者是被证实不患有以细胞增殖为特征的疾病的对照受试者。在一些实施例中，所述受试者是患有或可能患有免疫性疾病如自身免疫性疾病，或接受移植物治疗后。
术语“工程化”是指应用细胞生物学和分子生物学的原理和方法，通过某种工程学手段，在细胞整体水平或细胞器水平上，按照人们的意愿来改变细胞内的遗传物质或获得细胞产品的一门综合科学技术。术语“遗传修饰”、“基因修饰”、“基因工程化”或“经修饰的”是指修饰细胞的方法，包括但不限于通过基因编辑的方法在基因编码或非编码区或其表达调控区；或通过核酸内切酶和/或反义RNA技术；或增加引入外源的蛋白和/或复合体、小分子抑制剂对基因的蛋白表达水平进行改变来造成基因缺陷。在具体实施例中，“修饰”是指本发明的蛋白或多肽的状态或结构的改变。修饰的方式可以是化学的、结构的和功能上的。在一些实施方案中，经修饰的细胞为干细胞(例如，造血干细胞(HSC)或祖细胞、胚胎干细胞(ES)、诱导性多能干(iPS)细胞)，淋巴细胞(例如T细胞)，其可以是从受试者或供体获得。可以修饰细胞以表达外源构建体，例如pre-TCRα蛋白，嵌合抗原受体(CAR)或T细胞受体(TCR)，其可以整合到细胞基因组中。
术语“多能干细胞”具有分化成三个胚层中的任一个的潜力：内胚层(例如，胃连接、胃肠道、肺等)，中胚层(例如，肌肉、骨骼、血液，泌尿生殖组织等)或外胚层(例如表皮组织和神经系统组织)。如本文所用，术语“多能干细胞”还涵盖“诱导多能干细胞”或“iPSC”，是来源于非多能细胞的一种多能干细胞。在一实施例中，所述多能干细胞来自于通过对体细胞进行重新编程转化而来具有多能干细胞特征的细胞。这样的“iPS”或“iPSC”细胞可以通过诱导某些调节基因的表达或通过外源施加某些蛋白质来产生。
“多能干细胞特征”是指将多能干细胞与其他细胞区分开的细胞特征。例如，人多能干细胞表达至少几种以下标志物：SSEA-3、SSEA-4、TRA-1-60、TRA-1-81、TRA-2-49/6E、 ALP、Sox2、E-钙粘蛋白、UTF-1、Oct4、Rex1和Nanog。具有与多能干细胞相关的细胞形态也是多能干细胞特征。
术语“非内源性多肽”是指通常并非由供体免疫细胞表达的多肽，优选为由已经导入免疫细胞基因组的外源多核苷酸表达的多肽。示例性的，非内源性多肽是靶向CS1和CD3的双特异性抗体构建体。
术语“免疫细胞”是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞等。在一些实施方案中，所述的免疫细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞是同种异体NK细胞。
术语“经人工改造的具有免疫效应细胞功能的细胞”是指不具有免疫效应的细胞或细胞系经人工改造或接受刺激物刺激后，该细胞获得了免疫效应细胞功能。如293T细胞，经人工改造，使其具有免疫效应细胞的功能；如干细胞，经体外诱导，使其分化成免疫效应细胞。
本文所述的“T细胞”可以是PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织、肿瘤组织中获取的天然的T细胞，还可以是经过分选等获得的具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)和/或效应记忆T细胞(Tem)。在一些情况下，“T细胞”可以是某种特定亚型的T细胞，如γδT细胞。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离和/或单采术(apheresis)，从个体收集的血液获得T细胞。在一个实施方案中，T细胞来源于诱导的多能干细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。所述T细胞可以从健康供体，或者来自诊断患有癌症个体的衍生细胞。T细胞可以是自体T细胞或同种异体T细胞。T细胞可以是原代T细胞。
本文所述的“T细胞”还可以是携带识别靶抗原的外源蛋白的T细胞，如CAR T细胞、TCR-T细胞、T细胞抗原耦合器(TAC)、T细胞融合蛋白等。
术语“MHC”为组织相容性复合物，是所有编码生物相容复合体抗原的基因群一种统称，MHC抗原表达于所有高等脊椎动物的组织，在人类细胞中称为HLA抗原，在移植反应中发挥重要作用，由对所植入的组织的表面上的组织相容性抗原产生反应的T细胞介导排异。MHC蛋白质在T细胞刺激中发挥至关重要的作用，抗原呈递细胞(通常是树突状细胞)展示属于MHC上的细胞表面上的外源蛋白的降解产物的肽，在共刺激信号的存在下，T细胞被活化并作用于也展示相同肽/MHC复合体的靶细胞。例如，刺激的T辅助细胞会靶向巨噬细胞，该巨噬细胞展示与其MHC结合的抗原，或细胞毒性T细胞(CTL)会作用于展示外源病毒肽的病毒感染的细胞。MHC抗原分为NHC I类抗原和MHC II类抗原。
NKG2A(OMIM 161555，将其全部公开内容通过引用结合在此)是NKG2转录物组的成员，NKG2A与CD94一起形成在NK细胞、α/βT细胞、γ/δT细胞、和NKT细胞的亚群的表面上发现的异源二聚体抑制性受体CD94/NKG2A。如本文所使用的，“NKG2A”是指NKG2A基因或编码的蛋白以及任何变体、衍生物或同种型。
NKG2A/CD94为异源二聚体，为非经典HLA-I类分子HLA-E的受体，分布于绝大多数NK细胞表面，发挥抑制性作用。在与HLA-E结合后，NKG2A传递抑制性信号来抑制这些免疫细胞的细胞毒活性，从而减弱T细胞对病毒(如多瘤病毒或人类巨细胞病毒)的清除作用，或者抑制免疫细胞对肿瘤细胞的杀伤。
术语“人类白细胞抗原”(Human leukocyte antigen，HLA)是人类的主要组织相容性复合体的编码基因，位于6号染色体上(6p21.31)，与人类的免疫系统功能密切相关。HLA包括有I类、II类和III类基因部分。HLA的I类和II类基因所表达的抗原位于细胞膜上，为MHC-I(HLA-A、HLA-B、HLA-C位点编码)和MHC-II(HLA-D区编码)，HLA I类几乎分布于身体全部细胞表面，是一个异二聚体，由重链(α链)与β2微球蛋白组成(B2M)，II类主要是定位于巨噬细胞和B淋巴细胞表面的糖蛋白。
术语“B2M”为β-2微球蛋白，也称为B2M，是MHC I类分子的轻链。在人类中，B2M由位于15号染色体上的b2m基因编码，与6号染色体上的作为基因簇定位的其他MHC基因相对。有研究表明，当B2M基因发生突变，来自缺乏正常细胞表面MHC I表达的小鼠的造血移植物被正常小鼠中的NK细胞排斥，说明MHC I分子的缺陷性表达使细胞易于被宿主免疫系统排斥(Bix et al.1991)。
术语“嵌合受体”，即用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子，包括胞外域、跨膜域和胞内域。嵌合受体包括但不限于：嵌合抗原 受体(CAR)、嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)。
术语“嵌合抗原受体”(CAR)包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。胞内信号传导结构域包括刺激性分子和/或共刺激性分子的功能信号传导结构域，在一个方面，刺激性分子为与T细胞受体复合体结合的ζ链；在一个方面，细胞质信号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28。
术语“T细胞受体(T cell receptor，TCR)”介导T细胞对特异性主要组织相容性复合物(MHC)-限制性肽抗原进行识别，包括经典的TCR受体和优化的TCR受体。经典的TCR受体，由α、β两条肽链组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等，其抗原特异性存在于V区，V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3，在一个方面，表达经典的TCR的T细胞可以通过对T细胞采用如抗原刺激等方式，诱导T细胞的TCR对靶抗原的特异性。
术语“嵌合T细胞受体”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常共定位在T细胞表面。嵌合T细胞受体由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ、CD3z、TCR的α链、或TCR的β链，并且，该嵌合T细胞受体整合进T细胞上表达的TCR/CD3复合物。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、抗原结合结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
术语“信号传导结构域”也称为“细胞质信号传导结构域”,是指通过在细胞内传递信息而起作用的蛋白质的功能性部分，用来通过产生第二信使或通过响应这样的信使起效应物作用经由确定的信号传导途径调节细胞的活性。胞内信号传导结构域可以包括蛋白质的全部细胞内部分、或全部天然胞内信号传导结构域、或其功能片段或衍生 物。示例性，靶向CS1、靶向BCMA的CAR的信号传到结构域包括CD3ζ。在具体实施例中，CD3ζ为人CD3ζ分子，包含SEQ ID NO:5或SEQ ID NO:6所示序列。
术语“共刺激分子”是为T(或B)细胞完全活化提供共刺激信号的细胞表面分子及其配体，当与细胞刺激信号分子，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号，从而介导T细胞的共刺激应答。共刺激分子包括但不限于MHC I类分子、BTLA和Toll配体受体、以及OX40，CD2，CD27，CD28，CDS，ICAM-1，LFA-1(CD11a/CD18)和4-1BB(CD137)。示例性，靶向BCMA、靶向CS1的CAR的信号传到结构域包括4-BB。在具体实施例中，CD137为人CD137分子，包含SEQ ID NO:7或SEQ ID NO:8所示序列。
