WO2022206941A1 - Cs1 engineered cells and composition thereof - Google Patents

Cs1 engineered cells and composition thereof Download PDF

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Publication number
WO2022206941A1
WO2022206941A1 PCT/CN2022/084732 CN2022084732W WO2022206941A1 WO 2022206941 A1 WO2022206941 A1 WO 2022206941A1 CN 2022084732 W CN2022084732 W CN 2022084732W WO 2022206941 A1 WO2022206941 A1 WO 2022206941A1
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cells
antigen
binding unit
seq
antibody
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PCT/CN2022/084732
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French (fr)
Chinese (zh)
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李宗海
王鹏
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克莱格医学有限公司
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Priority to CN202280022824.1A priority Critical patent/CN117083292A/en
Publication of WO2022206941A1 publication Critical patent/WO2022206941A1/en

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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
    • A61K39/461—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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present application relates to an antigen binding unit capable of targeting CS1 protein and its application.
  • MM Multiple myeloma
  • MM is a cancer of plasma cells in the bone marrow, which normally produce antibodies and play a key role in immune function.
  • the uncontrolled growth of these cells leads to bone pain and fractures, anemia, infection and other complications.
  • multiple myeloma is the second most common hematological malignancy, accounting for 2% of all cancer deaths.
  • MM is a heterogeneous disease and is mostly caused by chromosomal translocation of t(11;14), t(4;14), t(8;14), del(13), del(17) (among others).
  • MM multiple myeloma
  • the primary pathology of multiple myeloma is the indefinite expansion and enrichment of plasma cells in the bone marrow, leading to osteonecrosis.
  • Patients affected by MM may experience a variety of disease-related symptoms due to bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychological burden of a cancer diagnosis.
  • the main treatment options are chemotherapy and stem cell transplantation.
  • the chemotherapy drugs are mainly steroids, thalidomide, lenalidomide, bortezomib or a combination of various cytotoxic agents. For younger patients, high-dose chemotherapy can be used. In conjunction with autologous stem cell transplantation.
  • CS1 is a lymphocyte signaling activation molecule (the seventh member of the SLAM family, CD319 antigen), and is a member of the CD2 family of cell surface glycoproteins. Based on the high expression of CS1 in multiple myeloma, it is used as a target for the preparation of antibodies or CAR-T cells for the treatment of multiple myeloma and CS1-positive tumors.
  • the purpose of this application is to provide an anti-CS1 antigen-binding unit that can specifically inhibit the growth of human multiple myeloma and other CS1-positive tumor cells, and T immune cells expressing a chimeric antigen receptor containing the antigen-binding unit sequence (CAR-T), they play a role in adoptive immunotherapy of tumors.
  • CAR-T chimeric antigen receptor containing the antigen-binding unit sequence
  • a first aspect provides an antigen binding unit targeting CS1, the antigen binding unit is selected from the group consisting of:
  • an antigen-binding unit comprising a heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 3, 13 or 23, and/or comprising SEQ ID NO: 4, 14, 24 or HCDR2 shown in 32, and/or comprising HCDR3 shown in SEQ ID NO: 5, 15, 25 or 41;
  • an antigen binding unit comprising a light chain variable region comprising LCDR1 set forth in SEQ ID NO: 8, 18 or 28, and/or comprising LCDR2 set forth in SEQ ID NO: 9, 19 or 29, and/or comprising LCDR3 set forth in SEQ ID NO: 10, 20 or 30;
  • an antigen-binding unit comprising (1) a heavy chain variable region of the antigen-binding unit and (2) a light chain variable region of the antigen-binding unit;
  • an antigen-binding unit comprising (1) to ( 3) A variant of the antigen-binding unit described in any one of (1) to (3), which has the same or similar activity as the antigen-binding unit described in any one of (1) to (3).
  • described antigen binding unit is selected from: (1) antigen binding unit, it comprises HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, SEQ ID NO: HCDR3 shown in 5 and LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:9, LCDR3 shown in SEQ ID NO:10; or (2) an antigen binding unit comprising SEQ ID NO: HCDR1 shown in 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15 and LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 19, SEQ ID NO: The LCDR3 shown in 20; or (3) an antigen binding unit comprising HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 25 and SEQ ID NO: LCDR1 shown in 28, LCDR2 shown in SEQ ID NO: 29, LCDR3 shown in SEQ ID NO: 30
  • the antigen binding unit is selected from:
  • an antigen-binding unit the heavy chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 1, 11, 21, 31, 36 or 40;
  • an antigen-binding unit the The light chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 34, 38 or 43;
  • the antigen-binding unit comprising (1) the heavy weight of the antigen-binding unit.
  • an antigen-binding unit a variant of the antigen-binding unit of any one of (1) to (3), and having The same or similar activity as the antibody described in any one of (1) to (3).
  • the antigen binding unit is selected from:
  • an antigen binding unit the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 6
  • an antigen binding unit the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 11 and the light chain variable region of the antigen binding unit has SEQ ID NO: : amino acid sequence shown in 16
  • antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antigen binding unit Has the amino acid sequence shown in SEQ ID NO: 26;
  • an antigen binding unit the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 31 and the light weight of the antigen binding unit
  • the chain variable region has the amino acid sequence shown in SEQ ID NO: 34
  • an antigen binding unit the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in S
  • the antigen-binding unit recognizes the same epitope as any of the above-mentioned antigen-binding units; or binds to the Ig-like V-type domain of CS1 protein; or binds to the Ig-like V-type domain of CS1 protein like C2-type domain.
  • the antigen binding unit is a hybridoma antibody, a humanized antibody, a chimeric antibody or a fully human antibody; or the antigen binding unit is a monoclonal antibody; or the antigen binding unit is a whole Anti, scFv, Fv fragment, Fab fragment, Fab' fragment, (Fab') 2 fragment, Fd fragment, dAb fragment, single domain antibody, multifunctional antibody or scFv-Fc antibody.
  • a second aspect provides an immunoconjugate, the immunoconjugate comprising: the antigen binding unit of the first aspect; and a functional molecule linked thereto.
  • a third aspect provides a chimeric receptor, the extracellular domain of the chimeric receptor comprises the antigen-binding unit of the first aspect, and the chimeric receptor includes: chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), or a combination thereof.
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the chimeric receptor comprises sequentially linked: the antigen binding unit of the first aspect, a transmembrane region and an intracellular signaling region.
  • the intracellular signal region of the chimeric receptor is selected from the group consisting of: CD3 ⁇ , Fc ⁇ RI ⁇ , CD27, CD28, CD137, CD134, MyD88, CD40 intracellular signal region sequences or combinations thereof; and/or
  • the transmembrane region comprises the transmembrane region of CD8 or CD28.
  • the chimeric receptor comprises: the antigen binding unit described in the first aspect, the transmembrane region of CD8/CD28 and CD3 ⁇ ; or the antigen binding unit described in the first aspect, CD8/CD28 The transmembrane region of CD28, the intracellular signal region of CD137 and CD3 ⁇ ; or the antigen binding unit of the first aspect, the transmembrane region of CD8/CD28, the intracellular signaling region of CD28 and CD3 ⁇ ; or the first aspect Antigen binding unit, transmembrane region of CD8/CD28, intracellular signaling region of CD28, CD137 and CD3 ⁇ .
  • amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 45, 46 or 47.
  • the fourth aspect provides nucleic acids encoding the antigen binding unit of the first aspect, the immunoconjugate of the second aspect, and the chimeric receptor of the third aspect.
  • the fifth aspect provides an expression vector comprising the nucleic acid of the fourth aspect.
  • a sixth aspect provides a virus comprising the expression vector of the fifth aspect.
  • the seventh aspect provides a composition comprising the antigen-binding unit of the first aspect, the immunoconjugate of the second aspect, and/or the chimeric receptor of the third aspect, wherein the combination is characterized in that The compound is cytotoxic to cells expressing CS1.
  • the CS1-expressing cells are tumor cells and/or pathogen cells.
  • the eighth aspect provides a host cell comprising the expression vector of the fifth aspect or the nucleic acid of the fourth aspect integrated into the genome.
  • the host cell expresses the chimeric receptor of the third aspect.
  • the host cells comprise T cells, cytotoxic T lymphocytes, NK cells, NKT cells, DNT cells, regulatory T cells, NK92 cells, stem cell-derived immune effector cells, or a combination thereof.
  • the T cells are T cells derived from natural T cells and/or induced by pluripotent stem cells; preferably, the T cells are autologous or allogeneic T cells; preferably , the T cells are primary T cells; preferably, the T cells are derived from human autologous T cells.
  • the T cells comprise memory stem-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory cells T cells (Tem), ⁇ T cells, ⁇ T cells, or a combination thereof.
  • the host cell comprises: knockout of genes encoding TCR proteins and/or low or no expression of endogenous TCR molecules, and/or knockout of genes encoding MHC proteins and/or Endogenous MHC is low or not expressed.
  • the host cell uses CRISPR/Cas9 technology to knock out the endogenous MHC molecule B2M and endogenous TCR.
  • the gRNA used for knocking out B2M in the host cell includes the sequences shown in SEQ ID NO: 84, 85, 86 and/or 87, and the gRNA used for knocking out TCR includes SEQ ID NO: 76 , 77, 78, 79, 80, 81, 82 and/or 83.
  • the host cell comprises a knockout of a gene encoding a CS1 protein and/or low or no expression of an endogenous CS1 molecule.
  • CRISPR/Cas9 technology is used to knock out the CS1 gene of the host cell, and the gRNA used is selected from SEQ ID NO: 88, 89, 90, 91, 92, 93, 94 and/or 95. display sequence.
  • the host cell binds cells that express CS1 and does not significantly bind cells that do not express CS1.
  • the host cell also carries an exogenous cytokine coding sequence; or it also expresses another chimeric receptor; or it also expresses a chemokine receptor; or it also expresses Safety switch.
  • the ninth aspect provides a combination drug, the antigen binding unit described in the first aspect, the immunoconjugate described in the second aspect, the chimeric receptor described in the third aspect, the composition described in the seventh aspect, and the eighth aspect.
  • the host cell of the aspect is administered in combination with an agent that enhances its function, preferably, in combination with a chemotherapeutic agent; and/or in combination with an agent that ameliorates one or more side effects associated therewith; and/or with expression targeting Co-administration of host cells with chimeric receptors other than CS1.
  • the tenth aspect provides a method for preparing the antigen binding unit of the first aspect, the immunoconjugate of the second aspect, the chimeric receptor of the third aspect, and/or the composition of the seventh aspect.
  • a method comprising culturing the host cell of the eighth aspect under conditions suitable for expression of the antigen binding unit, immunoconjugate, chimeric receptor, and isolating the host cell expressed by the host cell Antigen binding units, immunoconjugates, chimeric receptors, and/or compositions.
  • the eleventh aspect provides a pharmaceutical composition, comprising: the antigen-binding unit described in the first aspect or a nucleic acid encoding the antigen-binding unit; or the immunoconjugate described in the second aspect or a nucleic acid encoding the conjugate nucleic acid; or the chimeric receptor of the third aspect or a nucleic acid encoding the chimeric receptor; or the host cell of the eighth aspect; and optionally, a pharmaceutically acceptable carrier or excipient.
  • a twelfth aspect provides a method of treating/diagnosing a disease, comprising administering to a subject in need thereof an effective amount of the antigen-binding unit of the first aspect, or the immunoconjugate of the second aspect, or the host cell of the third aspect, or the composition of the seventh aspect; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors; preferably the tumor is multiple myeloid tumor; preferably, the subject is a human; preferably, wherein the host cells are autologous or allogeneic T cells to the subject.
  • a thirteenth aspect provides the antigen-binding unit of the first aspect, or the immunoconjugate of the second aspect, or the host cell of the eighth aspect, or the composition of the seventh aspect in the treatment and Use in diagnosing a disease, characterized in that the disease expresses CS1; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors, preferably the tumor is multiple myeloma.
  • the fourteenth aspect provides the antigen binding unit of the first aspect, or the immunoconjugate of the second aspect, or the host cell of the eighth aspect, or the composition of the seventh aspect for use in the preparation of Use of a drug that kills NK cells.
  • the use increases the persistence and/or engraftment survival of autologous or allogeneic immune cells in the presence of host immune cells.
  • the application also relates to:
  • the application provides an antigen binding unit targeting CS1, the antigen binding unit is selected from the group consisting of: (1) an antigen binding unit comprising a heavy chain variable region, the The chain variable region comprises HCDR1 set forth in SEQ ID NO: 3, 13 or 23, and/or comprises HCDR2 set forth in SEQ ID NO: 4, 14, 24 or 32, and/or comprises SEQ ID NO: 5, 15 HCDR3 shown in any one of , 25 or 41; (2) an antigen binding unit comprising a light chain variable region comprising LCDR1 shown in SEQ ID NOs: 8, 18, 28, and /or comprising the LCDR2 shown in SEQ ID NO: 9, 19, 29, and/or comprising the LCDR3 shown in any one of SEQ ID NO: 10, 20 or 30; (3) an antigen binding unit comprising (1) the The heavy chain variable region of the antigen-binding unit and (2) the light chain variable region of the antigen-binding unit; (4) the antigen-binding unit, the antigen-binding unit of any one
  • the antigen binding unit is selected from: (1) an antigen binding unit, which comprises HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, SEQ ID NO:5 The HCDR3 shown and the LCDR1 shown in SEQ ID NO:8, the LCDR2 shown in SEQ ID NO:9, and the LCDR3 shown in SEQ ID NO:10; (2) an antigen-binding unit comprising the SEQ ID NO:13 HCDR1 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20 (3) antigen binding unit, HCDR1 shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, HCDR3 shown in SEQ ID NO:25 and LCDR1 shown in SEQ ID NO:28 , LCDR2 shown in SEQ ID NO:29, LCDR3 shown in SEQ
  • the antigen-binding unit is selected from: (1) an antigen-binding unit, the heavy chain variable region of the antigen-binding unit has SEQ ID NO: 11, 11, 21, 31, 36 or The amino acid sequence shown in 40; (2) an antigen binding unit, the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 34, 38 or 43; (3 ) an antigen-binding unit, comprising (1) a heavy chain variable region of the antigen-binding unit and (2) a light chain variable region of the antigen-binding unit; (4) an antigen-binding unit, (1) to (3) A variant of the antigen-binding unit according to any one of the above, which has the same or similar activity as the antigen-binding unit according to any one of (1) to (3).
  • the antigen binding unit is selected from: (1) an antigen binding unit, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1 and the antigen binding unit The variable region of the light chain has the amino acid sequence shown in SEQ ID NO: 6; (2) the antigen binding unit, the variable region of the heavy chain of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 11 and the The light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 16; (3) the antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 21 amino acid sequence and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 26; (4) antigen binding unit, the heavy chain variable region of the antigen binding unit has SEQ ID NO: The amino acid sequence shown in 31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 34; (5) the antigen binding unit,
  • the application provides an antigen-binding unit that recognizes the same epitope as the antigen-binding unit described herein; or an Ig-like V-type domain that binds to CS1 protein; or an Ig that binds to CS1 protein -like C2-type domain.