术语“激活”和“活化”可互换使用，可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指T细胞逐步活化的过程。该活化过程受第一刺激信号和共刺激信号的共同调控。T细胞的活化是一个动态变化的过程，其持续时间和活化程度的强弱均受到外界条件刺激的影响。“T细胞活化”或“T细胞激活”指被刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。使用CD3/CD28磁珠，体外抗原刺激或者体内抗原刺激都会对T细胞的活化程度和持续时间造成影响。在一个实施例中，所述工程化T细胞与含特定靶抗原肿瘤细胞共孵育或病毒感染后活化。
术语“基因编辑(Genome editing，Gene editing)”，是指利用部位特异性核酸酶在生物体基因组中的特定位置进行DNA插入、敲除、修改或替换的基因工程技术，会改变DNA序列。这种技术有时称作“基因剪辑”或“基因组工程。基因编辑可以用来实现精确的、高效的基因敲除或者基因敲入。
核酸酶指导的基因组靶向修饰技术通常由一个DNA识别结构域和一个非特异性核酸内切酶结构域构成，由DNA识别结构域识别靶位点，把核酸酶定位到需要进行编辑的基因组区域，然后由非特异性核酸内切酶切断DNA双链，引起DNA断裂自我修复机制，从而引发基因序列的突变和促进同源重组的发生。所述核酸内切酶可以是巨型核酸酶(Meganuclease)、锌指核酸酶、CRISPR/Cas9核酸酶、MBBBD-核酸酶或TALEN-核酸酶。在优选的实施方式中，所述核酸内切酶是CRISPR/Cas9核酸酶、TALEN-核酸酶。利用核酸酶进行基因敲除技术包括CRISPR/Cas9技术、ZFN技术、TALE技术和TALE-CRISPR/Cas9技术。在一些是实施例中，所述的基因编辑技术选 自单碱基编辑(Base Editor)技术、引导编辑(Prime Editor)技术归巢核酸内切酶(Meganuclease)技术。
术语“人工锌指核酸酶(Zinc Finger Nucleases，ZFN)”技术，是第一代核酸酶定点修饰技术，利用能够特异性识别三联体DNA片段的锌指基序(motif)而不是碱基作为识别特定DNA序列的基本单位。最经典的锌指核酸酶是将一个非特异性的核酸内切酶FokI与含有锌指的结构域进行融合，其目的自然是对特定序列进行切割。
术语“转录激活样效应因子(transcription activator-like effector,TALE)”，具有DNA结合特异性，具有能够特异性识别碱基的模块，操作简洁和方便。TALE-DNA结合结构域由串联重复单元组成，大部分单元含3 4个氨基酸，把单元的第12和13位氨基酸设计为可变区(repeat variable residues,RVD)。TALE的RVD识别DNA序列的4个碱基具有高度专一性，第13位氨基酸直接与DNA的碱基特异结合。能够根据DNA序列，在任意位点构建特异的TALEDN识别结合域，可以广泛用于基因序列突变修饰和基因打靶等。设定DNA靶序列，组装TALE-DNA结合域，融合Fok I内切酶的非特异性DNA切割域，组装成TALE核酸酶(tanscription activator-like effector nucleases，TALENs)。TALENs靶向结合DNA，产生DNA双链断裂(DNA double-srand breaks,DSBs)。
CRISPER/Cas9是第三代基因编辑技术。“CRISPR系统”统称为转录物和涉及CRISPR相关(“Cas”)基因的表达或指导其活性的其他元件，包括编码Cas基因的序列、tracr(反式激活CRISPR)序列(例如，tracrRNA或活性部分tracrRNA)、tracr配对序列(涵盖“同向重复”和在内源CRISPR系统背景下的tracrRNA加工的部分同向重复)、指导序列(在内源CRISPR系统背景下也称为“间隔子(spacer)”)、或来自CRISPR座位的其他序列和转录物。CRISPR系统的特征为促进在靶序列的位点处的CRISPR复合物(在内源CRISPR系统的背景下也称为前间区)的形成的元件。在CRISPR复合物形成的背景下，“靶序列”是指指导序列被设计为对其具有互补性的序列，其中在靶序列与指导序列之间的杂交促进CRISPR复合物的形成。完全互补性不是必需的，条件是存在足够互补性以引起杂交并且促进一种CRISPR复合物的形成。CRISPR复合物形成后在cas9酶的作用下可以对基因组特定位点进行切割，引入基因突变；也可以对基因的表达进行调控，如激活或抑制。一个靶序列可以包含任何多核苷酸，如DNA或RNA多核苷酸。在一些实施例中，靶序列位于细胞的细胞核或细胞质中。
术语“基因沉默”，是指由于各种原因，使基因不表达或低表达的现象。基因沉默 可以是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默，也可以是转录后基因沉默，即在基因转录后的水平上通过对靶标RNA进行特异性抑制而使基因失活，包括反义RNA、RNA干扰和微小RNA介导的翻译抑制等。
所述“TCR沉默”是指内源性的TCR不表达或低表达。
所述“MHC沉默”是指内源性的MHC不表达或低表达。
术语“CRISPR(Clustered regularly interspaced short palindromicrepeats)”是指规律成簇间隔短回文重复。
术语“Cas9(CRISPRassociated nuclease)”是CRISPR相关核酸酶，是一种由RNA指导的，利用Cas9核酸酶对靶向基因进行编辑的技术。
一般而言，指导序列(gRNA)是与靶多核酸序列具有足够互补性以便与该靶序列杂交并且指导CRISPR复合物与该靶序列的序列特异性结合的任何多核酸序列。gRNA用于引导，结合或者识别Cas酶。在一些实施例中，当使用适合的比对算法进行最佳比对时，在指导序列与其相应的靶序列之间的互补程度是约或多于约50％、60％、75％、80％、85％、90％、95％、97.5％、99％、或更多。可以使用用于比对序列的任何适合的算法来确定最佳比对，其非限制性实例包括史密斯-沃特曼(Smith-Waterman)算法、尼德曼-翁施(Needleman-Wunsch)算法、基于伯罗斯-惠勒变换(Burrows-Wheeler Transform)的算法(例如伯罗斯-惠勒比对工具(Burrows Wheeler Aligner))、ClustalW、Clustal X、BLAT、Novoalign(Novocraft技术公司)、ELAND(亿明达公司(Illumina)，圣地亚哥，加利福尼亚州)、SOAP(在soap.genomics.org.cn可获得)、以及Maq(在maq.sourceforge.net可获得)。
在一些实施例中，该CRISPR酶是包含一个或多个异源蛋白结构域(例如除了该CRISPR酶之外的约或多于约1、2、3、4、5、6、7、8、9、10个或更多个结构域)的融合蛋白的一部分。CRISPR酶融合蛋白可以包含任何其他蛋白质，以及任选地在任何两个结构域之间的连接序列。可以融合到CRISPR酶上的蛋白质结构域的实例包括但不限于，表位标签、报告基因序列、以及具有下列活性的一者或多者的蛋白质结构域：甲基酶活性、脱甲基酶活性、转录激活活性、转录阻抑活性、转录释放因子活性、组蛋白修饰活性、RNA切割活性和核酸结合活性。表位标签的非限制性实例包括组氨酸(His)标签、V5标签、FLAG标签、流感病毒血凝素(HA)标签、Myc标签、VSV-G标签、和硫氧还蛋白(Trx)标签。
术语“Cas9酶”可以是野生型Cas9或人工改造Cas9。
术语“sgRNA”指短小的gRNA。
在进行基因编辑时，给予的gRNA、tracr配对序列、及tracr序列可以单独给予，也可以一条完整的RNA序列。
Cas9蛋白与gRNA结合能够实现在特异位点处切割DNA，来源于Streptococcus pyogenes的CRISPR/Cas系统识别序列为23bp，并能靶向20bp，其识别位点最末3位NGG序列被称作PAM(protospacer adjacent motif)序列。
CRISPR/Cas9转基因可以通过载体(例如AAV、腺病毒、慢病毒)、和/或粒子和/或纳米粒子、和/或电转来递送。
在一实施例中，分别对在CS1、B2M、TCR的α和β链中的一种或两种链的恒定区域的相应编码基因的外显子用CRISPER/Cas技术敲除，使内源性CS1、B2M或TCR不具有活性；优选为定点敲除内源性TCRα链恒定区的第一外显子,使用的gRNA选自如SEQ ID NO:17所示序列。在具体实施例中，采用CRISPR/Cas9技术敲除所述工程化T细胞内源性CS1基因，使用的gRNA选自如SEQ ID NO:19、20、40、41、42、43、44和/或45所示序列。采用CRISPR/Cas9技术敲除所述工程化T细胞内源性B2M基因，使用的gRNA选自如SEQ ID NO:18所示序列。
“抑制”或“遏制”B2M或TCR或CS1的表达是指细胞中B2M或TCR或CS1的表达减少至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。更具体而言，“抑制”或“遏制”B2M的表达是指细胞中B2M的含量降低至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。可以通过本领域内已知的任何合适的方法，如ELISA、免疫组织化学、免疫印迹(Western Blotting)或流式细胞术使用B2M、TCR、CS1的特异性抗体测定细胞中蛋白的表达或含量。
如本文所用的“RNA干扰剂”被定义为通过RNA干扰(RNAi)干扰或抑制靶基因表达的任何制剂。此类RNA干扰剂包括但不限于与靶基因或其片段同源的RNA分子的核酸分子、短干扰RNA(siRNA)、shRNA或miRNA和通过RNA干扰(RNAi)干扰或抑制靶基因的表达的小分子。
本文所述的“抗体”可以是全长的抗体，也可以是保留抗原结合能力的抗体片段。抗体片段是指包含从该分子所衍生于的抗体的抗原结合部分(例如，CDR)的任何分子。抗体片段的实例包括但不限于，从抗原结合分子形成的Fab、Fab'、F(ab')2和Fv 片段、dAb、线性抗体、scFv抗体和多特异性抗体。在某些实施方案中，抗体是指是特异性结合抗原的抗体片段，包括其一个或多个互补决定区(CDR)，在进一步的实施方案中，抗体可以是单链抗体(scFv)。在一些实施方案中，抗体包含高亲和性多聚体(avimer)或由高亲和性多聚体组成。示例性的，识别CS1的抗体或其功能片段含有SEQ ID NO:21所示的HCDR1，和/或SEQ ID NO:22所示的HCDR2，和/或SEQ ID NO:23所示的HCDR3，和/或SEQ ID NO:24所示的LCDR1，和/或SEQ ID NO:25所示的LCDR2，和/或SEQ ID NO:26所示的LCDR3。示例性的，识别CS1的抗体或其功能片段含有SEQ ID NO:27所述的重链可变区和/或SEQ ID NO:28所述的轻链可变区。示例性的，识别CS1的抗体或其功能片段含有SEQ ID NO:11或SEQ ID NO:12所述的scFV序列。示例性的，识别BCMA的抗体或其功能片段含有SEQ ID NO:15或16所示的scFV序列。示例性的，识别NKG2A的抗体或其功能片段含有SEQ ID NO:13或14所示的scFV序列。
术语“抗体构建体”是指结构和/或功能基于抗体(例如全长或完整免疫球蛋白分子)的结构和/或功能的分子。因此，抗体构建体能够结合到其特异性靶标或抗原。此外，根据本发明的抗体构建体包含抗体的允许靶抗原结合的最小结构需要。此最小需要可例如通过存在至少三个轻链CDR(即VL区的CDR1、CDR2和CDR3)和/或三个重链CDR(即VH区的CDR1、CDR2和CDR3)，优选所有六个CDR来定义。根据本发明的构建体所基于的抗体包括例如单克隆抗体、重组抗体、嵌合抗体、去免疫化(deimmunized)抗体、人源化抗体和人抗体。
全长或完整抗体在根据本发明的“抗体构建体”的定义内，也包括骆驼抗体和通过生物技术或蛋白质工程改造方法或过程产生的其他免疫球蛋白抗体。这些全长抗体可为例如单克隆抗体、重组抗体、嵌合抗体、去免疫化抗体、人源化抗体和人抗体。全长抗体的片段(诸如VH、VHH、VL、(s)dAb、Fv、Fd、Fab、Fab′、F(ab′)2或“r IgG”(“半抗体”))也在“抗体构建体”的定义内。根据本发明的抗体构建体也可为抗体的经修饰片段，也称为抗体变体，诸如scFv；di-scFv或bi(s)-scFv；scFv-Fc；scFv拉链(scFv-zipper)；scFab；Fab2；Fab3；双功能抗体(diabody)；单链双功能抗体；串联双功能抗体(Tandab)；串联di-scFv；串联tri-scFv；“微型抗体”，其由如下结构例示：(VH-VL-CH3) 2、(scFv-CH3) 2、((scFv) 2-CH3+CH3)、((scFv) 2-CH3)或(scFv-CH3-scFv) 2；多功能抗体，诸如三功能抗体或四功能抗体；和单结构域抗体，诸如纳米抗体，或仅含一个可变结构域(其可为VHH、VH或VL)的独立于其他V区或结构域而特异性结合抗原或表位 的单可变结构域抗体。
结合结构域通常可包含抗体轻链可变区(VL)和抗体重链可变区(VH)；然而，无须同时包含这两者。例如，Fd片段具有两个VH区且通常保留完整抗原结合结构域的一些抗原结合功能。抗体片段、抗体变体或结合结构域的形式的另外实例包括(1)Fab片段，其为具有VL、VH、CL和CH1结构域的单价片段；(2)F(ab′) 2片段，其为具有两个由二硫桥在铰链区连接的Fab片段的二价片段；(3)Fd片段，其具有两个VH和CH1结构域；(4)Fv片段，其具有抗体的单个臂的VL和VH结构域；(5)dAb片段(Ward等人(1989)Nature 341：544-546)，其具有VH结构域；(6)分离的互补决定区(CDR)和(7)单链Fv(scFv)，后者是优选的(例如来源于scFV文库)。术语“抗体构建体”的定义包括单价、二价和多价构建体，且因此包括特异性结合到仅一种抗原结构的单特异性构建体以及经由不同结合结构域特异性结合超过一种抗原结构(例如两种、三种或更多种)的双特异性和多特异性构建体。此外，术语“抗体构建体”的定义包括仅由一条多肽链组成的分子以及由超过一条多肽链组成的分子，这些链可以相同(均二聚体、均三聚体或均寡聚物)或不同(杂二聚体、杂三聚体或杂寡聚物)。
本发明的抗体构建体优选为“体外产生的抗体构建体”。该术语是指根据上述定义的抗体构建体，其中所有或一部分可变区(例如至少一个CDR)在非免疫细胞选择(例如体外噬菌体展示)蛋白芯片或任何其他可测试候选序列结合到抗原的能力的方法中产生。因此，该术语优选排除仅通过在动物中在免疫细胞中进行基因组重排而产生的序列。“重组抗体”是通过使用重组DNA技术或遗传工程改造而制备的抗体。
在具体实施例中，抗体构建体是靶向CS1和CD3的双特异性抗体BITE(bispecific T cell engage antibody)。本发明中表达BITE的T细胞也称为T-BITE细胞。
如本文所用，术语“单克隆抗体”(mAb)或单克隆抗体构建体是指从基本上均质的抗体的群获得的抗体，即构成该群的各个抗体除可以少量出现的可能天然存在的突变和/或翻译后修饰(例如异构化、酰胺化)以外是相同的。与通常包括针对不同决定簇(或表位)的不同抗体的常规(多克隆)抗体制备物相比，单克隆抗体对抗原上的单一抗原位点或决定簇具有高度特异性。除其特异性之外，单克隆抗体的有利之处还在于其通过杂交瘤培养而合成，因此不受其他免疫球蛋白污染。修饰语“单克隆”指示抗体的特征是从基本上均质的抗体群获得，且不应解释为需要通过任何特定的方法产生该抗体。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、 电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
术语“个体”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
术语“外周血单个核细胞”(peripheral blood mononuclear cell,PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。
当用于指核酸序列时，本文所用的术语“序列”可以包括DNA或RNA，并且可以是单链或双链。
本文所用的术语“有效量”是指提供治疗或预防益处的量。
本文所用的术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核酸序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域所有已知的那些，如质粒、病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。还可以包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。
本文使用的术语序列“同一性”通过在比较窗口(例如至少20个位置)上比较两个经最佳匹配的序列来确定同一性百分比，其中比较窗口中多核苷酸或多肽序列的部分可以包含添加或缺失(即间隙)，例如对于最佳匹配的两个序列而言与参考序列(其不包含添加或缺失)相比20％或更少的间隙(例如5至15％、或10至12％)。通常通过确定在两个序列中发生相同的核酸碱基或氨基酸残基的位置的数目来计算百分比，以产生正确匹配的位置的数目，将正确匹配位置的数目除以参考序列中的位置总数(即窗口大小)，并将结果乘以100，以产生序列同一性的百分比。
术语“外源”指的是一个核酸分子或多肽、细胞、组织等没有在生物体自身内源性表达，或表达水平不足以实现过表达时具有的功能。
术语“内源”是指一个核酸分子或多肽等来自生物体自身。
术语“嵌合受体”，即用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子，包括胞外域、跨膜域和胞内域。嵌合受体包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)。在一些实施方案中，本发明嵌合受体是嵌合抗原受体。
术语“嵌合抗原受体”(CAR)是指两组或以上多肽；当免疫效应细胞表达所述CAR时，那么所述免疫细胞能特异性针对靶细胞(本发明包括肿瘤细胞、NK细胞)，并且产生细胞内信号。在某些实施例中，CAR包括至少一个胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域(也称为“细胞质信号传导结构域”)。胞内信号传导结构域包括刺激性分子和/或共刺激性分子的功能信号传导结构域，在一个方面，刺激性分子为与T细胞受体复合体结合的ζ链；在一个方面，细胞质信号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28。在某些实施例中，多肽组彼此邻接。示例性，靶向CS1的CAR包含SEQ ID NO:37序列。表达靶向CS1的CAR且表达膜结合型NKG2A抗体的细胞包括SEQ ID NO:37和38所示的氨基酸序列。表达靶向BCMA的CAR且表达膜结合型NKG2A抗体的细胞包括SEQ ID NO:38和39所示的氨基酸序列。