  • the antigen binding unit is a humanized antibody, chimeric antibody or fully human antibody; or the antigen binding unit is a monoclonal antibody; or the antigen binding unit is scFv, Fv, Fab or (Fab) 2 .
  • the antigen-binding unit is a humanized antibody selected from: (1) an antigen-binding unit, and the variable region of the heavy chain of the antigen-binding unit is shown in SEQ ID NO: 31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 34; (2) the antigen binding unit, the heavy chain variable region of the antigen binding unit has SEQ ID NO.
  • the amino acid sequence shown in 36 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 38;
  • the antigen binding unit, the heavy chain variable region of the antigen binding unit Has the amino acid sequence shown in SEQ ID NO: 40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 43
  • the application provides nucleic acids encoding the antibodies described herein.
  • the application provides an expression vector comprising the nucleic acid described herein.
  • the present application provides a host cell comprising the expression vector described herein or the nucleic acid described herein integrated into the genome.
  • the present application provides an antigen-binding unit of the antibody described in the present application, which is used to prepare a targeted drug, an antibody-drug conjugate or a multifunctional antibody that specifically targets CS1 tumor cells; or
  • the tumor expresses CS1; or for preparing immune cells modified with chimeric antigen receptors; preferably, the immune cells include: T lymphocytes, NK cells or NKT lymphocytes.
  • the application provides a multifunctional immunoconjugate
  • the multifunctional immunoconjugate includes:
  • the antigen binding unit described in this application; and the functional molecule connected to it; the functional molecule is selected from the group consisting of: a molecule targeting tumor surface markers, a tumor-inhibiting molecule, and a surface marker targeting immune cells. Molecular or detectable label.
  • the tumor-inhibiting molecules are anti-tumor cytokines or anti-tumor toxins, preferably, the cytokines include: IL-12, IL-15, type I interferon, TNF-alpha.
  • the molecules targeting immune cell surface markers are antibodies or ligands that bind immune cell surface markers; preferably, the immune cell surface markers include: CD3, CD16 , CD28, 4-1BB, more preferably, the antibody that binds to immune cell surface markers is an anti-CD3 antibody.
  • the molecule targeting the surface marker of immune cells is an antibody that binds to the surface marker of T cells.
  • the application provides nucleic acids encoding the multifunctional immunoconjugates described herein.
  • the present application provides the use of the multifunctional immunoconjugate described in the present application, for the preparation of anti-tumor drugs, or for the preparation of reagents for diagnosing tumors, which express CS1; or for the preparation of chimeric antigen receptors body-modified immune cells; preferably, the immune cells include: T lymphocytes, NK cells or NKT lymphocytes.
  • the present application provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular domain comprises the antibody described in the present application, and the antibody is preferably a single-chain antibody or a domain antibody .
  • the intracellular signaling domain comprises one or more costimulatory signaling domains and/or primary signaling domains.
  • the chimeric antigen receptor further comprises a hinge domain.
  • the transmembrane domain is selected from the alpha, beta, zeta chains of TCR, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the transmembrane region of PD1; and/or the costimulatory signaling domain is selected from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, Intracellular signaling regions of OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C SLP76, TRIM, Fc ⁇ RI ⁇ , MyD88, and 41BBL; and/or the The primary signaling domain is selected from the group consisting of TCR ⁇ , FcR ⁇ , FcR ⁇ , CD
  • the chimeric antigen receptor comprises the following sequentially linked antigen binding units, transmembrane regions and intracellular signaling regions: the antigen binding units described in this application, the CD8 transmembrane region and CD3 ⁇ ; the antigen binding unit, the transmembrane region of CD8, the intracellular signal region of CD137 and CD3 ⁇ described in the present application; the antigen binding unit, the transmembrane region of CD28, the intracellular signal region of CD28 and CD3 ⁇ described in the present application; or The antigen binding unit, the transmembrane region of CD28, the intracellular signal region of CD28, CD137 and CD3 ⁇ described in this application.
  • the extracellular domain of the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 50, 51 or 52; the transmembrane domain is selected from the group shown in SEQ ID NO: 54 CD8 transmembrane domain, CD28 transmembrane domain shown in SEQ ID NO: 72; the costimulatory signal domain is selected from CD28 intracellular domain shown in SEQ ID NO: 73, CD3 ⁇ intracellular shown in SEQ ID NO: 56 The signaling domain and the intracellular domain of CD137 set forth in SEQ ID NO: 55 or a mixture thereof.
  • the chimeric antigen receptor is selected from the group consisting of: chimeric antigen receptor one, having the extracellular domain shown in SEQ ID NO:50, the hinge domain shown in SEQ ID NO:53, : a transmembrane domain shown in 54, a costimulatory signaling domain shown in SEQ ID NO: 55, and a primary signaling domain shown in SEQ ID NO: 56 (hu32A12BBz); or a chimeric antigen receptor one with SEQ ID NO : the extracellular domain shown in 51, the hinge domain shown in SEQ ID NO:53, the transmembrane domain shown in SEQ ID NO:54, the costimulatory signal domain shown in SEQ ID NO:55, and the costimulatory signal domain shown in SEQ ID NO:56
  • the primary signal domain (hu37A3BBz) shown; or the chimeric antigen receptor one, with the extracellular domain shown in SEQ ID NO: 52, the hinge domain shown in SEQ ID NO: 53, and the transmembrane
  • the application provides nucleic acids encoding the chimeric antigen receptors described herein.
  • the application provides an expression vector comprising the nucleic acid described herein.
  • the present application provides a virus comprising the vector described herein.
  • the virus is a lentivirus.
  • the present application provides the use of the chimeric antigen receptor described in the present application, or the nucleic acid described in the present application, or the expression vector described in the present application, or the virus described in the present application, for preparing targeted Genetically modified immune cells from tumor cells expressing CS1,
  • the CS1-expressing tumor is multiple myeloma.
  • the present application provides a genetically modified immune cell transduced with the nucleic acid described in the present application, or the expression vector described in the present application or the virus described in the present application; chimeric antigen receptor,
  • Said immune cells are preferably selected from T lymphocytes, NK cells or NKT cells.
  • the genetically modified immune cells also express other sequences other than the chimeric antigen receptors described in the present application, the other sequences include cytokines, or another chimeric antigen receptor body, or chemokine receptor, or siRNA that reduces PD-1 expression, or protein that blocks PD-L1, or TCR, or safety switch; preferably, the cytokines include IL-12, IL- 15. IL-21, or type I interferon; preferably, the chemokine receptor includes CCR2, CCR5, CXCR2, or CXCR4; preferably, the safety switch includes iCaspase-9, Truancated EGFR or RQR8 .
  • the other sequences include cytokines, or another chimeric antigen receptor body, or chemokine receptor, or siRNA that reduces PD-1 expression, or protein that blocks PD-L1, or TCR, or safety switch; preferably, the cytokines include IL-12, IL- 15. IL-21, or type I interferon; preferably,
  • the present application provides the use of the gene-modified immune cells described in the present application, which is characterized in that it is used to prepare a drug for inhibiting tumors, and the tumor is a tumor expressing CS1.
  • the tumor is a tumor expressing CS1.
  • the The CS1-expressing tumor described above is multiple myeloma.
  • the present application provides a pharmaceutical composition comprising: the antibody described in the present application or a nucleic acid encoding the antibody; or the immunoconjugate described in the present application or a nucleic acid encoding the conjugate; or the present The chimeric antigen receptor described in the application or the nucleic acid encoding the chimeric antigen receptor; or the genetically modified immune cell described in the application.
  • the present application provides a kit comprising: a container, and a pharmaceutical composition of the present application in the container; or a container, and an antibody of the present application or a nucleic acid encoding the antibody in the container; or the present The immunoconjugate of the application or the nucleic acid encoding the conjugate; or the chimeric antigen receptor of the application or the nucleic acid encoding the chimeric antigen receptor; or the genetically modified immune cell described in the application.
  • Figure 1 shows the binding of CS1 hybridoma antibodies 32A12MAb, 37A3MAb and 48G9MAb to recombinant protein hSLAMF7-avi-His determined by ELISA.
  • Figure 2 shows the binding of CS1 hybridoma antibody to the multiple myeloma cell line MM.1S.
  • Figure 3 shows the binding of CS1 hybridoma antibody to SLAMF7 recombinant proteins of human, murine and monkey species, respectively.
  • Figure 4 shows the binding of CS1 hybridoma antibody to MM.1S, NCI H929, RPMI 8226 and WI38, HEK293 cells.
  • Figure 5 shows the binding of humanized CS1 antibodies hu32A12, hu37A3 and hu48G9 to recombinant protein hSLAMF7-avi-His.
  • Figure 6 shows the binding of humanized CS1 antibody to multiple myeloma cell lines.
  • Figure 7 shows the binding of humanized CS1 antibody to SLAMF7 recombinant proteins of human, murine and monkey species, respectively.
  • Figure 8 shows the binding of humanized CS1 antibody to MM.1S, NCI H929 and WI38, HEK293 cells.
  • Figure 9 shows the affinity determination of humanized CS1 antibody hu37A3 with human SLAMF7.
  • Figure 10 shows the affinity determination of humanized CS1 antibody hu48G9 with human SLAMF7.
  • Figure 11 shows the affinity determination of humanized CS1 antibody huLuc63 with human SLAMF7.
  • Figure 12 shows the affinity determination of CS1 antibody Luc90 with human SLAMF7.
  • Figure 13 shows the affinity determination of humanized CS1 antibody hu37A3 with monkey SLAMF7.
  • Figure 14 shows the affinity determination of humanized CS1 antibody hu48G9 with monkey SLAMF7.
  • Figure 15 shows the affinity determination of humanized CS1 antibody hu32A12 with human SLAMF7.
  • Figure 16 shows the aggregation results of humanized CS1 antibody hu37A3 as determined by SEC.
  • Figure 17 shows the aggregation results of humanized CS1 antibody hu32A12 as determined by SEC.
  • Figure 18 shows the aggregation results of the humanized CS1 antibody hu48G9 as determined by SEC.
  • Figure 19 shows the positive rate of CS1 CAR T cell CAR.
  • Figure 20 shows the positive rate of CS 1 CAR T cell CAR at different time points.
  • Figure 21 shows the in vitro killing results of CS1 CAR T on CS1 positive and negative cells under different effector-target ratios.
  • Figure 22 shows the secretion of IFN- ⁇ after CS1 CAR T co-incubated with CS1-expressing positive and negative target cells.
  • Figure 23 shows the secretion of TNF- ⁇ after CS1 CAR T co-incubated with CS1-expressing positive and negative target cells.
  • Figure 24 shows the secretion of IL-2 after CS1 CAR T co-incubated with CS1-expressing positive and negative target cells.
  • Figure 25 shows the effect of soluble CS1 on CS1 CAR T cell killing in vitro.
  • Figure 26 shows the expression of PD-1 in CS1 CAR T cells after stimulation with CS1-positive multiple myeloma cells.
  • Figure 27 shows the expression of Tim-3 in CS1 CAR T cells after stimulation with CS1-positive multiple myeloma cells.
  • Figure 28 shows the expression of LAG-3 in CS1 CAR T cells after stimulation with CS1-positive multiple myeloma cells.
  • Figure 29 shows western blot results of CS1 CAR T cells phosphorylated CD3- ⁇ (CAR).
  • Figure 30 shows the secretion of IL-6 after CS1 CAR T cells co-incubated with monocytes and CS1-positive multiple myeloma cells.
  • Figure 31 shows the in vitro expansion of CS1 CAR T cells following target cell stimulation.
  • Figure 32 shows the viability of CS1 CAR T cells at different time points after stimulation with target cells.
  • Figure 33 shows the in vitro expansion and viability of UTD after stimulation of target cells.
  • Figure 34 shows the in vitro expansion and viability of CS1 CAR T cells under IL-2 stimulation.
  • Figure 35 shows the effect of CS1 CAR T cells on tumor volume over time in the in vivo treatment of multiple myeloma NPG mouse subcutaneous xenograft tumor model and the comparison of tumor photos.
  • Figure 36 shows the subcutaneous anti-tumor and anti-tumor effect of CS1 CAR T cells on human multiple myeloma cells RPMI-8226-CS1 in NPG mice.
  • Figure 37 shows the anti-tumor effect of CS1-UCAR-T cells and CS1-UCAR-CS1-/-T cells on the subcutaneous transplanted tumors of human multiple myeloma cells RPMI 8226-CS1 in NPG mice.
  • antigen-binding units that specifically bind to CS1
  • these antigen-binding units can be used to prepare various targeted antitumor drugs and drugs for tumor diagnosis. This application is completed on this basis.
  • 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
  • CS1 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+ T cells, activated B cells and mononuclear dendritic cells are basically not expressed in hematopoietic lineage progenitor cells and other human tissues.
  • CS1 is a type I transmembrane protein with an extracellular segment consisting of serine 23 (S) - methionine (M) 226, which contains two domains Ig-like V region (23 serine S-124 Val amino acid V, distal membrane end) and Ig-like C region (131 proline P-206 serine S, proximal membrane end).
  • S serine 23
  • M methionine
  • the amino acid sequence of the extracellular segment of human SLAMF7 is shown in SEQ ID NO: 57
  • the amino acid sequence of the extracellular segment of mouse SLAMF7 is shown in SEQ ID NO: 58
  • the amino acid sequence of the extracellular segment of cynomolgus monkey SLAMF7 is shown in SEQ ID NO: 58.
  • NO: 59 shown.
  • CS1 molecule is highly expressed in multiple myeloma (MM) cells, and its monoclonal antibody Elotuzumab (huLuc63) has good efficacy in combination with immunomodulators and proteasome inhibitors in the treatment of relapsed or refractory MM patients. , has been approved by the FDA for the treatment of MM.
  • the hybridoma antibody recognizing CS1 and its modified humanized antibody provided in the present application can be used for the treatment of multiple myeloma.
  • the CAR-T cells containing the CS1 antibody prepared in this application can significantly kill multiple myeloma cells in vitro and in vivo.
  • CAR-T cells comprising antibodies recognizing CS1 of the present application can resist the killing of autologous or allogeneic T cells by NK cells in the host, thereby increasing autologous or allogeneic immune cells (exemplary, Persistence and/or transplantation survival of CS1-CAR-T cells, CS1-UCAR-T cells, CS1-UCAR-CS1-/-T cells) in the presence of host immune cells.
  • the bispecific antibody comprising the CS1 antibody prepared in the present application and the antibody recognizing T cells can resist the killing of autologous or allogeneic T cells by NK cells in the host, thereby increasing the autologous or allogeneic immune cells (exemplary).