嵌合抗原受体通常包含胞外抗原结合区。在一些实施方案中，胞外抗原结合区可以是全人的，人源化，鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。
胞外抗原结合区的例子可以是scFv、Fv、Fab、Fab'、Fab'-SH、F(ab') 2、单结构域片段、或与接合其同源受体的天然配体，以及它们的任何衍生物。
在一些方面，细胞外抗原结合区(例如scFv)，可以包含对抗原特异性的轻链CDR。在一些情况下，轻链CDR可以包含两个或更多个轻链CDR，其可以被称为轻链CDR-1，CDR-2等。在一些情况下，轻链CDR可以包含三个轻链CDR，其可分别称为轻链CDR-1，轻链CDR-2和轻链CDR-3。在一实施方式中，存在于普通轻链上的一组CDR可统称为轻链CDR。
在一些方面，细胞外抗原结合区(例如scFv)，可以包含对抗原特异的重链CDR。重链CDR可以是抗原结合单元例如scFv的重链互补决定区。在一些情况下，重链 CDR可以包含两个或更多个重链CDR，其可以称为重链CDR-1，CDR-2等。在一些情况下，重链CDR可以包含三个重链CDR，其可分别称为重链CDR-1，重链CDR-2和重链CDR-3。在一实施方式中，存在于共同重链上的一组CDR可统称为重链CDR。
通过使用基因工程，可以以各种方式修饰细胞外抗原结合区。在一些情况下，可以突变细胞外抗原结合区域，从而可以选择细胞外抗原结合区域以对其靶抗原具有更高的亲和力。在一些情况下，细胞外抗原结合区域对其靶抗原的亲和力可针对可在正常组织上以低水平表达的靶抗原进行优化。在其他情况下，对靶抗原的膜结合形式具有更高亲和力的细胞外抗原结合区域的克隆可以优于其可溶形式的对应物。
在一些情况下，细胞外抗原结合区域还包括铰链或间隔区，术语铰链和间隔区可以互换使用。铰链可以被认为是用于向细胞外抗原结合区提供柔性的CAR的一部分。例如，铰链可以是CD8α分子的天然铰链区。
术语“跨膜域”可以将嵌合蛋白锚定在细胞的质膜上。例如，可以采用CD28、CD8α的跨膜域(也称为CD8跨膜域)。
术语“调控”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。在一具体实施方式中，是指负向改变。
术语“治疗”是指在试图改变疾病过程的干预措施，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
术语“预防”是指在试图在疾病(如细胞移植产生的排斥反应)产生前进行的干预措施。术语“移植免疫排斥”是指宿主进行同种异体的组织、器官、或细胞等移植物移植后，外源的移植物作为一种“异己成分”被宿主的免疫系统识别，并发起针对移植物的攻击、破坏和清除的免疫学反应。本发明提供一种抗移植免疫排斥的细胞及抗抑制排斥的方法。
本发明的所提供的工程化T细胞可用于治疗、预防或改善自身免疫性疾病或炎性疾病，特别是自身免疫疾病相关的的炎症疾病，诸如关节炎(例如类风湿性关节炎、慢性进行性关节炎(arthritis chronica progrediente)和变形性关节炎)和风湿性疾病，包括牵涉骨损失、炎症性疼痛的炎性病况和风湿性疾病、脊椎关节病变(包括强直性脊柱炎)、赖特尔综合征、反应性关节炎、银屑病关节炎、幼年型特发性关节炎和肠病性关节炎、起止点炎、超敏反应(包括气道超敏反应和皮肤超敏反应)和过敏。本发明 所提供的工程化T细胞用于治疗及预防包括自身免疫性血液学障碍(包括例如溶血性贫血、再生障碍性贫血、纯红细胞贫血和特发性血小板减少)、系统性红斑狼疮(SLE)、狼疮性肾炎、炎性肌肉疾病(皮肌炎)、牙周炎、多软骨炎、硬皮病、韦格纳肉芽肿、皮肌炎、慢性活动性肝炎、重症肌无力、银屑病、史蒂芬约翰逊综合征、自发性口炎性腹泻、自身免疫性炎性肠病(包括例如溃疡性结肠炎、克罗恩病和肠易激综合症)、内分泌性眼病、格雷夫斯病、结节病、多发性硬化、系统性硬化病、纤维变性疾病、原发性胆汁性肝硬化、幼年型糖尿病(I型糖尿病)、葡萄膜炎、干燥性角结膜炎和春季角结膜炎、间质性肺纤维化、假体周围骨溶解、肾小球肾炎(有和无肾病综合症，例如包括特发性肾病综合征或微小病变性肾病)、多发性骨髓瘤、其他类型的肿瘤、皮肤和角膜的炎性疾病、肌炎、骨植入物的松动、代谢紊乱(诸如肥胖、动脉粥样硬化和其它心血管疾病，包括扩张型心肌病、心肌炎、II型糖尿病和血脂异常)和自身免疫性甲状腺疾病(包括桥本甲状腺炎)、中小血管原发性血管炎、大血管血管炎包括巨细胞性动脉炎、化脓性汗腺炎、视神经脊髓炎、斯耶格伦氏综合征、白塞氏病、特应性和接触性皮炎、细支气管炎、炎性肌肉疾病、自身免疫性外周神经病、免疫性肾脏、肝脏和甲状腺疾病、炎症和动脉粥样硬化、自身炎症发热综合征、免疫血液学紊乱以及皮肤和粘膜的大疱性疾病。
本发明所提供的工程化T细胞可用于治疗、预防或改善哮喘、支气管炎、细支气管炎、特发性间质性肺炎、尘肺、肺气肿以及气道的其它阻塞性或炎性疾病。
可将本发明的工程化T细胞作为唯一的活性成分或与其它药物例如免疫抑制剂或免疫调节剂或其它抗炎剂或其它细胞毒性剂或抗癌剂结合(例如作为其佐剂或与其组合)施用，例如以治疗或预防免疫紊乱相关疾病。例如，可将本发明的抗体与如下药物组合使用：DMARD，例如金盐、柳氮磺吡啶、抗疟药、甲氨蝶呤、D-青霉胺、硫唑嘌呤、麦考酚酸、他克莫司、西罗莫司、二甲胺四环素、来氟米特、糖皮质激素；钙调神经磷酸酶抑制剂，例如环孢菌素A或FK 506；淋巴细胞再循环的调节剂，例如FTY720和FTY720类似物；mTOR抑制剂，例如雷帕霉素，40-O-(2-羟基乙基)-雷帕霉素、CCI779、ABT578、AP23573或TAFA-93；具有免疫抑制性质的子囊霉素，例如ABT-281、ASM981等；皮质类固醇；环磷酰胺；硫唑嘌呤；来氟米特；咪唑立宾；吗替麦考酚酯；15-脱氧精胍菌素或其免疫抑制同系物、类似物或衍生物；免疫抑制单克隆抗体，例如，针对白细胞受体，例如，MHC、CD2、CD3、CD4、CD7、CD8、CD25、CD28、CD40。CD45、CD58、CD80、CD86或其配体的单克隆抗体； 其它免疫调节化合物。
“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。本发明的过度增生性病症是指癌症。本发明所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。
本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1 (MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。优选的，所述肿瘤抗原为BCMA或者CD19。
病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。
本发明提供的用途可以联合一种或多种抗癌疗法的治疗，所述抗癌疗法选自抗体治疗、化疗、细胞因子治疗、树突细胞治疗、基因治疗、激素治疗、激光治疗和放射治疗组中的组。
本发明提供的工程化免疫细胞的用途可以任何方便的方式进行，包括通过雾化吸入、注射、摄取、输血、植入或移植。本文所述的组合物可通过静脉内或淋巴管内注射或腹膜内皮下、皮内、瘤内、结内，髓内、肌内给予患者。在一个实施方式中，优选通过静脉注射给予本发明的细胞组合物。
可以一种或多种剂量施用起本发明用途的细胞或细胞群。在另一个实施方式中，以单一剂量形式给予所述有效量的细胞。在另一个实施方式中，在一段时间内以多于一种剂量施用所述有效量的细胞。施用时间在管理医师的判断之内，并且取决于患者的临床状况。可从任何来源获得细胞或细胞群，例如血库或供者。虽然个体需要变化，但确定用于特定疾病或病况的给定细胞类型的有效量的最佳范围在本领域的技术范围内。有效量是指提供治疗或预防益处的量。施用的剂量取决于受体的年龄、健康和体重，同期治疗(如有的话) 的种类、治疗频率和所需效果的性质。
在本发明的某些实施方式中，可结合任何数量的相关治疗方式(例如，之前、同时或之后)将细胞施用于患者，其包括但不限于用诸如抗肿瘤治疗、抗病毒治疗、西多福韦和白介素-2、阿糖胞苷(也称为ARA-C)的治疗或针对MS患者的那他丽珠单抗(nataliziimab)治疗或针对银屑病患者的依法丽珠单抗(efaliztimab)治疗或针对PML患者的其它治疗。在另外的实施方式中，可联合化学疗法、辐射、免疫抑制剂如环孢菌素、硫唑嘌呤、甲氨蝶呤、霉酚酸酯和FK506抗体或其它免疫烧蚀剂如CAM PATH、抗-CD3抗体或其它抗体疗法，细胞毒素、氟达拉滨、环孢菌素、FK506、雷帕霉素、霉酚酸、类固醇、FR901228、细胞因子和辐照使用本发明的T细胞。这些药物抑制钙依赖性磷酸酶钙调磷酸酶(环孢菌素和FK506)或抑制对生长因子诱导的信号传导(雷帕霉素)重要的p70S6激酶(Liu等人，Cell 66:807-815,11；Henderson等人，Immun.73:316-321,1991；Bierer等人，Citrr.Opin.mmn.5:763-773,93)。在另一个实施方式中，联合骨髓移植(例如，之前、同时或之后)、使用化疗剂如氟达拉滨、体外放射线治疗(XRT)、环磷酰胺或抗体如OKT3或阿仑单抗(CAMPATH)的T细胞消除治疗将本发明的细胞组合物施用于患者。在另一个实施方式中，在B细胞消除治疗后施用本发明的细胞组合物，例如与CD20反应的药剂如利妥昔单抗。例如，在一个实施方式中，受试者可通过高剂量化疗，随后进行外周血干细胞移植进行标准治疗。在某些实施方式中，在移植后，受试者接受本发明的扩增的免疫细胞的输注。在另外的实施方式中，在手术之前或之后施用扩增的细胞。通过本文所述的任何一种方法获得的所述修饰的细胞可用于本发明的特定方面，用于治疗有需要的患者的宿主抗移植物(HvG)排斥和移植物抗宿主病(GvHD)；因此在本发明的范围内的是一种治疗有需要的患者的宿主抗移植物(HvG)排斥和移植物抗宿主病(GvHD)的方法，其包括通过向所述患者施用有效量的修饰的细胞来治疗所述患者，所述修饰的细胞包含失活的TCRα和/或TCRβ基因。
本文提供了一种T细胞被基因工程化表达CS1-CAR或CS1-CD3 BITE(bispecific T-cell engager)用于杀伤NK细胞；提供了所述基因功能化T细胞制备方法、用于杀伤NK细胞的用途、及其增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率的方法。在一些实施方案中，工程化T细胞被基因工程化以表达CS1-CAR或CS1-CD3 BITE。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR或CS1-CD3 BITE，还减少或消除内源性CS1的表达或活性。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR或CS1-CD3 BITE，还减少或消除内源性CS1的表达或活性,还被基因工程化表达NKG2A结合蛋白，优选地为NKG2A膜结合抗体。在一些实施方案中，工程化T细胞被基 因工程化表达CS1-CAR或CS1-CD3 BITE，还减少或消除内源性CS1的表达或活性,还被基因工程化表达NKG2A结合蛋白，优选地为NKG2A膜结合抗体；所述细胞也被基因工程化表达至少非靶向CS1的嵌合受体(CAR、修饰的TCR、TFP、TAC、aTCR或其组合)，所述嵌合受体特异性结合肿瘤抗原和/或病毒抗原。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR或CS1-CD3 BITE，还减少或消除B2M和TCR的表达或活性,所述细胞也被基因工程化表达至少一个非靶向CS1的嵌合受体(CAR、修饰的TCR、TFP、TAC、aTCR或其组合)。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR或CS1-CD3 BITE，还减少或消除B2M和TCR的表达或活性,所述细胞也被基因工程化表达至少非靶向CS1的嵌合受体(CAR、修饰的TCR、TFP、TAC、aTCR或其组合)，所述嵌合受体特异性结合肿瘤抗原和/或病毒抗原。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR或CS1-CD3 BITE,还减少或消除B2M和TCR的表达或活性,所述细胞也被基因工程化表达非靶向CS1的嵌合受体(CAR、修饰的TCR、TFP、TAC、aTCR或其组合)，其中，至少一个所述嵌合受体特异性结合肿瘤抗原或病毒抗原，还被基因工程化表达NKG2A结合蛋白，优选地为NKG2A膜结合抗体。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR或CS1-CD3 BITE，还减少或消除B2M、TCR和内源性CS1的表达或活性，所述细胞也被基因工程化表达非靶向CS1的嵌合受体(CAR、修饰的TCR、TFP、TAC、aTCR或其组合)，其中，至少一个所述嵌合受体特异性结合肿瘤抗原或病毒抗原，还被基因工程化表达NKG2A结合蛋白，优选地为NKG2A膜结合抗体。在一个具体的实施方式中，CS1-CD3 BITE的氨基酸序列如SEQ ID NO.31所示，核酸序列如SEQ ID NO.36所示。
本发明所提供的基因工程化T细胞能增强在先、同时、在后导入受试者的T细胞和/或携带靶向靶抗原的CAR-T细胞对肿瘤细胞的杀伤、以及增强所述T细胞和/或CAR-T细胞的存活、增殖。
本文提供了药物联用方案：1.本发明所提供的基因工程化T细胞与非靶向CS1的嵌合抗原受体2的T细胞联合施用，优选地，所述嵌合抗原受体2靶向肿瘤或病原体抗原，更优选地，所述嵌合抗原受体2靶向CD19、GPC3、Claudin18.2、EGFR、EGFRvIII或其组合。
2.本发明所提供的基因工程化T细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；或与改善其相关的一种或多种副作用的药剂联合施用。
本专利包括，例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、 CN 108884459 A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN 109908176 A、CN109880803A、CN 110055275 A、CN110123837A、CN 110438082 A、CN 110468105 A国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306 A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018/219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/219029中公开的那些CAR-T细胞及其制备方法。
本发明的优点：
本发明提供的工程化T细胞对NK细胞有杀伤作用，为抗NK细胞肿瘤提供一种新的治疗手段。增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率的方法
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1.表达CS1或CS1-mscFv的CAR-T细胞的制备
采用本领域常规分子生物学方法构建表达载体(图1)。将由CD8α信号肽(SEQ ID NO：1)、CS1-scfv(SEQ ID NO：11)、CD8α铰链和跨膜域(SEQ ID NO：32)、CD137胞内信号传导结构域(SEQ ID NO：7)以及CD3ζ(SEQ ID NO：5)组成的CS1-BBZ片段插入PRRLsin载体构建载体PRRLsin-CS1-BBZ；将包含CD8α信号肽(SEQ ID NO：1)、NKG2A-scfv(SEQ ID NO：13)、CD8α铰链和跨膜域(SEQ ID NO：32)的片段插入PRRLsin载体构建载体PRRLSIN-NKG2A；将CS1-BBZ片段、F2A(SEQ ID NO：9)、NKG2A-scfv(SEQ ID NO：13)、CD8α铰链和跨膜域(SEQ ID NO：32)组成的片段插入PRRLsin载体构建载体PRRLsin-CS1-BBZ-F2A-NKG2A(图1)。
将由CD8α信号肽(SEQ ID NO：1)、BCMA-scfv(SEQ ID NO：15)、CD8α铰链和跨膜域(SEQ ID NO：32)、CD137胞内信号传导结构域(SEQ ID NO：7)以及CD3ζ(SEQ ID NO：5)组成的BCMA-BBZ片段插入PRRLsin载体构建载体PRRLsin-BCMA-BBZ； 将BCMA-BBZ片段、F2A(SEQ ID NO：9)、NKG2A-scfv(SEQ ID NO：13)、CD8α铰链和跨膜域(SEQ ID NO：32)组成的片段插入PRRLsin载体构建载体PRRLsin-BCMA-BBZ-F2A-NKG2A(图1)
将上述载体PRRLsin-CS1-BBZ、PRRLSIN-NKG2A、PRRLsin-CS1-BBZ-F2A-NKG2A、PRRLsin-BCMA-BBZ、PRRLsin-BCMA-BBZ-F2A-NKG2A分别转染293T细胞，包装慢病毒，得到相应的慢病毒CS1-BBZ、NKG2A和CS1-BBZ-F2A-NKG2A、BCMA-BBZ和BCMA-BBZ-F2A-NKG2A。感染方法为本领域表达嵌合抗原受体的T细胞制备过程中常规的感染方法。
利用Ficoll-Paque(GE bioscience)进行密度梯度离心，从人外周血中分离出单个核细胞(PBMC)，加入抗CD3/CD28的磁珠分选得到T细胞，体外进行活化与扩增。将上述慢病毒CS1-BBZ、NKG2A、CS1-BBZ-F2A-NKG2A、BCMA-BBZ和BCMA-BBZ-F2A-NKG2A分别感染活化后的T细胞并培养扩增至需要的数量，得到CS1 CAR-T细胞、NKG2A-T细胞、CS1 CAR-NKG2A-T细胞、BCMA CAR-T和BCMA CAR-NKG2A-T细胞。经流式检测，分别筛选出CS1 CAR-T细胞、NKG2A-T细胞、CS1 CAR-NKG2A-T细胞、BCMA CAR-T细胞和BCMA CAR-NKG2A-T细胞阳性细胞。
实施例2、基因敲除T细胞的制备