  • CS1-CAR-T cells, CS1-UCAR-T cells, CS1-UCAR-CS1-/-T cells persistence and/or transplantation survival in the presence of host immune cells.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • protein protein
  • proteins polymers of amino acids of any length.
  • the polymer may be linear, cyclic or branched, it may contain modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids.
  • amino acid polymers such as those that have been processed by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, elimination Amino acid polymers modified by spination, selenylation, transfer-RNA mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation to labeling components.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • a polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide.
  • the term also includes polypeptides expressed from the specified nucleic acid sequence.
  • antigen-binding unit refers to immunoglobulin molecules (or “antibodies”) and immunologically active portions of immunological molecules, ie, molecules that contain an antigen-binding site that specifically binds ("immunoreactively") an antigen.
  • immunoglobulin molecules derived from various species including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibodies (eg, IgG) comprise four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Immunoglobulins represent a large family of molecules including several types of molecules, such as IgD, IgG, IgA, IgM and IgE.
  • immunoglobulin molecule includes, for example, hybrid antibodies or altered antibodies and fragments thereof. It has been shown that the antigen-binding function of antibodies can be carried out by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen binding units". Also included in the term “antigen-binding unit” is any polypeptide chain-containing molecular structure having a specific shape that conforms to and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the relationship between the molecular structure and the epitope. Complex.
  • antigen binding units examples include Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains, bivalent fragments (F(ab) comprising two Fab fragments linked by a disulfide bridge on the hinge region 2 fragments); Fd fragments composed of VH and CH1 domains, Fv fragments composed of the VL and VH domains of the one-armed antibody; dAb fragments composed of VH domains (Ward et al., Nature, 341:544- 546, 1989); and an isolated complementarity determining region (CDR) or any fusion protein comprising such an antigen binding unit.
  • Fab fragments monovalent fragments consisting of VL, VH, CL and CH1 domains
  • bivalent fragments F(ab) comprising two Fab fragments linked by a disulfide bridge on the hinge region 2 fragments
  • Fd fragments composed of VH and CH1 domains Fv fragments composed of the VL and VH domains of the one-armed antibody
  • antibody is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the indicated desired antigen-binding activity.
  • antibody refers to an antigen binding protein of the immune system.
  • antibody as referred to herein includes an intact full-length antibody having an antigen-binding region and any fragment thereof in which the "antigen-binding portion" or “antigen-binding region” remains, or a single chain thereof, such as a single-chain variable fragment ( scFv).
  • “Native antibody” refers to a naturally occurring immunoglobulin molecule with various structures, and refers to a carbohydrate comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding fragments thereof, interconnected by disulfide bonds protein.
  • the term “antibody” also includes all recombinant forms of antibodies, particularly those described herein, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and antigen-binding antibody fragments and derivatives described below.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • variable region or variable domain refers to a domain of an antibody heavy or light chain that is involved in antibody antigen binding.
  • VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, usually arranged in the following order from amino-terminus to carboxy-terminus: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • antibodies that bind a particular antigen can be isolated by screening a library of complementary VL or VH domains using the VH or VL domains, respectively, from antibodies that bind to the antigen. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • hypervariable region or “complementarity determining region” or “CDR” refers to an antibody variable domain that is hypervariable in sequence and/or forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting regions of residues ("antigen contacts").
  • an antibody typically contains six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).
  • Antibody fragments include, but are not limited to: (i) Fab fragments composed of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments composed of VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) F(ab')2 fragments , a bivalent fragment comprising two linked Fab fragments; (vi) an antigen-binding site for a single-chain Fv molecule (Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc.Natl.Acad.Sci.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • Fc or "Fc region” are used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • CDR residues and other residues in the variable domains are numbered according to Kabat et al. above.
  • full antibody full length antibody
  • whole antibody refers to having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region as defined herein or including having antigen binding region of the complete full-length antibody.
  • the application provides full-length antibodies, the heavy and light chains of which can be full-length (eg, an antibody can include at least one, preferably two, full heavy chains, and at least one, preferably two, whole light chain) or may include an antigen binding portion (Fab, F(ab')2, Fv or scFv).
  • the antibody heavy chain constant region is selected from, eg, IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE.
  • the choice of antibody type will depend on the immune effector function the antibody is designed to elicit.
  • appropriate amino acid sequences for the constant regions of the various immunoglobulin isotypes and methods for generating a wide variety of antibodies are known to those skilled in the art.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker such as a short flexible polypeptide linker
  • a scFv may have the VL and VH variable regions described in any order (eg, with respect to the N-terminus and C-terminus of the polypeptide), the scFv may include a VL-linker-VH or VH-linker-VL can be included.
  • flexible amino acids (G4S) 3 are introduced as linkers for the VH and VL fragments of the antibody to form a single-chain variable fragment (scFv) whose amino acid sequence confers specificity to the molecule against CS1 and forms the present The basis for all of the application's antigen-binding units.
  • the scFv can be used to design a range of different "antibody” molecules, including, for example, full-length antibodies, fragments thereof such as Fab and F(ab')2, scFvs, fusion proteins (including scFv-Fc), multivalent antibodies, i.e. with Antibodies with more than one specificity against the same antigen or different antigens, e.g., bispecific T-cell binding antibodies (BiTE), tri-antibodies, etc. (Cuesta et al., Multivalent antibodies: when design surpasses evolution, Trends in Biotechnology 28:355 -362, 2010).
  • the application includes antibodies having scFv sequences fused to one or more heavy chain constant regions to form antibodies having human immunoglobulin Fc regions or murine immunoglobulin Fc regions to generate dual valent protein, thereby increasing the overall affinity and stability of the antibody.
  • the Fc portion allows for the direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to antibodies, eg, used in antigen quantification studies, to immobilize antibodies for affinity measurements, for targeted delivery of therapeutics drug, the use of immune effector cells to test Fc-mediated cytotoxicity and many other applications.
  • single domain antibody refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also called nanobody (Nanobody).
  • single domain antibody refers to an antibody comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain.
  • the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Pat. No. 6,248,516).
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical and/or bind the same epitope, except where possible
  • variant antibodies eg, containing naturally-occurring mutations or produced during the preparation of monoclonal antibody preparations
  • variants are usually present in small amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the antibody is of the nature obtained from a substantially homogeneous population of antibodies, and is not considered to require that the antibody be prepared by any particular method.
  • it can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals that contain all or a portion of human immunoglobulin loci.
  • the monoclonal antibodies of the present application can be produced by hybridoma methods, which can be formed by isolating stimulated immune cells, such as those from the spleen of a vaccinated animal. These cells, such as myeloma cells or transformed cells, can then be fused with immortalized cells capable of replicating indefinitely in cell culture, thereby producing immortal, immunoglobulin-secreting cell lines.
  • the immortal cell lines utilized are selected (for lack of enzymes necessary to utilize certain nutrients).
  • TK thymidine kinase
  • HGPRT hypoxanthine-guanine phosphoribosyltransferase
  • HAT hypoxanthine aminopterin thymidine medium
  • the present application screened 3 hybridoma antibodies 32A12, 37A3, and 48G9 that bind hSLAMF7.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain of an antibody is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • chimeric antibodies comprise non-human variable regions (eg, variable regions derived from mouse, rat, hamster, rabbit, or non-human primates such as monkeys) and human constant regions.
  • the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from the class or subclass of the parent antibody.
  • Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a "humanized antibody.”
  • humanized is used for non-human antibodies, such as rodents or primates, etc., to be hybrid immunoglobulins, immunoglobulin chains or fragments thereof containing minimal sequence derived from non-human immunoglobulins.
  • Humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one (generally two) variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of the non-human antibody, and all or substantially all of the CDRs correspond to those of the non-human antibody All the FRs above correspond to the FRs of human antibodies.
  • a humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody.
  • "humanized antibodies” may include mutations, such as those introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo.
  • the hybridoma antibodies 32A12, 37A3, and 48G9 were humanized by the method of CDR transplantation to obtain hu32A12, hu37A3, and hu48G9.
  • parent antibody or "parent immunoglobulin” includes unmodified antibodies that have been subsequently modified to produce variants.
  • the parent antibody can be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody.
  • a parent antibody may refer to the antibody itself, a composition comprising said parent antibody, or its encoded amino acid sequence.
  • parent antibody or parent immunoglobulin includes murine or chimeric antibodies that are subsequently modified to produce humanized antibodies.
  • variable antibody or “antibody variant” include antibody sequences that differ from the parent antibody sequence due to at least one amino acid modification compared to the parent.
  • Variant antibody sequences herein preferably have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity.
  • An antibody variant may refer to the antibody itself, a composition comprising the parent antibody, or the amino acid sequence encoding it.
  • Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis.
  • amino acid modification includes amino acid substitutions, additions and/or deletions
  • amino acid substitution means replacing an amino acid at a particular position in the parent polypeptide sequence with another amino acid.
  • amino acid insertion means the addition of an amino acid at a specific position in the parent polypeptide sequence.
  • amino acid deletion or “deletion” means the removal of an amino acid at a particular position in the parent polypeptide sequence. Any combination of deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, eg: binding to antigen.
  • Modification refers to a change in the state or structure of a protein or polypeptide of the present application. Modifications can be chemical, structural and functional.
  • conservative modification or “conservative sequence modification” means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged acute side chains eg , glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains eg, alanine, valine, leucine, isoleucine
  • proline e.g, phenylalanine, methionine
  • beta branched side chains eg, threonine, valine, isoleucine
  • aromatic side chains eg, tyrosine, benzene alanine, tryptophan, histidine.
  • one or more amino acid residues in the CDR regions or in the framework regions of the antibodies of the present application can be replaced with other amino acid residues of the same side chain family, and the altered antibodies (variant antibodies) can be tested for retained function.
  • anti-CS1 antibody refers to an antibody capable of binding CS1 with sufficient affinity for use as a diagnostic agent for targeting CS1 and /or therapeutic agent.
  • the anti-CS1 antibody binds to an unrelated, non-CS1 protein to less than about 10% of the extent of the antibody to CS1, as determined by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the antigen binding unit targeting CS1 of the present application binds to the Ig-like V-type domain at the distal membrane end of the extracellular domain of hCS1, or the proximal Ig-like C2-type domain of the extracellular domain. domain.
  • the hybridoma antibody obtained by immunizing mice with the immunogen hSLAMF7-avi-His recombinant protein using conventional hybridoma antibody preparation technology in the art is described.
  • the hybridoma antibody is also humanized by the method of CDR transplantation to obtain a humanized antibody.
  • These molecules exhibit specificity.
  • the antibody recognizes CS1 protein.
  • the CS1 antibody recognizes cells expressing CS1, such as tumor cells MM.1S cells, RPMI 8226 cells, NCI-H929 cells, NK cells.
  • the CS1 antibody does not recognize CS1 negative cells, such as HEK293, WI38 cells.
  • CS1 herein refers to human CS1, murine CS1 or monkey CS1.
  • the present application provides an antigen-binding unit that recognizes CS1, comprising a heavy chain CDR1 comprising the amino acid sequence of any of SEQ ID NOs: 3, 13, 23, and/or comprising any of the amino acids of SEQ ID NO: 4, 14, 24, 32
  • the heavy chain CDR2 of the sequence, and/or the heavy chain CDR3 comprising the amino acid sequence of any of SEQ ID NOs: 5, 15, 25, 41.
  • the application provides an antigen-binding unit or fragment thereof that binds CS1, comprising a light chain CDR1 comprising the amino acid sequences of SEQ ID NOs: 8, 18, 28, and/or comprising SEQ ID NOs: 9, 19 , the light chain CDR2 of the amino acid sequence of 29, and/or the light chain CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 10, 20, and 30.
  • the application provides an antigen-binding unit or fragment thereof that binds CS1, comprising a heavy chain CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 3, 13, 23, and/or comprising SEQ ID NO: 4,
  • the antibody or fragment thereof that binds to CS1 comprises the heavy chain CDR1 comprising any of the amino acid sequences of SEQ ID NOs: 3, 13, 23, and the heavy chain CDR1 comprising any of the amino acid sequences of SEQ ID NOs: 4, 14, 24, and 32 Heavy chain CDR2, and heavy chain CDR3 comprising the amino acid sequence of any of SEQ ID NOs: 5, 15, 25, 41, and/or light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, 18, 28, and comprising The light chain CDR2 of the amino acid sequence of SEQ ID NO: 9, 19, 29, and the light chain CDR3 comprising the amino acid sequence of any of SEQ ID NO: 10, 20, 30.
  • the antibody or fragment thereof that binds to CS1 comprises the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 3, 13, 23, and the heavy chain comprising the amino acid sequence of SEQ ID NO: 4, 14, 24, 32.
  • Chain CDR2, and heavy chain CDR3 comprising the amino acid sequence of any of SEQ ID NOs: 5, 15, 25, 41, and light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, 18, 28, and comprising SEQ ID NO : the light chain CDR2 of the amino acid sequence of 9, 19, 29, and the light chain CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 10, 20, 30.
  • the application provides an antigen binding unit recognizing CS1 comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21, 31, 36 or 40.
  • the application provides an antigen binding unit recognizing CS1 comprising a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 6, 16, 26, 34, 38 or 43.
  • the heavy and light chain variable region sequences can recognize CS1
  • the heavy and light chain variable region sequences can be "mixed and matched" to generate the anti-CS1 binding molecules of the present application.
  • the application provides an antigen binding unit that recognizes CS1, comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region of the antigen binding unit having SEQ ID
  • the chain variable region has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 26;
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 31
  • the amino acid sequence shown and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 34; the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 36 and the antigen binding
  • the application provides variants of antigen binding units that recognize CS1.
  • the application thus provides antigen binding units having heavy and/or light chain variable regions that are at least 80% identical in sequence to the variable region sequences of the heavy or light chains.
  • the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98% , most particularly 99%, including e.g. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
  • Variants can be obtained by using the antibody described in the present application as the parent antibody by yeast library screening, phage library screening, point mutation and other methods.
  • the present application provides an antigen-binding unit that recognizes the same epitope as the aforementioned anti-CS1 antigen-binding unit; or an Ig-like V-type domain that binds CS1; or an Ig-like C2 that binds CS1 -type field. All the antigen-binding units that recognize CS1 of the present application can specifically bind to CS1-positive cells, but do not bind to CS1-negative cells, showing their good therapeutic potential. Part of the scFv of the present application that recognizes the antigen-binding unit of CS1 is structurally stable and does not easily aggregate.
  • the application provides an antigen binding unit that recognizes CS1, the antigen binding unit is a hybridoma antibody, a humanized antibody, a chimeric antibody or a fully human antibody; or the antigen binding unit is a monoclonal antibody; Alternatively the antigen binding unit is a whole antibody, scFv, single domain antibody, Fv fragment, Fab fragment, Fab' fragment, (Fab')2 fragment, dAb fragment or multifunctional antibody.