按照试剂说明书(GeneArt TM Precision gRNA Synthesis Kit，Thermo Tisher，体外合成分别靶向TCR、B2M和CS1的gRNA序列分别如SEQ ID NO：17，18，19所示。慢病毒CS1-BBZ、NKG2A、CS1-BBZ-F2A-NKG2A、BCMA-BBZ、BCMA-BBZ-F2A-NKG2A转染T细胞后第3天，分别对UTD(未感染的T细胞)，感染后的CS1 CAR-T细胞、NKG2A-T细胞、CS1 CAR-NKG2A-T细胞、BCMA CAR-T细胞、BCMA CAR-NKG2A-T细胞进行TRAC/B2M双敲除、或TRAC/B2M/CS1三敲除。Cas9酶和gRNA按1:4比例进行室温孵育，将细胞与Cas9酶和gRNA复合物(RNP)进行混合，利用maxcyte电转仪将RNP复合物导入到CAR-T细胞中。体外扩增TCR/B2M敲除的T细胞、TCR/B2M/CS1敲除的T细胞，用B2M抗体、CD3抗体对双敲除细胞进行标记，或用B2M抗体、CD3抗体、CS1抗体对三敲除细胞进行标记，再用偶联藻红素(PE)的二抗进行标记，标记后的细胞用抗PE的磁珠经分选柱分选后，收集CD3、B2M双阴性的细胞，或CD3、B2M、CS1三阴性的细胞(分选试剂盒购自美天旎)，即得到TCR、 B2M缺失的双敲除T细胞(也称为DKO-T细胞)：CS1 CAR-DKO-T细胞、NKG2A-DKO-T细胞、CS1 CAR-NKG2A-DKO-T细胞、BCMA-CAR-DKO-T细胞、BCMA CAR-NKG2A-DKO-T；TCR、B2M、CS1缺失的三敲除T细胞(也称为TKO-T细胞)：CS1 CAR-TKO-T细胞、NKG2A-TKO-T细胞、CS1 CAR-NKG2A-TKO-T细胞、BCMA-CAR-TKO-T细胞、BCMA CAR-NKG2A-TKO-T；经流式细胞术检测显示双敲或三敲除效率均能达到90％以上，且经过磁珠阴性富集后CD3阴性或B2M阴性的T细胞能达到97％以上。以表达CS1-CAR的T细胞为例，敲除效率如图2A所示，T细胞或CS1 CAR-T细胞进行双敲或三敲除，其敲除效率都能达到90％以上；且经过磁珠阴性富集后CD3阴性或B2M阴性的T细胞比例如图2B所示，能达到97％以上。
以表达CS1-CAR的T细胞为例，如图3A所示，CS1-CAR T、CS1 CAR-TKO-T细胞，CAR阳性率达到90％以上。流式检测CAR或膜结合的NKG2A scFv表达情况如图3 B所示。
实施例3、体外肿瘤细胞杀伤功能检测
采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)检测细胞毒性，具体参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
靶细胞：分别接种多发性骨髓瘤细胞RPMI-8226(上海中科院细胞库)、NCI-H929(上海中科院细胞库)、MM.1R(上海中科院细胞库)和MM.1S细胞(上海中科院细胞库)于96孔板，其中，RPMI-8226和NCI-H929细胞为低表达CS1细胞，MM.1R和MM.1S为高表达CS1细胞；
效应细胞：按效靶比3:1，1:1，0.3:1加UTD(未感染病毒的T细胞)、CS1 CAR-T细胞、CS1 CAR-DKO-T细胞和CS1 CAR-TKO-T细胞孵育；
各组均设3复孔，取3个复孔的平均值。实验结果如图4所示，相对UTD,CS1 CAR-T细胞、CS1 CAR-DKO-T细胞和CS1 CAR-TKO-T细胞显示显著的体外抗肿瘤活性。
实施例4、CAR-T细胞对人原代NK细胞的毒性检测
1.CS1在各免疫细胞上的表达情况检测
CS1特异的表达在免疫细胞上，包括NK细胞、T细胞、浆细胞及活化的单核细胞。在静息的NK细胞上(rNK)，CS1的表达比例超过95％，细胞因子IL2和IL15体外活化的NK细胞(aNK)CS1表达比例达到98％(图5)(图5中NK-1、NK-2、NK-3、NK-4分别来自4个供体)。
2.CS1 CAR-T细胞对人原代NK细胞杀伤能力的检测
采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)检测细胞毒性。
靶细胞：分别接种rNK或aNK细胞于96孔板；
效应细胞：按效靶比3:1，1:1，0.3:1加UTD、CS1 CAR-T细胞孵育6小时；
各组均设3复孔，取3个复孔的平均值。实验结果如图6A、6B所示，相对UTD，CS1 CAR-T对原代NK细胞里的rNK和原代NK细胞经IL2和IL15体外活化的aNK都显示出显著的效杀伤活性。此外在效靶比1:1的条件下通过流式技术检测发现，在共培养24小时后，CS1 CAR-T细胞能够完全清除共培养体系中的rNK和aNK细胞(图7A，7B)。由此可见，CS1 CAR-T细胞在体外显示出显著的杀伤NK细胞的活性，说明CS1CAR-T细胞可以有效清除静息和活化状态的NK细胞。
实施例5、基因敲除的CAR-T细胞抵抗NK细胞杀伤能力的检测
1.NK细胞对TCR/B2M缺失的T细胞的杀伤
采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)检测NK细胞毒性。
靶细胞：分别接种UTD、DKO-T细胞于96孔板；
效应细胞：按效靶比2:1，0.4:1加rNK细胞、体外细胞因子活化的aNK细胞孵育18小时；
各组均设3复孔，取3个复孔的平均值。实验结果如图8所示，相对UTD细胞，aNK细胞对DKO-T细胞显示出显著杀伤活性；而rNK对DKO-T细胞无显著的杀伤活性。上述结果说明TCR/B2M缺失的T细胞会被活化的NK细胞杀伤。
2.CS1 CAR-TKO-T细胞抵抗NK细胞杀伤能力的检测
效应细胞：分别接种aNK-1细胞(NKG2A高表达)和aNK-2细胞(NKG2A低表达)(图9)，于96孔板；
靶细胞：按照效靶比1:1加TKO-T细胞(未转染的UTD细胞)、BCMA CAR-TKO-T细胞、CS1 CAR-TKO-T细胞、NKG2A-TKO-T细胞、BCMA CAR-NKG2A-TKO-T细胞、CS1 CAR-NKG2A-TKO-T细胞，共培养24、48小时通过流式技术检测T细胞和NK细胞共培养体系中T细胞比例及数量。
实验结果如图10所示，aNK-1细胞或aNK-2细胞对TCR/B2M/CS1缺失的T细胞如TKO-T细胞、BCMA CAR-TKO-T细胞都有明显杀伤活性；当TKO-T细胞中表达CS1CAR，如CS1 CAR-TKO-T细胞能显著抑制NKG2A低表达的aNK-2细胞对TKO-T细胞 的杀伤但对NKG2A高表达的aNK-1细胞抵抗能力不佳；当TKO-T细胞中表达NKG2A，如NKG2A-TKO-T细胞、BCMA CAR-NKG2A-TKO-T细胞能抑制NKG2A高表达的aNK-1细胞对TKO-T细胞的杀伤，但对NKG2A低表达的aNK-2细胞抵抗能力不佳；当TKO-T同时表达CS1 CAR和NKG2A，如CS1 CAR-NKG2A-TKO-T细胞能够同时非常有效抵抗aNK-1、aNK-2的杀伤。上述结果说明表达CS1-CAR或NKG2A能抵抗NK细胞对TCR/B2M、TCR/B2M/CS1缺失的T细胞的杀伤作用，尤其是共表达CS1-CAR和NKG2A能显著地抵抗NK细胞对TCR/B2M、TCR/B2M/CS1缺失的T细胞的杀伤作用。
实施例6、T-BITE细胞的制备
1.CS1-CD3 BITE载体构建
采用本领域常规分子生物学方法构建表达载体CS1-CD3 BITE。将由CD8α信号肽(SEQ ID NO：1)、CS1-scfv(SEQ ID NO：11)、BITE接头核酸(SEQ ID NO：34)、CD3 scFv(SEQ ID NO：35)、F2A(SEQ ID NO：9)、EGFP(SEQ ID NO：3)组成的片段插入PRRLsin载体，构建载体PRRLsin-CS1-CD3 BITE(图11)。
2.表达CS1BITE T细胞的制备
采用本领域病毒感染的常规技术，将载体PRRLsin-CS1-CD3 BITE转染293T细胞，包装慢病毒，得到相应的慢病毒CS1BITE。
利用Ficoll-Paque(GE bioscience)进行密度梯度离心，从人外周血中分离出PBMC，加入抗CD3/CD28的磁珠，体外进行活化与扩增得到T细胞。
将上述慢病毒CS1BITE感染活化后的T细胞培养扩增至需要的数量，得到T-BITE细胞。因BITE是分泌蛋白，所以本实验中以EGFP的表达去检测BITE的表达，经流式检测，EGFP的表达如图12所示。
实施例7.T-BITE细胞对共培养的NK细胞的杀伤检测
同一供体来源的活化NK细胞(aNK)和T-BITE或UTD细胞按照1:1比例共培养，24小时候通过流式技术检测培养体系中CD56 +NK细胞比例。结果如图13所示，T-BITE的加入清除或杀伤了培养体系中的NK细胞。说明了构建的BITE具有生物学活性。
实施例8.T-BITE细胞培养上清对共培养的NK细胞的杀伤检测
制备上清液：T-BITE细胞和未感染的T细胞(UTD)分别培养过夜后离心取上清备用。
同一供体来源的活化的NK细胞和T细胞按照1:1的比例共培养，并加入50％体积的上述UTD或T-BITE培养上清，24小时后通过流式技术检测培养体系中NK细胞的比例。
实验结果如图14示，培养体系中同时有T细胞以及T-BITE培养上清组中，NK细胞(CD56 +)几乎被完全清除。培养体系中有T-BITE培养上清但没有T细胞组中，NK细胞(CD56 +)几乎没有被清除。
由此可见，可溶性的CS1-CD3 BITE在T细胞存在的情况下能够有效的清除NK细胞。
本发明所用的序列总结于下表：
Figure PCTCN2021121016-appb-000001
Figure PCTCN2021121016-appb-000002
Figure PCTCN2021121016-appb-000003
Figure PCTCN2021121016-appb-000004
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (54)

  1. 一种表达靶向CS1的嵌合受体1的工程化免疫细胞用于制备杀伤NK细胞的药物的用途。
  2. 如权利要求1所述的用途，其特征在于，所述嵌合受体1包括：嵌合抗原受体1(CAR1)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)、aTCR-T或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述CAR1包括：
    (i)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3δ；和/或
    (ii)特异性识CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3δ；和/或
    (iii)特异性识CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3δ；
    (iv)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区和CD3δ；
    优选的，所述特异性识别CS1的抗体的核酸分子与SEQ ID NO：11具有至少80％、90％、或95％的同一性；或所述特异性识别CS1的抗体的氨基酸序列与SEQ ID NO：12具有至少80％、90％、或95％的同一性。
  4. 如权利要求1-3任一所述的用途，其特征在于，所述免疫细胞还表达膜结合型NK细胞抑制性受体的配体或抗体片段。
  5. 如权利要求4所述的用途，其特征在于，所述免疫细胞还表达膜结合型NKG2A抗体或抗体片段；
    优选的，所述膜结合型NKG2A抗体或抗体片段包括与SEQ ID NO：14或38具有至少80％、90％、或95％的同一性序列；
    或者所述膜结合型NKG2A抗体或抗体片段的核酸分子与SEQ ID NO：13具有至少80％、90％、或95％的同一性。
  6. 如权利要求1-5所述的用途，其特征在于，所述免疫细胞的内源性CS1基因被敲除，优选采用CRISPR/Cas9技术敲除所述免疫细胞的内源性CS1基因。
  7. 如权利要求6所述的用途，其特征在于，CRISPR/Cas9技术使用的gRNA如SEQ ID NO:19、20、40、41、42、43、44和/或45所示。
  8. 如权利要求1-7任一所述的用途，其特征在于，所述免疫细胞和表达非靶向CS1的嵌合抗原受体的T细胞联合施用或者所述免疫细胞还表达非靶向CS1的嵌合抗原受体，
    优选地，所述非靶向CS1的嵌合抗原受体靶向肿瘤或病原体抗原，
    更优选地，所述非靶向CS1的嵌合抗原受体靶向BCMA、CD19、GPC3、Claudin18.2、EGFR、EGFRvIII或它们的组合。
  9. 如权利要求1-8任一所述的用途，其特征在于，所述免疫细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；
    优选地，所述T细胞为自体/同种异体T细胞；
    优选地，所述T细胞为原代T细胞；
    优选地，所述T细胞来源于人的自体T细胞。
  10. 如权利要求9所述的用途，其特征在于，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。
  11. 如权利要求1-10任一所述的用途，其特征在于，所述免疫细胞的内源性MHC和内源性TCR被敲除，优选采用CRISPR/Cas9技术敲除内源性MHC和内源性TCR。
  12. 如权利要求11所述的用途，其特征在于，采用CRISPR/Cas9技术敲除B2M使用的gRNA如SEQ ID NO:18所示，和/或敲除TRAC使用的gRNA如SEQ ID NO:17所示。
  13. 如权利要求1-12任一所述的用途，其特征在于，所述免疫细胞能增强在先、同时、在后导入受试者的T细胞和/或携带靶向靶抗原的CAR-T细胞对肿瘤细胞的杀伤、以及增强所述T细胞和/或CAR-T细胞的存活、增殖。
  14. 如权利要求1-13任一所述的用途，其特征在于，所述免疫细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；
    或所述免疫细胞与改善其相关的一种或多种副作用的药剂联合施用。
  15. 增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率的方法，包括：
    a)提供同种异体免疫细胞；
    b)任选通过编码参与针对自体和非自体抗原识别的响应的多肽的至少一种内源基因表达、活性和/或信号传导被降低或抑制来修饰所述细胞；以及
    c)利用编码靶向CS1的嵌合受体1多核苷酸来修饰所述细胞。
  16. 如权利要求15所述的方法，其特征在于，采用CRISPR/Cas9技术敲除内源性MHC 和内源性TCR。
  17. 如权利要求15或16所述的方法，其特征在于，采用CRISPR/Cas9技术敲除B2M使用的gRNA如SEQ ID NO:18示序列，和/或敲除TRAC使用的gRNA如SEQ ID NO:17示序列。
  18. 根据权利要求15-17任一所述的方法，其特征在于，所述嵌核受体1包括：嵌合抗原受体1(CAR1)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)、aTCR-T或其组合；
    优选地，所述CAR1包括：
    (i)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3δ；和/或
    (ii)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3δ；和/或
    (iii)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3δ；
    (iv)特异性识别CS1的抗体或其功能片段、CD28或CD8的跨膜区和CD3δ；
    优选的，所述特异性识别CS1的抗体的核酸分子与SEQ ID NO：11具有至少80％、90％、或95％的同一性；或所述特异性识别CS1的抗体的氨基酸序列与SEQ ID NO：12具有至少80％、90％、或95％的同一性。
  19. 如权利要求15-18任一所述的方法，其特征在于，还包括步骤d)编码膜结合型NK细胞抑制性受体的配体或抗体片段的非内源性多核苷酸来修饰所述细胞。
  20. 如权利要求19所述的方法，其特征在于，还包括步骤d)编码所述免疫细胞膜结合型NKG2A抗体或抗体片段的非内源性多核苷酸来修饰所述细胞；
    优选的，所述NKG2A抗体或抗体片段包括与SEQ ID NO：14或38具有至少80％、90％、或95％的同一性序列；
    或者所述膜结合型NKG2A抗体或抗体片段的核酸分子与SEQ ID NO：13具有至少80％、90％、或95％的同一性。
  21. 如权利要求15-20任一所述的方法，其特征在于，还包括步骤b)采用CRISPR/Cas9技术敲除所述工程化T细胞内源性CS1基因。
  22. 如权利要求21所述的方法，其特征在于，CRISPR/Cas9技术使用的gRNA如SEQ ID NO:19、20、40、41、42、43、44和/或45所示序列。
  23. 如权利要求15-22任一所述的方法，其特征在于，还包括步骤e)编码靶向肿瘤抗 原、和/或病原体抗原、和/或病毒抗原的嵌合抗原受体非内源性的多核苷酸来修饰所述细胞；
    优选的，所述肿瘤抗原包括BCMA、CD19、GPC3、Claudin18.2、EGFR、EGFRvIII或其组合。
  24. 如权利要求15-23任一所述的方法，其特征在于，所述免疫细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；
    优选地，所述T细胞为自体/同种异体T细胞；
    优选地，所述T细胞为原代T细胞；
    优选地，所述T细胞来源于人的自体T细胞。
  25. 如权利要求15-24任一所述的方法，其特征在于，当所述方法制备的免疫细胞与宿主NK细胞共培养时，所述免疫细胞能杀伤宿主NK细胞。
  26. 如权利要求15-25任一所述的方法，其特征在于，所述方法制备的免疫细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；
    或所述方法制备的免疫细胞与改善其相关的一种或多种副作用的药剂联合施用。
  27. 一种双特异性抗体构建体，其特征在于，所述抗体构建体包含结合到靶细胞表面上的CS1的第一结合结构域和结合到T细胞表面上的CD3的第二结合结构域。
  28. 如权利要求27所述的抗体构建体，其特征在于，所述第一结合结构域结合到人或猕猴CS1；和/或所述第二结合结构域结合到人CD3ε、普通狨、棉顶狨或松鼠猴CD3ε。
  29. 如权利要求27或28所述的抗体构建体，其特征在于，所述抗体构建体选自以下的形式：scFv、(scFv) 2、scFv-单结构域mAb、双功能抗体和它们的寡聚物。
  30. 根据权利要求27-29任一所述的抗体构建体，其特征在于，所述第一结合结构域包含的VH区含有SEQ ID NO：21所示的CDR1、SEQ ID NO：22所示的CDR2和SEQ ID NO：23所示的CDR3，第一结合结构域包含的VL区含有SEQ ID NO：24所示的CDR1、SEQ ID NO：25所示的CDR2和SEQ ID NO：26所示的CDR3；
    优选所述第一结合结构域的VH区如SEQ ID NO：27所示和VL区如SEQ ID NO：28所示。
  31. 根据权利要求27至30中任一项所述的抗体构建体，其特征在于，所述抗体构建体以从N端到C端的次序包含：
    如SEQ ID NO.12所示的氨基酸序列的第一结合结构域：
    如SEQ ID NO.29所示的氨基酸序列的肽接头；
    如SEQ ID NO.30所示的氨基酸序列的第二结合结构域。
  32. 根据权利要求31所述的抗体构建体，其特征在于，所述抗体构建提包含如SEQ ID NO：31所示的氨基酸序列。