  • anti-CS1 antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art. These include, for example, ELISA, biacore, microplate reader and flow cytometric analysis. Suitable assays are described in detail in the Examples.
  • antigen refers to a substance that is recognized and specifically bound by an antigen-binding unit.
  • Antigens can include peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens.
  • Antigen can also refer to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunologically-competent cells, or both.
  • any macromolecule including virtually any protein or peptide, can serve as an antigen.
  • Antigens used in this application include human, mouse and monkey SLAMF7 recombinant proteins.
  • affinity refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the use of Biacore to determine the affinity of an antibody as described herein.
  • the "affinity" of an antibody for CS1 herein is expressed as the KD of the antibody.
  • the KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction.
  • the present application determined the affinity of CS1 antibodies to SLAMF7 recombinant proteins of different species (exemplarily, human, mouse, monkey), with KD values ranging from 940pM to 107nM.
  • EC50 concentration for 50% of maximal effect (EC50) refers to the concentration that elicits 50% of the maximal effect.
  • concentration for 50% of maximal effect EC50
  • concentration for 50% of maximal effect refers to the concentration that elicits 50% of the maximal effect.
  • CS1 antibody and recombinant protein hSLAMF7-avi-His were detected by ELISA, in the range of 0.01-0.058 ⁇ g/ml; the EC50 values of CS1 antibody and CS1 positive cells MM.1S cells were detected by flow cytometry , in the range of 0.05-1.76 ⁇ g/ml.
  • the present application also uses a microplate reader to detect the binding of CS1 antibodies to CS1 of different species (exemplarily, human, mouse, monkey), and the antibodies all bind to human CS1, but not to mouse CS1 Binding, antibodies 37A3 and 48G9 also bound to monkey CS1.
  • CS1 antibodies to CS1 of different species exemplarily, human, mouse, monkey
  • the antibodies all bind to human CS1, but not to mouse CS1 Binding, antibodies 37A3 and 48G9 also bound to monkey CS1.
  • antigenic epitope also known as “antigenic epitope”, or “epitope” or “antigenic determinant” includes any determinant or region capable of being bound by an antibody.
  • An epitope is a region of an antigen that is bound by an antibody targeting the antigen and includes specific amino acids that are in direct contact with the antibody.
  • the epitope may consist of a contiguous sequence of CS1 protein sequences, or it may consist of a three-dimensional structure in which CS1 protein sequences are discontinuous.
  • the antigen used herein is human, murine or monkey CS1.
  • the binding epitope of CS1 antibody and human CS1 is analyzed, and the binding epitope is obtained in the Ig-like V-type domain of human CS1, at the far membrane end; or in the Ig-like C2-type domain of human CS1, proximal end.
  • the application also provides immunoconjugates comprising the antibodies described herein, and functional molecules linked thereto.
  • the antibody and the functional molecule can form a conjugate by covalent connection, coupling, attachment, cross-linking and the like.
  • the functional molecule is selected from the group consisting of: a molecule targeting a tumor surface marker, a tumor-inhibiting molecule, a molecule targeting an immune cell surface marker, or a detectable marker.
  • the molecule targeting the surface marker of an immune cell is an antigen-binding unit (eg, an antibody) that binds to a T-cell surface marker, which forms a T-cell participation with the antigen-binding unit described herein.
  • Bifunctional antigen binding units eg, diabodies).
  • in-frame fusion refers to a longer ORF that joins two or more ORFs to form contiguous in a manner that maintains the correct reading frame of the original open reading frame (ORF).
  • ORF open reading frame
  • the resulting recombinant fusion protein is a single protein containing two or more fragments corresponding to the polypeptide encoded by the original ORF (the fragments are not normally so linked in their natural state).
  • the reading frame is thus contiguous throughout the fusion fragment, the fragments may be physically or spatially separated by, for example, in-frame linking sequences (eg "flexon").
  • nucleic acid molecules encoding at least one antibody, functional variant or immunoconjugate thereof of the present application.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • the present application also provides nucleic acid molecules encoding the aforementioned antibodies, preferably, the nucleic acid molecules of the present application are selected from SEQ ID NO: 2, 12, 22, 33, 37 or 42 encoding the variable region of the heavy chain, and/or selected from SEQ ID NO: 7, 17, 27, 35, 39 or 44 encoding the light chain variable region.
  • nucleic acid molecule comprising the heavy chain variable region sequence of SEQ ID NO:2, and the light chain variable region sequence comprising SEQ ID NO:7; or comprising SEQ ID NO:12
  • one or more vectors comprising the above-described nucleic acids are provided.
  • the present application also relates to vectors comprising the appropriate DNA sequences described above together with appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • cell refers to cells of animal origin, human or non-human.
  • 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.
  • patient is a subject suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include transformed primary cells and progeny derived therefrom (regardless of the number of passages).
  • the nucleic acid content of the progeny may not be identical to the parental cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.
  • a method of making an anti-CS1 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expressing the antibody as described above, and optionally extracting the antibody from the host cell (or host cell culture medium) to recover the antibody.
  • host cells comprising nucleic acids encoding the above-described antibodies.
  • the host cell comprises (e.g., transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of the antibody, or (2) comprising a nucleic acid encoding a VL comprising the antibody A first vector of nucleic acid comprising an amino acid sequence, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH.
  • the host cell is a eukaryotic cell, eg, a 293T cell.
  • the host cell may also express a chemokine receptor.
  • the host cell may also express a safety switch.
  • the host cell is administered in combination with an agent that enhances its function, preferably, in combination with a chemotherapeutic drug; and/or the host cell is administered in combination with an agent that improves one or more side effects associated therewith and/or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor targeting a chimeric antigen other than CS1.
  • the host cell is an immune effector cell.
  • immune effector cell refers to a cell involved in an immune response that produces an immune effector, 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 effector 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 may be autologous NK cells or allogeneic NK cells.
  • CIK cells that is, cytokine-induced killer cells (Cytokine-Induced Killer, CIK) is a new type of immune active cells, CIK has strong proliferation ability, strong cytotoxicity, and has certain immune characteristics. Because this cell expresses both CD3 and CD56 membrane protein molecules, it is also called NK cell (natural killer cell)-like T lymphocyte, which has both strong antitumor activity of T lymphocyte and non-MHC restriction of NK cell. Tumor-killing advantages.
  • NK cell natural killer cell
  • 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.
  • the T cells described herein can be obtained from a number of sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and naive T cells obtained from tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors, and also It can be a cell population with specific phenotypic characteristics obtained by sorting, etc., or a mixed cell population with different phenotypic characteristics, such as "T cells” can be cells that contain at least one T cell subset: memory stem cell-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).
  • sources including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and naive T cells obtained from tissue from sites of infection, ascites, ple
  • a "T cell” may be a specific subtype of T cell, such as ⁇ T cells, ⁇ 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.
  • T cells can be of any type and of any developmental stage, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells such as Th1 and Th2 cells, CD8+ T cells (e.g. cytotoxic T cells) , tumor infiltrating cells, memory T cells, naive T cells, etc.
  • the T cells may be CD8+ T cells or CD4+ T 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.
  • cells can be derived from a healthy donor, or from an individual diagnosed with cancer.
  • 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.
  • 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. Using CD3/CD28 magnetic beads, antigen stimulation in vitro or in vivo will affect the degree and duration of T cell activation.
  • the engineered T cells are co-incubated with tumor cells containing a specific target antigen or activated after viral infection.
  • peripheral blood mononuclear cells refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, and the like.
  • 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.
  • the term "engineering” 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. A comprehensive science and technology of products.
  • the engineering refers to one or more alterations of nucleic acids, such as nucleic acids within the genome of an organism.
  • the engineering refers to changes, additions and/or deletions of genes.
  • the engineered cell or the engineered cell may also refer to a cell with added, deleted and/or altered genes.
  • 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 noncoding 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.
  • 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 chimeric antigen receptors (CARs) or T cell receptors (TCRs), which can be integrated into the cell genome.
  • CARs chimeric antigen receptors
  • TCRs T cell receptor
  • TCR silencing refers to no or low expression of endogenous TCR.
  • MHC silencing refers to no or low expression of endogenous MHC.
  • Low expression as used herein means that the protein and/or RNA level of the target gene expressed in the engineered cell is lower than the expression level before the cell engineering treatment.
  • low expression of B2M or TCR or CS1 refers to a decrease in 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%.
  • 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 or TCR or CS1.
  • B2M beta-2 microglobulin, also known as B2M, the light chain of an MHC class I molecule. Playing an important role in the transplant response, rejection is mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.
  • 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.
  • TCRs are divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, ⁇ and ⁇ , and TCR2 is composed of two chains, ⁇ and ⁇ .
  • TCR refers to the constant region of the TCR ⁇ chain.
  • 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 to alter DNA sequences. This technique is sometimes called “gene clipping” or “genome engineering.” Gene editing can be used to achieve precise and 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, Base Editor technology, guide editing technology and/or homing endonuclease technology.
  • CRISPR Clustered regularly interspaced short palindromicrepeats
  • Cas9 CRISPR associated nuclease
  • 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.
  • a target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide.
  • a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
  • gRNA When the application involves the sequence of gRNA, it can be a targeted DNA sequence, or it can also be a complete Cas9 guide sequence formed by the ribonucleotides corresponding to the DNA, crRNA and TracrRNA. In some embodiments, 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.
  • sgRNA refers to short gRNAs.
  • CRISPR/Cas 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.
  • This application obtains universal T cells or universal CAR-T cells by knocking out the genes TRAC and B2M.
  • the exons of the corresponding genes encoding the constant regions of one or both of the alpha and beta chains of B2M, TCR are knocked out using CRISPR/Cas technology, respectively.
  • the gRNA used for knocking out TCR is selected from the sequences shown in SEQ ID NO: 76, 77, 78, 79, 80, 81, 82 and/or 83.
  • the engineered T cell B2M gene is knocked out using CRISPR/Cas9 technology, and the gRNA used is selected from the sequences shown in SEQ ID NOs: 84, 85, 86 and/or 87.
  • knocking out the cellular CS1 gene reduces self-killing by CS1-CAR-T cells.
  • the CS1 gene is knocked out using CRISPR/Cas9 technology, and the gRNA used is selected from the sequences shown in SEQ ID NOs: 88, 89, 90, 91, 92, 93, 94 and/or 95.
  • “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%.
  • 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 or TCR or CS1.
  • specific CAR-T cells are constructed first, and then CRISPER/Cas9 technology is used to knock out the endogenous TRAC, B2M and/or CS1 of the CAR-T cells to construct the corresponding UCAR-T.
  • CRISPER/Cas9 technology is used to knock out endogenous TRAC, B2M and/or CS1 to construct universal T cells, and then express specific CAR to construct UCAR-T cells.
  • CRISPER/Cas9 technology knocks out endogenous TRAC, B2M and/or CS1 and expresses specific CAR simultaneously to construct UCAR-T cells.
  • transfection refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell.
  • a "transfected” or “transformed” or “transduced” cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cells include primary subject cells and their progeny. 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.
  • sequence when used in reference to a nucleotide sequence or a polynucleotide sequence may include DNA or RNA, and may be single-stranded or double-stranded.
  • 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.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleotide 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).
  • viruses eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses.
  • the nucleic acid encoding the antigen binding unit of the present application can be integrated into an expression vector.
  • Expression vectors for use in this application include, but are not limited to, expression vectors that enable proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.
  • 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.
  • 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 corresponding cDNA or polypeptide fragments of proteins 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 T cell receptor consists of a TCR subunit combined with an antigen binding domain (such as an antibody domain), wherein the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, TCR The stimulatory domain of the intracellular signaling domain; the TCR subunit is operably linked to the antibody domain.
  • the antigen binding domain of the chimeric T cell receptor comprises the CS1 antigen recognition unit described herein.
  • 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 bringing the TAC receptor close to the TCR receptor; (3) the transmembrane domain and the intracellular CD4 co-receptor Area.
  • the TAC antigen binding domain includes the CS1 antigen recognition unit described in this application.
  • the present application provides a T cell that can significantly kill target cells.
  • the application provides CAR-T cells comprising the CS1 antigen recognition unit, such as hu32A12 CAR-T cells, hu37A3 CAR-T cells, hu48G9 CAR-T cells.
  • the CS1-CAR-T cells have any of the following advantages:
  • the CS1-CAR-T cells can specifically kill CS1-positive target cells.
  • the CS1-CAR-T cells can specifically kill CS1-positive tumor cells.
  • the CS1-CAR-T cells specifically kill CS1-positive multiple myeloma.
  • CS1 is a member of the lymphocyte activating molecule family and participates in the activation function of NK cells
  • CS1 is expressed on NK cells; for example, in in vitro and in vivo experiments, the CS1-CAR-T cells specifically kill host NK cells, or all
  • the CS1-CAR-T cells can resist the killing of autologous or allogeneic immune cells (such as T cells, CAR-T cells) by host NK cells, and improve the persistence of autologous or allogeneic immune cells in the presence of host immune cells. Sex and/or transplant survival.
  • soluble CS1 did not affect the killing effect of CS1-CAR-T cells on CS1-positive target cells.
  • Cytokine secretion In in vitro and in vivo experiments, the CS1-CAR-T cells can be significantly activated after co-incubation with CS1 positive target cells to secrete cytokines TNF- ⁇ , IL-2 or IFN- ⁇ ; in vivo In vitro and in vivo experiments, the CS1-CAR-T cells did not produce significant non-specific cytokine TNF- ⁇ , IL-2 or IFN- ⁇ secretion after co-incubation with CS1-negative cells; After co-incubation of CAR-T cells with monocytes and CS1-positive target cells, the secretion of cytokine IL-6 was lower.
  • the present application provides universal T cells comprising the CS1-CAR and double knockout of endogenous TCR and B2M (CS1-UCAR-T), and endogenous CS1, TCR and B2M Triple knockout CS1-UCAR-T cells.
  • the double-knockout or triple-knockout CS1-UCAR-T cells have any of the following advantages: specifically kill CS1-positive target cells.
  • the CS1-UCAR-T cells can specifically kill CS1-positive tumor cells.
  • the CS1-UCAR-T cells specifically kill CS1-positive multiple myeloma cells.
  • the CS1-UCAR-T cells specifically kill host NK cells, or the CS1-UCAR-T cells can resist host NK cells against autologous or allogeneic immune cells (such as T cells). , CAR-T cells, CS1-UCAR-T) killing, improving the persistence and/or transplantation survival rate of autologous or allogeneic immune cells in the presence of host immune cells.
  • autologous or allogeneic immune cells such as T cells.
  • chimeric antigen receptor includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the extracellular antigen-binding domain includes an antigen-binding unit of the present application that recognizes CS1, or includes an Fv, Fab, Fab', Fab'-SH, F(ab')2, scFv, or derived from an antigen-binding unit of the present application that recognizes CS1. multispecific antibodies.
  • Intracellular signaling domains include functional signaling domains of stimulatory and/or costimulatory molecules; or entire intracellular portions, or entire native intracellular signaling structures including stimulatory and/or costimulatory molecules domain, or a functional fragment or derivative thereof.
  • the stimulatory molecule comprises a CD3 ⁇ chain bound to a T cell receptor complex; in one aspect, the intracellular signaling domain further comprises a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (ie CD137), CD27 and/or CD28.
  • 4-1BB ie CD137
  • CD27 CD27
  • CD28 CD28
  • groups of polypeptides are linked to each other.
  • a CAR targeting CS1 comprises the antigen binding domain set forth in SEQ ID NO: 50, 51 or 52, and exemplary, a CAR targeting CS1 comprises the sequence set forth in SEQ ID NO: 45, 46 or 47.
  • the transmembrane domains and intracellular domains of the above-mentioned SEQ ID NOs: 45, 46, and 47 can be replaced by those skilled in the art by selecting conventional transmembrane domains and intracellular domains, and all fall within the protection scope of the present application.
  • the functional signaling domain of the stimulatory molecule modulates the initial activation of the TCR complex in a stimulatory manner.
  • the primary signaling domain is initiated by, for example, binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.).
  • Primary signaling domains that act in a stimulatory manner may contain immunoreceptor tyrosine activation motifs or signaling motifs of ITAMs.
  • ITAM-containing primary signaling domains examples include, but are not limited to, those derived from TCR ⁇ , CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also known as "" ICOS") and CD66d sequences.
  • the intracellular signaling domain in any one or more of the CARs of the present application comprises an intracellular signaling sequence, such as the primary signaling domain of CD3 ⁇ .
  • costimulatory signaling domain refers to "costimulatory molecule” and refers to a signal that binds to a cell stimulatory signaling molecule, such as TCR/CD3, in combination resulting in T cell proliferation and/or up- or down-regulation of key molecules. is an cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, including but not limited to cell proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response.
  • 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 ).
  • the signaling domain of a CAR targeting CS1 includes CD3 ⁇ .
  • the signaling domains of a CAR targeting CS1 include CD28 intracellular domain, CD137 intracellular domain.
  • the CD28 intracellular domain comprises the sequence shown in SEQ ID NO:73
  • the CD137 intracellular domain comprises the sequence shown in SEQ ID NO:55.
  • CD3 ⁇ is used interchangeably with “CD3z” and “CD3Z” in this application.
  • CD3 ⁇ is a human CD3 ⁇ molecule comprising the sequence shown in SEQ ID NO:56.
  • the CAR includes an optional leader sequence.
  • the CAR further comprises a leader sequence N-terminal to the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane.
  • the leader sequence comprises the sequence shown in SEQ ID NO:71.
  • the extracellular antigen-binding region of the CAR includes an antigen-binding unit and a linker fragment (also referred to as a hinge, spacer, or linker) of the present application that recognizes CS1.
  • Linked fragments can be considered as part of a CAR used to provide flexibility to the extracellular antigen binding region.
  • Linked fragments can be of any length.
  • the CAR includes a hinge domain, which is a CD8 ⁇ hinge, preferably, the CD8 ⁇ hinge domain comprises the amino acid sequence set forth in SEQ ID NO:53.
  • the transmembrane (TM) domain (or domain) of a CAR can anchor the CAR to the plasma membrane of cells.
  • a TM is a CD8 or CD28 transmembrane domain.
  • the TM is a human CD8 transmembrane domain or a human CD28 transmembrane domain.
  • the CD8 transmembrane domain comprises the amino acids of SEQ ID NO:54 and the CD28 transmembrane domain comprises the amino acids of SEQ ID NO:72.
  • interferon is a cytokine produced by the immune system, mainly divided into three types: ⁇ , ⁇ , and ⁇ , and has anti-viral, anti-tumor and immunomodulatory effects.
  • interleukin-2 (interleukin-2, IL-2) is a cytokine in the chemokine family, which plays an important role in the body's immune response and antiviral infection.
  • interleukin-6 is a broadly functional pleiotropic cytokine. IL-6 is a biomarker of cytokine storm.
  • T cell exhaustion is a state of T cell dysfunction manifested by progressive loss of function, changes in gene expression profiles, and sustained secretion of inhibitory cytokines.
  • Biomarkers of T cell exhaustion include PD-1, TIM-3, LAG-3, etc.
  • the antibodies, immunoconjugates, chimeric receptors, and host cells comprising the antibodies of the present application can be applied to the preparation of pharmaceutical compositions or diagnostic reagents.
  • the composition may also comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • the pharmaceutically acceptable carrier can be one of those conventionally used and is limited only by chemical physical considerations, such as solubility and inactivity with the active agent, and the route of administration.
  • a pharmaceutically acceptable carrier such as adjuvants, excipients and diluents, are well known to those skilled in the art and are readily available to the public.
  • a pharmaceutically acceptable carrier is one that is innocuous under the conditions of use and has no toxic side effects.
  • suitable dosage forms for the pharmaceutical compositions of the present application Methods of preparing administrable (eg, parenterally) compositions are known or apparent to those skilled in the art.
  • the composition comprises a chemotherapeutic agent.
  • composition of the present application can be prepared into various dosage forms as required, and the physician can determine the dosage beneficial to the patient according to factors such as the type, age, weight and general disease state of the patient, and the mode of administration.
  • the mode of administration can be, for example, parenteral administration (eg, injection) or other therapeutic modes.
  • parenteral administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
  • the engineered cells of the present application can be administered to a subject in any suitable manner.
  • the antibodies of the present application, immunoconjugates comprising the antibodies, chimeric receptors, host cells are administered intravenously.
  • Suitable pharmaceutically acceptable carriers for injectable antibodies of the present application, immunoconjugates comprising the antibodies, chimeric receptors, host cells may include any isotonic carrier, for example, physiological saline (about 0.90% w in water). /v NaCl, about 300mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), normal temperature or electrolyte solution.
  • the pharmaceutically acceptable carrier is supplemented with human serum protein.
  • an “effective amount” or “therapeutically effective amount” refers to a dose sufficient to prevent or treat a disease (cancer) in a subject. Effective doses for therapeutic or prophylactic use depend on the stage and severity of the disease being treated, the age, weight and general health of the subject, and the judgment of the prescribing physician. The size of the dose will also depend on the active substance selected, the method of administration, the timing and frequency of administration, the presence, nature and extent of adverse side effects that may accompany administration of the particular active substance, and the desired physiological effect. According to the judgment of the prescribing physician or those skilled in the art, one or more rounds, or multiple administrations of the antibody of the present application, the immunoconjugate comprising the antibody, the chimeric receptor, and the host cell may be required.
  • Embodiments of the present application also include depletion of mammalian lymphocytes prior to administration of the antibodies of the present application, immunoconjugates comprising the antibodies, chimeric receptors, host cells, including but not limited to non-myeloablative lymphoid depletion chemotherapy, Myeloablative lymphatic depletion chemotherapy, total body irradiation, etc.
  • 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.
  • the engineered T cells provided in the present application can inhibit tumor cell proliferation, and/or inhibit tumor cell proliferation and increase tumor volume in vivo.
  • prevention refers to interventions that are attempted prior to the development of a disease such as rejection of a cell transplant.
  • the application provides the antibodies, CARs, nucleic acids, recombinant expression vectors, host cells, cell collections or pharmaceutical compositions of the present application for use in the treatment or prevention of mammalian tumors.
  • the engineered T cells provided in this application 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)
  • rheumatic diseases including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthropathies (including anky
  • the engineered T cells provided herein 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) ), 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
  • the engineered T cells provided herein can be used to treat, prevent or ameliorate asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema, and other obstructive or inflammatory diseases of the airways.
  • the engineered T cells of the present application 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 anticancer 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 anticancer agents (eg, as adjuvants thereof or in combination with them).
  • the tumor described herein can be any tumor, including acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (eg, medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal, eye, intrahepatic bile duct, joint, neck, gallbladder or pleura, nose, nasal cavity or middle ear, oral cavity, vulvar, chronic lymphocytic leukemia (CLL), chronic myeloid Cell cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid, head and neck cancer (such as head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer Cancer, leukemia, liver cancer, lung cancer (eg, non-small cell lung cancer), lymphoma, malignant mesothelioma, mast cell tumor,
  • 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 application refers to cancer.
  • the application provides a T cell that is genetically engineered to express CS1-CAR for significantly killing target cells; and a method for preparing the genetically engineered T cell.
  • the engineered T cells are genetically engineered to express CS1-CAR.
  • the present application also provides a T cell that can not only kill tumor cells significantly, but also resist the killing of autologous or allogeneic NK cells.
  • the engineered T cells are genetically engineered to express CS1-CAR, and gene editing techniques are used to knock out endogenous CS1.
  • the engineered T cells are genetically engineered to express CS1-CAR, and gene editing techniques are used to knock out endogenous B2M and TCR.
  • the engineered T cells are genetically engineered to express CS1-CAR, and gene editing techniques are used to knock out endogenous CS1, B2M, and TCR.
  • hSLAMF7-avi-His hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7-huFc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc expression plasmids were transfected into well-growing HEK 293F cells (from Shanghai Cancer Institute), 37°C, 5% CO 2 , 125 rpm shaker for continuous cultivation for 7 days, centrifugation at 4000 rpm for 10 min, remove the precipitate, collect the supernatant, and filter it with a 0.45 ⁇ m filter membrane.
  • the purified recombinant proteins hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7-huFc, h1m2D-SLAMF7-huFc and m1h2D-SLAMF7-huFc were finally obtained by purifying with a chromatography column.
  • mice aged 6-8 weeks Female Balb/c mice aged 6-8 weeks were selected, the immunogen hSLAMF7-avi-His recombinant protein and Freund's complete adjuvant were mixed at 1:1 (volume ratio), and the primary immunization was performed by subcutaneous injection at multiple points. The dosage is 100 ⁇ g/only. Then, the immunogen hSLAMF7-avi-His recombinant protein and incomplete Freund's adjuvant were mixed at 1:1 (volume ratio), followed by subcutaneous injection at multiple points for subsequent immunization, once every two weeks, for a total of 2 immunizations. The dosage is 100 ⁇ g/only. Three days before fusion and spleen harvesting, booster immunization was performed by intraperitoneal injection of 100 ⁇ g/hSLAMF7-avi-His recombinant protein.
  • Splenocytes from immunized mice were isolated and mixed with mouse myeloma cells SP2/0 (ATCC, CRL 1581) at a ratio of 2:1.
  • the isolated splenocytes were mixed with SP2/0 using 50% PEG (Sigma) as published Fusion was performed using standard protocols (Kohler, Milstein, 1975).
  • HAT medium Sigma, H0262
  • ELISA enzyme-linked immunosorbent assay
  • flow cytometry three hybridoma antibodies 32A12MAb (IgG1/k), 37A3MAb (IgG1/k) and 48G9MAb (IgG2b/k) that bind hSLAMF7 were obtained.
  • control antibodies Luc90 and huLuc63 are from patent US9175081B2, Luc90 heavy chain variable region sequence (SEQ ID NO:61), Luc90 light chain variable region sequence (SEQ ID NO:62), heavy chain variable region sequence of huLuc63 ( SEQ ID NO: 63), the light chain variable region sequence of huLuc63 (SEQ ID NO: 64); by conventional recombinant expression technology, a control antibody was prepared for subsequent detection.
  • Example 3 Determination of the binding activity of anti-CS1 hybridoma antibody and recombinant protein hSLAMF7-avi-His by ELISA
  • CS1-positive MM.1S cells human multiple myeloma cells, from the cell bank of the Type Culture Collection, Chinese Academy of Sciences 2 ⁇ 10 5 cells/well in a 96-well round-bottom culture plate, and add purified antibodies after washing.
  • Fig. 2 The results (Fig. 2) showed that the hybridoma antibodies 37A3MAb and 48G9MAb had strong binding ability to MM.1S cells, with EC50 values of 0.05 ⁇ g/ml and 0.18 ⁇ g/ml, respectively, while the hybridoma antibody 32A12MAb had a weaker binding ability to MM.1S cells.
  • the EC50 value was 1.76 ⁇ g/ml.
  • Example 5 Detecting the binding of anti-CS1 hybridoma antibodies to SLAMF7 recombinant proteins of different species by ELISA
  • FIG. 4 The results ( FIG. 4 ) showed that the three CS1 hybridoma antibodies 32A12MAb, 37A3MAb, and 48G9MAb specifically bound to CS1-positive cells (MM.1S, RPMI8226, NCI-H929), but did not bind to CS1-negative cells (HEK293, WI38).
  • hybridoma antibodies 32A12, 37A3 and 48G9 were humanized by the method of CDR transplantation (document Jones et al. 1986). After sequence similarity comparison, the antibody germline with the highest similarity was selected as the antibody template.
  • the germline sequence IGHV3-7*01 from the IMGT database was selected as the antibody template of the 32A12 heavy chain, and the germline sequence IGKV1-9*01 from the IMGT database was selected as the antibody template of the 32A12 light chain.
  • the CDR region of the 32A12 antibody was replaced with the CDR region of the antibody template, and at the same time, the threonine at position 61 of the heavy chain was mutated to alanine to remove the N-glycosylation site, thereby forming the light of the humanized antibody hu32A12. chain (SEQ ID NO:34) and heavy chain (SEQ ID NO:31).
  • the germline sequence IGHV1-69*02 from the IMGT database was selected as the antibody template of the 37A3 heavy chain, and the germline sequence IGKV1-33*01 from the IMGT database was selected as the antibody template of the 37A3 light chain.
  • the CDR regions of the 37A3 antibody were replaced with the CDR regions of the antibody template to form the light chain (SEQ ID NO:38) and heavy chain (SEQ ID NO:36) of the humanized antibody hu37A3.
  • the germline sequence IGHV1-2*02 from the IMGT database was selected as the antibody template of the 48G9 heavy chain, and the germline sequence IGKV1-33*01 from the IMGT database was selected as the antibody template of the 48G9 light chain.
  • the CDR region of the 48G9 antibody was replaced with the CDR region of the antibody template, and at the same time, the serine at position 102 of the heavy chain was mutated to alanine to remove the N-glycosylation site, thereby forming the light chain of the humanized antibody hu48G9 ( SEQ ID NO:43) and heavy chain (SEQ ID NO:40).
  • humanized antibodies hu32A12, hu37A3, and hu48G9 were prepared for subsequent detection.
  • Example 9 Determination of the binding activity of CS1 humanized antibody to recombinant protein hSLAMF7-avi-His by ELISA
  • Example 10 Determination of the binding activity of CS1 humanized antibody to human multiple myeloma cells
  • CS1 humanized antibodies hu37A3 and hu48G9 were obviously bound to CS1-positive cells (MM.1S, NCI-H929), and hu32A12 was weakly bound to CS1-positive cells (MM.1S, NCI-H929). None of the three humanized antibodies bound to CS1 negative cells (HEK293, WI38).