  33. 一种多核苷酸，其编码如权利要求1-26中任一所述用途中所述的嵌合受体1、嵌合抗原受体3、膜结合型NK细胞抑制性受体的配体或抗体片段或它们的组合。
  34. 一种载体，其包含如权利要求33所述的多核苷酸。
  35. 一种免疫细胞，其具有权利要求1-14中任一所述的用途，优选其是通过权利要求15-26中任一项所述的方法中获得的免疫细胞。
  36. 一种多核苷酸，其编码如权利要求27-32任一所述的抗体构建体。
  37. 一种载体，其包含如权利要求36所述的多核苷酸。
  38. 一种免疫细胞，其用如权利要求36所述多核苷酸或用如权利要求37所述载体转化或转染。
  39. 如权利要求38所述免疫细胞，其特征在于，所述免疫细胞能分泌所述权利要求27-32任一所述的抗体构建体。
  40. 如权利要求38或39所述的免疫细胞，其特征在于，所述免疫细胞还表达膜结合型NK细胞抑制性受体的配体或抗体片段。
  41. 如权利要求38-40任一所述的免疫细胞，其特征在于，所述免疫细胞还表达膜结合型NKG2A抗体或抗体片段；
    优选的，所述NKG2A抗体或抗体片段包括与SEQ ID NO：14或38具有至少80％、90％、或95％的同一性序列，
    或者所述膜结合型NKG2A抗体或抗体片段的核酸分子与SEQ ID NO：13具有至少80％、90％、或95％的同一性。
  42. 如权利要求38-41任一所述的免疫细胞，其特征在于，所述免疫细胞的内源性CS1基因被敲除，优选采用CRISPR/Cas9技术敲除所述免疫细胞的内源性CS1基因。
  43. 如权利要求42任一所述的免疫细胞，其特征在于，CRISPR/Cas9技术使用的gRNA如SEQ ID NO:19、20、40、41、42、43、44和/或45所示。
  44. 如权利要求38-43任一所述免疫细胞，其特征在于，所述细胞还表达非靶向CS1的嵌合抗原受体，所述非靶向CS1的嵌合抗原受体识别肿瘤抗原或病原体抗原；
    优选地，所述肿瘤抗原包括BCMA、CD19、GPC3、Claudin18.2、EGFR、EGFRvIII或它们的组合。
  45. 如权利要求38-44任一所述的免疫细胞，其特征在于，所述细胞为来源于天然的T 细胞和/或经多能干细胞诱导产生的T细胞；
    优选地，所述T细胞为自体/同种异体T细胞；
    优选地，所述T细胞为原代T细胞；
    优选地，所述T细胞来源于人的自体T细胞。
  46. 如权利要求38-45任一所述的免疫细胞，其特征在于，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。
  47. 如权利要求38-46任一所述的免疫细胞，其特征在于，所述免疫细胞的内源性MHC和内源性TCR被敲除，优选采用CRISPR/Cas9技术敲除内源性MHC和内源性TCR。
  48. 如权利要求47所述的免疫细胞，其特征在于，采用CRISPR/Cas9技术敲除B2M使用的gRNA如SEQ ID NO:18所示，和/或敲除TRAC使用的gRNA如SEQ ID NO:17所示。
  49. 一种药物组合物，其特征在于，包括如权利要求38-48任一所述的免疫细胞、和/或如权利要求35所述免疫细胞；还包括表达非靶向CS1的嵌合抗原受体的T细胞联合施用，
    优选地，所述非靶向CS1的嵌合抗原受体靶向肿瘤或病原体抗原，
    更优选地，所述非靶向CS1的嵌合抗原受体靶向BCMA、CD19、GPC3、Claudin18.2、EGFR、EGFRvIII或其组合。
  50. 一种用于产生根据权利要求27至32中任一项所述的抗体构建体的方法，其特征在于，所述方法包括在允许如权利要求27至32中任一所述抗体构建体表达的条件下培养如权利要求38-48任一所述免疫细胞和从所述培养物回收所产生的抗体构建体。
  51. 一种药物组合物，其特征在于，包含根据权利要求27至32中任一项所述的抗体构建体、根据权利要求50所述的方法产生的抗体构建体、权利要求35所述免疫细胞、权利要求38-48任一所述免疫细胞、权利要求36所述的多核苷酸、权利要求37所述的载体或其组合。
  52. 根据权利要求27至32中任一项所述的抗体构建体或根据权利要求50所述的方法产生的抗体构建体，其特征在于，用于增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率。
  53. 一种增加同种异体免疫细胞在有宿主免疫细胞存在时的持久性和/或移植成活率的方法，其包括向有需要的受试者施用根据权利要求27至32中任一项所述的抗体构建体、根据权利要求50所述的方法产生的抗体构建体、权利要求35所述免疫细胞及其组合的步骤。
  54. 一种试剂盒，其包含根据权利要求27至32中任一项所述的抗体构建体、根据权利要求50所述的方法产生的抗体构建体、权利要求35所述免疫细胞、如权利要求36所述的多核苷酸、如权利要求37所述的载体和/或如权利要求38-48任一所定义的免疫细胞。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
CN116785415A (zh) * 2023-05-04 2023-09-22 北京百普赛斯生物科技股份有限公司 一种特异性针对免疫细胞激活/增殖刺激的产品、制备及应用
WO2024114691A1 (en) * 2022-12-02 2024-06-06 Guangzhou Litai Biotechnology Co., Ltd. Human cs1 car-t cells
US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014179759A1 (en) * 2013-05-03 2014-11-06 Ohio State Innovation Foundation Cs1-specific chimeric antigen receptor engineered immune effector cells
CN104829730A (zh) * 2015-04-14 2015-08-12 苏静 一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用
CN107723275A (zh) * 2017-10-20 2018-02-23 重庆精准生物技术有限公司 通用型car‑t细胞及其制备方法和应用
WO2019178576A1 (en) * 2018-03-16 2019-09-19 Cytoimmune Therapeutics, LLC Bispecific antibody car cell immunotherapy

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014179759A1 (en) * 2013-05-03 2014-11-06 Ohio State Innovation Foundation Cs1-specific chimeric antigen receptor engineered immune effector cells
CN105377897A (zh) * 2013-05-03 2016-03-02 俄亥俄州创新基金会 Cs1-特异性嵌合抗原受体工程化的免疫效应细胞
CN104829730A (zh) * 2015-04-14 2015-08-12 苏静 一种能联合免疫细胞增强肿瘤杀伤能力的双特异性抗体及其制备方法和应用
CN107723275A (zh) * 2017-10-20 2018-02-23 重庆精准生物技术有限公司 通用型car‑t细胞及其制备方法和应用
WO2019178576A1 (en) * 2018-03-16 2019-09-19 Cytoimmune Therapeutics, LLC Bispecific antibody car cell immunotherapy

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LUM LAWRENCE G., THAKUR ARCHANA, ELHAKIEM ABDALLA, ALAMEER LENA, DINNING EMILY, HUANG MANLEY: "Anti-CS1 × Anti-CD3 Bispecific Antibody (BiAb)-Armed Anti-CD3 Activated T Cells (CS1-BATs) Kill CS1+ Myeloma Cells and Release Type-1 Cytokines", FRONTIERS IN ONCOLOGY, vol. 10, 31 May 2020 (2020-05-31), pages 1 - 11, XP055915665, DOI: 10.3389/fonc.2020.00544 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
WO2024114691A1 (en) * 2022-12-02 2024-06-06 Guangzhou Litai Biotechnology Co., Ltd. Human cs1 car-t cells
CN116785415A (zh) * 2023-05-04 2023-09-22 北京百普赛斯生物科技股份有限公司 一种特异性针对免疫细胞激活/增殖刺激的产品、制备及应用
CN116785415B (zh) * 2023-05-04 2024-03-22 北京百普赛斯生物科技股份有限公司 一种特异性针对免疫细胞激活/增殖刺激的产品、制备及应用

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