  • the target antibodies hu32A12, hu37A3, and hu48G9 were respectively fused and expressed with mFc (SEQ ID NO: 75) to obtain hu32A12-mFc, hu37A3-mFc, hu48G9-mFc fusion proteins as ligands, and recombinantly expressed huSLAMF7-avi-his or cySLAMF7-huFc 500nM A 3-fold serial dilution was used as the mobile phase. After the detection is completed, use the Evaluation Software to fit the affinity results.
  • Example 14 Analysis of the aggregation of CS1 humanized antibody-Fc fusion protein
  • the humanized single chain antibody fusion proteins hu37A3-mFc, hu48G9-mFc and hu32A12-mFc after affinity chromatography were analyzed by SEC for their aggregation.
  • the column type is XK16/70 (GE Healthcare)
  • the chromatography medium is 120 mL of Superdex200 prep grade
  • the mobile phase is PBS solution
  • the flow rate of 1 mL/min balances the column for more than 1.5 CV until the UV280, conductivity and pH baselines are stable.
  • the sample was loaded with a 0.5mL sample loop, the purification flow rate was 1mL/min, and the peak volume of the monomer peak was about 67mL.
  • VH and VL fragments of the humanized antibodies hu32A12, hu37A3, and hu48G9 constitute hu32A12 scFv, hu37A3 scFv, and hu48G9 scFv.
  • hu 32A12 scFv amino acid sequence SEO ID NO: 50
  • hu 37A3 scFv amino acid sequence SEO ID NO: 51
  • hu 48G9 scFv amino acid sequence SEO ID NO: 52.
  • PRRLSIN-cPPT.EF-1 ⁇ Using PRRLSIN-cPPT.EF-1 ⁇ as a vector, the chimeric antigen receptor sequences shown in Table 7 were inserted to construct the second-generation chimeric antigen receptor expressing humanized antibodies hu32A12, hu37A3, hu48G9, huLuc63, Luc90.
  • Viral plasmids including PRRL-hu32A12 BBz, PRRL-hu37A3 BBz, PRRL-hu48G9 BBz, PRRL-huLuc63 BBz, PRR-Luc90 BBz. The specific method is described in the literature (Berahovich R, 2018).
  • the above lentiviral plasmids were transfected into 293T cells to obtain the corresponding lentiviruses hu32A12 BBz CAR, hu37A3 BBz CAR, hu48G9 BBz CAR, huLuc63 BBz CAR, and Luc90 BBz CAR.
  • CD8 ⁇ signal peptide sequence (SEQ ID NO: 71); scFv (hu32A12) sequence (SEQ ID NO: 50); scFv (hu37A3) sequence (SEQ ID NO: 51); scFv (hu48G9) sequence (SEQ ID NO: 51) 52); huLuc63 VH sequence (SEQ ID NO: 63), (G4S)3linker (SEQ ID NO: 74), huLuc63 VL sequence (SEQ ID NO: 64); Luc90 VH sequence (SEQ ID NO: 61), Luc90 VL sequence (SEQ ID NO:62); CD8 ⁇ hinge region sequence (SEQ ID NO:53); CD8 ⁇ transmembrane domain sequence (SEQ ID NO:54), CD137 intracellular signal domain sequence (SEQ ID NO:55), CD3Zeta intracellular signaling domain sequence (SEQ ID NO:56), hu32A12 BBz sequence (SEQ ID NO:45), hu37A3 BBz
  • T lymphocyte activation PBMC cells were isolated from human peripheral blood, cultured in lymphocyte culture medium at a density of about 1 ⁇ 10 6 cells/mL, and magnetic beads coated with both anti-CD3 and CD28 antibodies were added and the final concentration 300U/mL recombinant human IL-2 was stimulated and cultured at 37°C, 5% CO 2 for 48h;
  • Example 16 T lymphocyte chimeric antigen receptor expression
  • the CAR-T cells prepared in Example 15 were washed and suspended in flow cytometry buffer (phosphate buffered saline (PBS) containing 1 % fetal bovine serum (FBS)) at a cell density of 1x10 cells/well .
  • flow cytometry buffer phosphate buffered saline (PBS) containing 1 % fetal bovine serum (FBS)
  • CS1 antigen labeled with Biotin (10 ⁇ g/mL, diluted with FACS buffer) was added and incubated at 4°C for 45 minutes.
  • Streptavidin-PE Conjugate antibody eBiosciences, 1:200, diluted with FACS buffer
  • the killing effect of CS1 CAR-T cells on tumor cell lines in vitro was detected by lactate dehydrogenase (LDH) assay.
  • LDH lactate dehydrogenase
  • CytoTox Non-Radioactive Cytotoxicity Assay Kit detects cytotoxicity, and the specific steps refer to the kit instructions.
  • Target cells CS1 expressing multiple myeloma cells MM.1S, NCl-H929, RPMI-8226 and HEK293 cells not expressing CS1.
  • Effector cells UTD (virus-uninfected T cells), hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, Luc90 BBz CAR-T cells.
  • the effector cells and target cells were successively plated into 96-well plates (200ul 1640+10% FBS system per well) according to the effector-target ratio of 3:1, 1:1, 1:3 respectively. Co-incubate for 16 hours at °C, 5% CO2 , while setting up spontaneous release wells and maximal release wells according to the instructions. The next day, 20 ⁇ l of lysate was added to the maximum release well, and the reaction was carried out for 30 minutes.
  • Cytotoxicity % [The amount of LDH released by experimental group (Avg.) – The amount of spontaneous LDH released by effector cells (Avg.) – The amount of spontaneous LDH released by target cells (Avg.)]/[The maximum amount of LDH released by target cells (Avg.)- Target cell spontaneous LDH release (Avg.) – volume calibration (Avg.)] ⁇ 100%
  • hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, and hu48G9 BBz CAR-T cells had obvious killing effects on target cells expressing CS1 in vitro, and there was an obvious dose-effect relationship with the effect-target ratio.
  • CS1 CAR T had almost no killing effect at different effector-target ratios (Figure 21). It shows that hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, and hu48G9 BBz CAR-T cells can better specifically recognize and kill CS1-positive tumor cells.
  • Example 18 CS1 CAR T cells induce cytokine release experiment in vitro
  • Example 17 The cytokine secretion of hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, and Luc90 BBz CAR-T cells under stimulation by target cells in Example 17 was further examined .
  • the supernatants of the hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, and Luc90 BBz CAR-T cells incubated with tumor cells in Example 17 were collected, and Cytokine detection was performed according to the instructions of BD TM CBA kit (BD Pharmingen).
  • Cytokine TNF- ⁇ secretion hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, Luc90 BBz CAR-T cells were incubated with MM.1S cells After that, the secretion of each CS1 CAR-T TNF- ⁇ was significantly enhanced (Fig. 23). After co-incubation with HEK293 cells that do not express CS1, each CS1 CAR T has no obvious TNF- ⁇ secretion, indicating that it does not produce non-specific TNF- ⁇ cytokine secretion.
  • Cytokine IL-2 secretion hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, Luc90 BBz CAR-T cells were incubated with MM.1S cells Afterwards, the secretion amount of IL-2 was negatively correlated with the effector-target ratio, and the lower the effector-target ratio was, the higher the IL-2 concentration was, which may be related to the consumption of IL-2 by T cell proliferation (Figure 24).
  • each CS1 CAR-T had no obvious IL-2 secretion, indicating that CS1 CAR-T did not produce non-specific IL-2 secretion after co-incubation with CS1-negative cells.
  • Example 19 Effect of soluble CS1 protein on cytotoxic killing of CS1 CAR T cells in vitro
  • soluble CS1 protein exists in patients with multiple myeloma (MM), and some literature (Mariko Ishibashi, 2018) pointed out that the concentration of soluble CS1 in serum of MM patients is mostly 0.091-14.7ng/mL. Therefore, we added different concentrations of recombinantly expressed CS1 protein (0, 1, 10, 100ng/mL) immediately after adding effector cells and target cells on the basis of the LDH method in Example 17 to detect the toxicity of CAR-T in vitro. Its effect on CS1 CAR T cytotoxic killing.
  • Example 20 Determination of the expression of exhaustion markers in CS1 CAR T cells stimulated by target antigens
  • depletion markers PD-1, TIM-3 and LAG-3 was detected by flow cytometry after CS1 CAR-T was stimulated by the target antigen CS1-positive multiple myeloma cell line NCl-H929.
  • the specific process is as follows: CS1-positive multiple myeloma cell line NCl-H929 (50,000 cells/well, 500 ⁇ l/well) and each CS1 CAR-T (50,000 cells/well, 500 ⁇ l/well) were added to 48-well cell culture Plate, and set UTD (untransfected CAR T cells) as a control at the same time. Incubate for 48 hours in a 37°C, 5% CO2 incubator. The 48-well plate was centrifuged to remove the cells.
  • Cells were washed and transferred to 96-well plates. Divided into two groups, one group added 50 ⁇ l/well PD-1 (BD HorizonTM, 562516)/TIM-3 (BD PharmingenTM, 563422)/CD3 (Invitrogen, 17-0028-42), the other group added LAG-3 (BD HorizonTM, 565720)/CD3 (BD, 1:100, diluted with FACS buffer) antibody mixed solution, incubated at 4°C for 30 minutes in the dark.
  • PD-1 BD HorizonTM, 562516
  • TIM-3 BD PharmingenTM, 563422
  • CD3 Invitrogen, 17-0028-42
  • LAG-3 BD HorizonTM, 565720
  • CD3 CD3
  • the depletion marker PD-1 Compared with UTD, the expression of each CS1 CAR-T PD-1 was significantly up-regulated after target cell stimulation, and the PD-1 expression level of CS1-CAR T provided in this application All were lower than the PD-1 expression level of the control luc90 CAR T ( Figure 26).
  • Tim-3 Compared with UTD, the expression of each CS1 CAR-T Tim-3 was significantly up-regulated after target cell stimulation, and the Tim-3 expression level of CS1-CAR T provided by the application was lower than Control luc90 CAR T ( Figure 27).
  • Depletion marker LAG-3 Compared with UTD, the expression of each CS1 CAR-T LAG-3 was significantly up-regulated after target cell stimulation, and the LAG-3 expression levels of hu37A3 CAR-T and hu48G9 CAR-T were lower than Control luc90 CAR T ( Figure 28).
  • the ratio of CD3- ⁇ phosphorylation in CS1 CAR-T cells was detected by Western-blot method using conventional molecular biology techniques, and the activation of antigen-independent signal transduction pathway was judged.
  • Example 22 Determination of IL-6 secretion after co-incubation of CS1 CAR T cells with monocytes and tumor cells
  • CS1 CAR-T was co-cultured with monocytes and CS1-positive multiple myeloma cells NCl-H929 at a ratio of 1:1:1 to the number of cells for 48 hours, and the culture supernatant was collected and carried out with the BD TM CBA kit according to the instructions. Cytokine detection.
  • Example 23 In vitro specific expansion test of CS1 CAR T cells
  • CS1-CAR-transduced T cells and untransduced CAR-transduced T cells were used as effector cells, which were associated with CS1-positive target cells MM.1S, NCl-H929, RPMI-8226 cells, and CS1-negative target cells, respectively HEK293 was co-incubated with an effector-target ratio of 2:1. The total number of cells was then counted every three days and the CD3 positivity and PI staining viability were determined every three days for 9 days.
  • mice Female NPG mice (from Beijing Weitongda Biotechnology Co., Ltd.) were inoculated with 3 ⁇ 10 6 human multiple myeloma cells MM.1S subcutaneously in the right axilla of 5-6 weeks old, and the tumor inoculation diary was D0.
  • the tumor volume grew to about 180mm 3 , they were randomly divided into 6 groups: UTD control group, hu37A3 CAR-T, and hu48G9 CAR T cell treatment groups, respectively, and the corresponding CAR T cells were injected into the tail vein at a dose of 4 ⁇ 10 6 cells /mouse.
  • Fig. 35 The experimental results (Fig. 35) showed that at D38, compared with UTD, the tumor inhibition rates of each group were hu37A3 CAR-T: 99.9% (4 tumors in 5 mice regressed), and hu48G9 CAR-T: 100% (5 mice). tumor regressed in all mice). It can be seen from the results that both the hu37A3 CAR T group and the hu48G9 CAR T group can significantly inhibit tumors.
  • the extracellular segment of human SLAMF7 (SEQ ID NO: 57) was transferred into RPMI-8226 cells by lentivirus-mediated method to construct a stable RPMI-8226 cell line RPMI-8226-overexpressing CS1 CS1.
  • Group1 Dose 2 ⁇ 10 6 UTD group; Group2: Dose 0.6 ⁇ 10 6 hu37A3 CAR-T group; Group3: Dose 1 ⁇ 10 6 hu37A3 CAR-T group; Group4: Dose 2 ⁇ 10 6 hu37A3 CAR-T group; Group5 : Dose 0.6 ⁇ 10 6 hu48G9 CAR-T group; Group6: Dose 1 ⁇ 10 6 hu48G9 CAR-T group; Group 7: Dose 2 ⁇ 10 6 hu48G9 CAR-T group.
  • Example 25 Preparation of CS1-UCAR-T cells and CS1 knockout UCAR-T cells
  • hu48G9 CAR-T cells were subjected to TRAC and B2M double-gene knockout, or triple-gene knockout (knockout TRAC, B2M and CS1), hu48G9-UCAR-T cells (TRAC and B2M double knockout), hu48G9-UCAR-CS1-/-T cells (TCR, B2M, CS1 triple knockout) were obtained.
  • the gRNA sequences targeting TRAC, B2M and CS1 were synthesized in vitro, wherein the nucleic acid sequence of TRAC-gRNA was shown in SEQ ID NO: 76, and the nucleic acid sequence of B2M-gRNA was shown in SEQ ID NO: 76. As shown in SEQ ID NO:84, the nucleic acid sequence of CS1 gRNA is shown in SEQ ID NO:89.
  • Example 26 Anti-tumor effect of CS1-UCAR-T cells and CS1-UCAR-CS1-/-T cells on subcutaneous transplanted tumors of human multiple myeloma cells RPMI 8226-CS1 in NPG mice
  • mice 5 ⁇ 10 6 human multiple myeloma cells RPMI-8226-CS1 overexpressing CS1 were inoculated subcutaneously in the right axilla of 5-6 week old female NPG mice (from Beijing Weitongda Biotechnology Co., Ltd.), tumor inoculation diary is D0.
  • tumor inoculation diary is D0.
  • the mice were inoculated with CAR T cells, and the mice were randomly divided into 3 groups:
  • Group1 Dose 1 ⁇ 10 6 UTD group
  • Group2 Dose 1 ⁇ 10 6 hu48G9-UCAR-T group
  • Group3 Dose 1 ⁇ 10 6 hu48G9-UCAR-CS1-/-T group;
  • CAR-T cells targeting CS1 can significantly resist the killing effect of NK cells on tumor antigen-targeting CAR-T or universal CAR-T cells, improve autologous or allogeneic immune cells in the presence of host immune cells Persistence and/or graft survival in the presence of tumor antigen-targeting CAR-T or universal CAR-T cells against tumor activity.

Abstract

Provided are an antigen-binding unit capable of targeting CS1, and the use thereof. The present invention relates to an immunoconjugate and a chimeric receptor comprising the CS1-targeting antigen-binding unit, and a nucleic acid encoding the CS1-targeting antigen-binding unit, the immunoconjugate and the chimeric receptor. The present invention further relates to the use of the CS1-targeting antigen-binding unit, the immunoconjugate, the chimeric receptor and a host cell in the treatment of diseases.

Description

CS1工程化细胞及其组合物CS1 engineered cells and compositions thereof
相关申请Related applications
本申请要求于2021年4月2日递交的申请号为202110363275.1的中国申请的优先权。This application claims the priority of the Chinese application with application number 202110363275.1 filed on April 2, 2021.
技术领域technical field
本申请涉及一种能靶向CS1蛋白的抗原结合单元及其应用。The present application relates to an antigen binding unit capable of targeting CS1 protein and its application.
背景技术Background technique
多发性骨髓瘤(MM)是骨髓中浆细胞的癌症，通常，浆细胞产生抗体并在免疫功能中起关键作用。然而，这些细胞的不受控制的生长导致骨痛和骨折、贫血、感染和其他并发症。尽管多发性骨髓瘤的确切原因尚不清楚，但多发性骨髓瘤是第二最常见的血液系统恶性肿瘤，占所有癌症死亡例的2％。MM是一种异质性疾病并且多数是由t(11；14)、t(4；14)、t(8；14)、del(13)、del(17)(除其他外)的染色体易位引起的(Drach等,(1998)Blood 92(3):802-809；Gertz等,(2005)Blood 106(8):2837-2840；Facon等.,(2001)Blood 97(6):1566-1571)。多发性骨髓瘤(MM)的主要病状是骨髓中的浆细胞无限度的扩增和富集，进而导致骨坏死。MM受累患者可能经历因骨髓浸润、骨质破坏、肾衰竭、免疫缺陷以及癌症诊断的心理负担而经历多种与疾病相关的症状。目前主要的治疗方案为化疗和干细胞移植，化疗药物主要是类固醇、沙利度胺、来那度胺、硼替佐米或多种细胞毒素试剂的组合，对于较年轻的患者可采用高剂量化疗理念配合自体干细胞移植。Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow, which normally produce antibodies and play a key role in immune function. However, the uncontrolled growth of these cells leads to bone pain and fractures, anemia, infection and other complications. Although the exact cause of multiple myeloma is unknown, multiple myeloma is the second most common hematological malignancy, accounting for 2% of all cancer deaths. MM is a heterogeneous disease and is mostly caused by chromosomal translocation of t(11;14), t(4;14), t(8;14), del(13), del(17) (among others). Bit induced (Drach et al., (1998) Blood 92(3):802-809; Gertz et al., (2005) Blood 106(8):2837-2840; Facon et al., (2001) Blood 97(6):1566 -1571). The primary pathology of multiple myeloma (MM) is the indefinite expansion and enrichment of plasma cells in the bone marrow, leading to osteonecrosis. Patients affected by MM may experience a variety of disease-related symptoms due to bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychological burden of a cancer diagnosis. At present, the main treatment options are chemotherapy and stem cell transplantation. The chemotherapy drugs are mainly steroids, thalidomide, lenalidomide, bortezomib or a combination of various cytotoxic agents. For younger patients, high-dose chemotherapy can be used. In conjunction with autologous stem cell transplantation.
CS1为淋巴细胞信号活化分子(SLAM家族第七成员，CD319抗原)，是细胞表面糖蛋白的CD2家族的成员。基于CS1在多发性骨髓瘤中的高表达，它被用作制备治疗多发性骨髓瘤及CS1阳性肿瘤抗体或CAR-T细胞的靶点。CS1 is a lymphocyte signaling activation molecule (the seventh member of the SLAM family, CD319 antigen), and is a member of the CD2 family of cell surface glycoproteins. Based on the high expression of CS1 in multiple myeloma, it is used as a target for the preparation of antibodies or CAR-T cells for the treatment of multiple myeloma and CS1-positive tumors.
发明内容SUMMARY OF THE INVENTION
本申请的目的在于提供一种能特异性抑制人多发性骨髓瘤及其他CS1阳性肿瘤细胞生长的抗CS1的抗原结合单元，以及表达含有该抗原结合单元序列的嵌合抗原受体的T免疫细胞(CAR-T)，它们在肿瘤过继免疫治疗中起作用。The purpose of this application is to provide an anti-CS1 antigen-binding unit that can specifically inhibit the growth of human multiple myeloma and other CS1-positive tumor cells, and T immune cells expressing a chimeric antigen receptor containing the antigen-binding unit sequence (CAR-T), they play a role in adoptive immunotherapy of tumors.
本申请提供的技术方案包括：The technical solutions provided in this application include:
第一方面提供一种靶向CS1的抗原结合单元，所述抗原结合单元选自下组：A first aspect provides an antigen binding unit targeting CS1, the antigen binding unit is selected from the group consisting of:
(1)抗原结合单元，其包含重链可变区，所述重链可变区包含SEQ ID NO:3、13或23所示的HCDR1，和/或包含SEQ ID NO:4、14、24或32所示的HCDR2，和/或包含SEQ ID NO:5、15、25或41所示的HCDR3；(2)抗原结合单元，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:8、18或28所示的LCDR1，和/或包含SEQ ID NO:9、19或29所示的LCDR2，和/或包含SEQ ID NO:10、20或30所示的LCDR3；(3)抗原结合单元，包含(1)所述抗原结合单元的重链可变区及(2)所述抗原结合单元的轻链可变区；(4)抗原结合单元，(1)～(3)中任一项所述的抗原结合单元的变体，且具备与(1)～(3)中任一项所述的抗原结合单元相同或相似的活性。(1) an antigen-binding unit comprising a heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 3, 13 or 23, and/or comprising SEQ ID NO: 4, 14, 24 or HCDR2 shown in 32, and/or comprising HCDR3 shown in SEQ ID NO: 5, 15, 25 or 41; (2) an antigen binding unit comprising a light chain variable region comprising LCDR1 set forth in SEQ ID NO: 8, 18 or 28, and/or comprising LCDR2 set forth in SEQ ID NO: 9, 19 or 29, and/or comprising LCDR3 set forth in SEQ ID NO: 10, 20 or 30; (3) an antigen-binding unit comprising (1) a heavy chain variable region of the antigen-binding unit and (2) a light chain variable region of the antigen-binding unit; (4) an antigen-binding unit, (1) to ( 3) A variant of the antigen-binding unit described in any one of (1) to (3), which has the same or similar activity as the antigen-binding unit described in any one of (1) to (3).
在一个具体的实施方式中，所述的抗原结合单元选自：(1)抗原结合单元，其包含SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:8所示的LCDR1、SEQ ID NO:9所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者(2)抗原结合单元，其包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3以及SEQ ID NO:18所示的LCDR1、SEQ ID NO:19所示的LCDR2、SEQ ID NO:20所示的LCDR3；或者(3)抗原结合单元，其包含SEQ ID NO:23所示的HCDR1、SEQ ID NO:24所示的HCDR2、SEQ ID NO:25所示的HCDR3以及SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2、SEQ ID NO:30所示的LCDR3；或者(4)抗原结合单元，其包含SEQ ID NO:3所示的HCDR1、SEQ ID NO:32所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:8所示的LCDR1、SEQ ID NO:9所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者(5)抗原结合单元，其包含SEQ ID NO:23所示的HCDR1、SEQ ID NO:24所示的HCDR2、SEQ ID NO:41所示的HCDR3以及SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2、SEQ ID NO:30所示的LCDR3；(6)抗原结合单元，(1)～(5)中任一项所述的抗原结合单元的变体，且具备与(1)～(5)中任一项所述的抗原结合单元相同或相似的活性。In a specific embodiment, described antigen binding unit is selected from: (1) antigen binding unit, it comprises HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, SEQ ID NO: HCDR3 shown in 5 and LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:9, LCDR3 shown in SEQ ID NO:10; or (2) an antigen binding unit comprising SEQ ID NO: HCDR1 shown in 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15 and LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 19, SEQ ID NO: The LCDR3 shown in 20; or (3) an antigen binding unit comprising HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 25 and SEQ ID NO: LCDR1 shown in 28, LCDR2 shown in SEQ ID NO: 29, LCDR3 shown in SEQ ID NO: 30; or (4) an antigen binding unit comprising HCDR1 shown in SEQ ID NO: 3, SEQ ID NO: HCDR2 shown in 32, HCDR3 shown in SEQ ID NO:5 and LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:9, LCDR3 shown in SEQ ID NO:10; or (5) An antigen binding unit comprising HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 41 and LCDR1 shown in SEQ ID NO: 28, SEQ ID NO: LCDR2 shown in 29, LCDR3 shown in SEQ ID NO: 30; (6) an antigen-binding unit, a variant of the antigen-binding unit described in any one of (1) to (5), and having and (1) The antigen-binding unit of any one of ~(5) has the same or similar activity.
在一个具体的实施方式中，所述的抗原结合单元选自：In a specific embodiment, the antigen binding unit is selected from:
(1)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：1、11、21、31、36或40所示的氨基酸序列；(2)抗原结合单元，所述的抗原结合单元的轻链可变区具有SEQ ID NO：6、16、26、34、38或43所示的氨基酸序列；(3)抗原结合单元，包含(1)所述抗原结合单元的重链可变区及(2)所述抗原结合单元的轻链可变区；(4)抗原结合单元，(1)～(3)中任一项所述的抗原结合单元的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。(1) an antigen-binding unit, the heavy chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 1, 11, 21, 31, 36 or 40; (2) an antigen-binding unit, the The light chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 34, 38 or 43; (3) the antigen-binding unit, comprising (1) the heavy weight of the antigen-binding unit. A chain variable region and (2) a light chain variable region of the antigen-binding unit; (4) an antigen-binding unit, a variant of the antigen-binding unit of any one of (1) to (3), and having The same or similar activity as the antibody described in any one of (1) to (3).
在一个具体的实施方式中，所述的抗原结合单元选自：In a specific embodiment, the antigen binding unit is selected from:
(1)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：1所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：6所示的氨基酸序列；(2)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：11所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：16所示的氨基酸序列；(3)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：26所示的氨基酸序列；(4)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：31所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：34所示的氨基酸序列；(5)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：36所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：38所示的氨基酸序列；(6)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：40所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：43所示的氨基酸序列；(7)抗原结合单元，(1)～(6)中任一项所述的抗原结合单元的变体，且具备与(1)～(6)中任一项所述的抗原结合单元相同或相似的活性。(1) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 6 (2) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 11 and the light chain variable region of the antigen binding unit has SEQ ID NO: : amino acid sequence shown in 16; (3) antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antigen binding unit Has the amino acid sequence shown in SEQ ID NO: 26; (4) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 31 and the light weight of the antigen binding unit The chain variable region has the amino acid sequence shown in SEQ ID NO: 34; (5) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 36 and the antigen The light chain variable region of the binding unit has the amino acid sequence shown in SEQ ID NO: 38; (6) the antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 40 And the light chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 43; (7) the antigen-binding unit, the variant of the antigen-binding unit described in any one of (1) to (6); and has the same or similar activity as the antigen-binding unit described in any one of (1) to (6).
在一个具体的实施方式中，抗原结合单元，与上面任一所述的抗原结合单元识别相同的抗原决定部位；或结合CS1蛋白的Ig-like V-type结构域；或结合CS1蛋白的Ig-like C2-type结构域。In a specific embodiment, the antigen-binding unit recognizes the same epitope as any of the above-mentioned antigen-binding units; or binds to the Ig-like V-type domain of CS1 protein; or binds to the Ig-like V-type domain of CS1 protein like C2-type domain.
在一个具体的实施方式中，所述抗原结合单元是杂交瘤抗体、人源化抗体、嵌合抗体或全人抗体；或者所述抗原结合单元是单克隆抗体；或者所述抗原结合单元是全抗、scFv、Fv片段、Fab片段、Fab’片段、(Fab’) 2片段、Fd片段、dAb片段、单域抗体、多功能抗体或scFv-Fc抗体。 In a specific embodiment, the antigen binding unit is a hybridoma antibody, a humanized antibody, a chimeric antibody or a fully human antibody; or the antigen binding unit is a monoclonal antibody; or the antigen binding unit is a whole Anti, scFv, Fv fragment, Fab fragment, Fab' fragment, (Fab') 2 fragment, Fd fragment, dAb fragment, single domain antibody, multifunctional antibody or scFv-Fc antibody.
第二方面提供一种免疫辍合物，所述免疫辍合物包括：第一方面所述的抗原结合单元；以及与之连接的功能性分子。A second aspect provides an immunoconjugate, the immunoconjugate comprising: the antigen binding unit of the first aspect; and a functional molecule linked thereto.
第三方面提供一种嵌合受体，所述嵌合受体的胞外域包含第一方面所述的抗原结合单元，所述嵌合受体包括：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。A third aspect provides a chimeric receptor, the extracellular domain of the chimeric receptor comprises the antigen-binding unit of the first aspect, and the chimeric receptor includes: chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), or a combination thereof.
在一个具体的实施方式中，所述嵌合受体包含顺序连接的：第一方面所述的抗原结合单元、跨膜区和胞内信号区。In a specific embodiment, the chimeric receptor comprises sequentially linked: the antigen binding unit of the first aspect, a transmembrane region and an intracellular signaling region.
在一个具体的实施方式中，所述嵌合受体的胞内信号区选自：CD3ζ、FcεRIγ、CD27、CD28、CD137、CD134、MyD88、CD40的胞内信号区序列或其组合；和/或所述的跨膜区包含CD8或CD28的跨膜区。In a specific embodiment, the intracellular signal region of the chimeric receptor is selected from the group consisting of: CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, MyD88, CD40 intracellular signal region sequences or combinations thereof; and/or The transmembrane region comprises the transmembrane region of CD8 or CD28.
在一个具体的实施方式中，所述的嵌合受体包括：第一方面所述的抗原结合单元、CD8/CD28的跨膜区和CD3ζ；或第一方面所述的抗原结合单元、CD8/CD28的跨膜区、CD137的胞内信号区和CD3ζ；或第一方面所述的抗原结合单元、CD8/CD28的跨膜区、CD28的胞内信号区和CD3ζ；或第一方面所述的抗原结合单元、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3ζ。In a specific embodiment, the chimeric receptor comprises: the antigen binding unit described in the first aspect, the transmembrane region of CD8/CD28 and CD3ζ; or the antigen binding unit described in the first aspect, CD8/CD28 The transmembrane region of CD28, the intracellular signal region of CD137 and CD3ζ; or the antigen binding unit of the first aspect, the transmembrane region of CD8/CD28, the intracellular signaling region of CD28 and CD3ζ; or the first aspect Antigen binding unit, transmembrane region of CD8/CD28, intracellular signaling region of CD28, CD137 and CD3ζ.
在一个具体的实施方式中，所述的嵌合受体的氨基酸序列如SEQ ID NO：45、46或47所示。In a specific embodiment, the amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 45, 46 or 47.
第四方面提供编码第一方面所述的抗原结合单元、第二方面所述的免疫缀合物、第三方面所述的嵌合受体的核酸。The fourth aspect provides nucleic acids encoding the antigen binding unit of the first aspect, the immunoconjugate of the second aspect, and the chimeric receptor of the third aspect.
第五方面提供一种表达载体，其包含第四方面所述的核酸。The fifth aspect provides an expression vector comprising the nucleic acid of the fourth aspect.
第六方面提供一种病毒，其包含第五方面所述的表达载体。A sixth aspect provides a virus comprising the expression vector of the fifth aspect.
第七方面提供包括第一方面所述的抗原结合单元、第二方面所述的免疫缀合物、和/或第三方面所述的嵌合受体的组合物，其特征在于，所述组合物对表达CS1的细胞具有细胞毒性。The seventh aspect provides a composition comprising the antigen-binding unit of the first aspect, the immunoconjugate of the second aspect, and/or the chimeric receptor of the third aspect, wherein the combination is characterized in that The compound is cytotoxic to cells expressing CS1.
在一个具体的实施方式中，所述表达CS1的细胞是肿瘤细胞和/或病原体细胞。In a specific embodiment, the CS1-expressing cells are tumor cells and/or pathogen cells.
第八方面提供一种宿主细胞，其包含第五方面所述的表达载体或基因组中整合有第四方面所述的核酸。The eighth aspect provides a host cell comprising the expression vector of the fifth aspect or the nucleic acid of the fourth aspect integrated into the genome.
在一个具体的实施方式中，所述的宿主细胞表达第三方面所述的嵌合受体。In a specific embodiment, the host cell expresses the chimeric receptor of the third aspect.
在一个具体的实施方式中，所述宿主细胞包括T细胞、细胞毒性T淋巴细胞、NK细胞、NKT细胞、DNT细胞、调节性T细胞、NK92细胞、干细胞衍生的免疫效应细胞或其组合。In a specific embodiment, the host cells comprise T cells, cytotoxic T lymphocytes, NK cells, NKT cells, DNT cells, regulatory T cells, NK92 cells, stem cell-derived immune effector cells, or a combination thereof.
在一个具体的实施方式中，所述T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细胞为自体或同种异体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。In a specific embodiment, the T cells are T cells derived from natural T cells and/or induced by pluripotent stem cells; preferably, the T cells are autologous or allogeneic T cells; preferably , the T cells are primary T cells; preferably, the T cells are derived from human autologous T cells.
在一个具体的实施方式中，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)、αβT细胞、γδT细胞或其组合。In a specific embodiment, the T cells comprise memory stem-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory cells T cells (Tem), αβ T cells, γδ T cells, or a combination thereof.
在一个具体的实施方式中，所述宿主细胞包含：编码TCR蛋白的基因的敲除和/或内源性TCR分子低表达或不表达，和/或编码MHC蛋白的基因的敲除和/或内源性MHC低表达或不表达。In a specific embodiment, the host cell comprises: knockout of genes encoding TCR proteins and/or low or no expression of endogenous TCR molecules, and/or knockout of genes encoding MHC proteins and/or Endogenous MHC is low or not expressed.
在一个具体的实施方式中，所述的宿主细胞采用CRISPR/Cas9技术敲除内源性MHC分子B2M和内源性TCR。In a specific embodiment, the host cell uses CRISPR/Cas9 technology to knock out the endogenous MHC molecule B2M and endogenous TCR.
在一个具体的实施方式中，所述的宿主细胞敲除B2M所使用的gRNA包括SEQ ID NO:84、85、86和/或87所示序列，敲除TCR使用的gRNA包括SEQ ID NO:76、77、78、79、80、81、82和/或83所示序列。In a specific embodiment, the gRNA used for knocking out B2M in the host cell includes the sequences shown in SEQ ID NO: 84, 85, 86 and/or 87, and the gRNA used for knocking out TCR includes SEQ ID NO: 76 , 77, 78, 79, 80, 81, 82 and/or 83.
在一个具体的实施方式中，所述宿主细胞包含编码CS1蛋白的基因的敲除和/或内源性CS1分子低表达或不表达。In a specific embodiment, the host cell comprises a knockout of a gene encoding a CS1 protein and/or low or no expression of an endogenous CS1 molecule.
在一个具体的实施方式中，采用CRISPR/Cas9技术敲除所述宿主细胞CS1基因，所使用的gRNA选自SEQ ID NO:88、89、90、91、92、93、94和/或95所示序列。In a specific embodiment, CRISPR/Cas9 technology is used to knock out the CS1 gene of the host cell, and the gRNA used is selected from SEQ ID NO: 88, 89, 90, 91, 92, 93, 94 and/or 95. display sequence.
在一个具体的实施方式中，所述宿主细胞结合表达CS1的细胞，不显著结合不表达CS1的细胞。In a specific embodiment, the host cell binds cells that express CS1 and does not significantly bind cells that do not express CS1.
在一个具体的实施方式中，所述的宿主细胞还携带外源的细胞因子的编码序列；或其还表达另一种嵌合受体；或其还表达趋化因子受体；或其还表达安全开关。In a specific embodiment, the host cell also carries an exogenous cytokine coding sequence; or it also expresses another chimeric receptor; or it also expresses a chemokine receptor; or it also expresses Safety switch.
第九方面提供联合用药，第一方面所述的抗原结合单元、第二方面所述的免疫缀合物、第三方面所述的嵌合受体、第七方面所述的组合物、第八方面所述的宿主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；和/或与改善其相关的一种或多种副作用的药剂联合施用；和/或与表达靶向CS1之外的嵌合受体的宿主细胞联合施用。The ninth aspect provides a combination drug, the antigen binding unit described in the first aspect, the immunoconjugate described in the second aspect, the chimeric receptor described in the third aspect, the composition described in the seventh aspect, and the eighth aspect. The host cell of the aspect is administered in combination with an agent that enhances its function, preferably, in combination with a chemotherapeutic agent; and/or in combination with an agent that ameliorates one or more side effects associated therewith; and/or with expression targeting Co-administration of host cells with chimeric receptors other than CS1.
第十方面提供一种制备第一方面所述的抗原结合单元、第二方面所述免疫缀合物、第三方面所述的嵌合受体、和/或第七方面所述的组合物的方法，所述方法包含在适于表达所述抗原结合单元、免疫缀合物、嵌合受体的条件下培养第八方面所述的宿主细胞，以及分离出由所述宿主细胞表达的所述抗原结合单元、免疫缀合物、嵌合受体、和/或组合物。The tenth aspect provides a method for preparing the antigen binding unit of the first aspect, the immunoconjugate of the second aspect, the chimeric receptor of the third aspect, and/or the composition of the seventh aspect. A method comprising culturing the host cell of the eighth aspect under conditions suitable for expression of the antigen binding unit, immunoconjugate, chimeric receptor, and isolating the host cell expressed by the host cell Antigen binding units, immunoconjugates, chimeric receptors, and/or compositions.
第十一方面提供一种药物组合物，其包括：第一方面所述的抗原结合单元或编码该抗原结合单元的核酸；或第二方面所述的免疫辍合物或编码该辍合物的核酸；或第三方面所述的嵌合受体或编码该嵌合受体的核酸；或第八方面所述的宿主细胞；以及任选的，药学上可接受的载体或赋型剂。The eleventh aspect provides a pharmaceutical composition, comprising: the antigen-binding unit described in the first aspect or a nucleic acid encoding the antigen-binding unit; or the immunoconjugate described in the second aspect or a nucleic acid encoding the conjugate nucleic acid; or the chimeric receptor of the third aspect or a nucleic acid encoding the chimeric receptor; or the host cell of the eighth aspect; and optionally, a pharmaceutically acceptable carrier or excipient.
第十二方面提供一种治疗/诊断疾病的方法，其包括向有需要的受试者给予有效量的如第一方面所述的抗原结合单元、或第二方面所述的免疫缀合物、或第三方面所述的宿主细胞、或如第七方面所述的组合物；优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤；优选所述肿瘤是多发性骨髓瘤；优选地，所述受试者是人；优选地，其中所述宿主细胞对于所述受试者是自体的或同种异体的T细胞。A twelfth aspect provides a method of treating/diagnosing a disease, comprising administering to a subject in need thereof an effective amount of the antigen-binding unit of the first aspect, or the immunoconjugate of the second aspect, or the host cell of the third aspect, or the composition of the seventh aspect; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors; preferably the tumor is multiple myeloid tumor; preferably, the subject is a human; preferably, wherein the host cells are autologous or allogeneic T cells to the subject.
第十三方面提供如第一方面所述的抗原结合单元、或第二方面所述的免疫缀合物、或第八方面所述的宿主细胞、或第七方面所述的组合物在治疗和/或诊断疾病中的应用，其特征在于，所述疾病表达CS1；优选地，所述疾病选自炎性病症、感染、自身免疫性疾 病和肿瘤，优选所述肿瘤是多发性骨髓瘤。A thirteenth aspect provides the antigen-binding unit of the first aspect, or the immunoconjugate of the second aspect, or the host cell of the eighth aspect, or the composition of the seventh aspect in the treatment and Use in diagnosing a disease, characterized in that the disease expresses CS1; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors, preferably the tumor is multiple myeloma.
第十四方面提供如第一方面所述的抗原结合单元、或第二方面所述的免疫缀合物、或第八方面所述的宿主细胞、或第七方面所述的组合物用于制备杀伤NK细胞的药物的用途。The fourteenth aspect provides the antigen binding unit of the first aspect, or the immunoconjugate of the second aspect, or the host cell of the eighth aspect, or the composition of the seventh aspect for use in the preparation of Use of a drug that kills NK cells.
在一个具体的实施方式中，所述用途提高自体或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。In a specific embodiment, the use increases the persistence and/or engraftment survival of autologous or allogeneic immune cells in the presence of host immune cells.
本申请还涉及：在一个方面，本申请提供一种靶向CS1的抗原结合单元，所述抗原结合单元选自下组：(1)抗原结合单元，其包含重链可变区，所述重链可变区包含SEQ ID NO:3、13或23所示的HCDR1，和/或包含SEQ ID NO:4、14、24或32所示的HCDR2，和/或包含SEQ ID NO:5、15、25或41中任一所示的HCDR3；(2)抗原结合单元，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:8、18、28所示的LCDR1，和/或包含SEQ ID NO:9、19、29所示的LCDR2，和/或包含SEQ ID NO:10、20或30中任一所示的LCDR3；(3)抗原结合单元，包含(1)所述抗原结合单元的重链可变区及(2)所述抗原结合单元的轻链可变区；(4)抗原结合单元，(1)～(3)中任一项所述的抗原结合单元的变体，且具备与(1)～(3)中任一项所述的抗原结合单元相同或相似的活性。The application also relates to: In one aspect, the application provides an antigen binding unit targeting CS1, the antigen binding unit is selected from the group consisting of: (1) an antigen binding unit comprising a heavy chain variable region, the The chain variable region comprises HCDR1 set forth in SEQ ID NO: 3, 13 or 23, and/or comprises HCDR2 set forth in SEQ ID NO: 4, 14, 24 or 32, and/or comprises SEQ ID NO: 5, 15 HCDR3 shown in any one of , 25 or 41; (2) an antigen binding unit comprising a light chain variable region comprising LCDR1 shown in SEQ ID NOs: 8, 18, 28, and /or comprising the LCDR2 shown in SEQ ID NO: 9, 19, 29, and/or comprising the LCDR3 shown in any one of SEQ ID NO: 10, 20 or 30; (3) an antigen binding unit comprising (1) the The heavy chain variable region of the antigen-binding unit and (2) the light chain variable region of the antigen-binding unit; (4) the antigen-binding unit, the antigen-binding unit of any one of (1) to (3) , and has the same or similar activity as the antigen-binding unit described in any one of (1) to (3).
在具体的实施方式中，所述的抗原结合单元选自：(1)抗原结合单元，其包含SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:8所示的LCDR1、SEQ ID NO:9所示的LCDR2、SEQ ID NO:10所示的LCDR3；(2)抗原结合单元，其包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3以及SEQ ID NO:18所示的LCDR1、SEQ ID NO:19所示的LCDR2、SEQ ID NO:20所示的LCDR3；(3)抗原结合单元，SEQ ID NO:23所示的HCDR1、SEQ ID NO:24所示的HCDR2、SEQ ID NO:25所示的HCDR3以及SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2、SEQ ID NO:30所示的LCDR3；(4)抗原结合单元，其包含SEQ ID NO:3所示的HCDR1、SEQ ID NO:32所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:8所示的LCDR1、SEQ ID NO:9所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者(5)抗原结合单元，其包含SEQ ID NO:23所示的HCDR1、SEQ ID NO:24所示的HCDR2、SEQ ID NO:41所示的HCDR3以及SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2、SEQ ID NO:30所示的LCDR3；(6)抗原结合单元，(1)～(5)中任一项所述的抗原结合单元的变体，且具备与(1)～(5)中任一项所述的抗原结合单元相同或相似的活性。In a specific embodiment, the antigen binding unit is selected from: (1) an antigen binding unit, which comprises HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, SEQ ID NO:5 The HCDR3 shown and the LCDR1 shown in SEQ ID NO:8, the LCDR2 shown in SEQ ID NO:9, and the LCDR3 shown in SEQ ID NO:10; (2) an antigen-binding unit comprising the SEQ ID NO:13 HCDR1 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20 (3) antigen binding unit, HCDR1 shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, HCDR3 shown in SEQ ID NO:25 and LCDR1 shown in SEQ ID NO:28 , LCDR2 shown in SEQ ID NO:29, LCDR3 shown in SEQ ID NO:30; (4) antigen binding unit, which comprises HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:32, HCDR3 shown in SEQ ID NO:5 and LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:9, LCDR3 shown in SEQ ID NO:10; or (5) an antigen binding unit comprising HCDR1 shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, HCDR3 shown in SEQ ID NO:41, LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, LCDR3 shown in SEQ ID NO: 30; (6) an antigen-binding unit, a variant of the antigen-binding unit described in any one of (1) to (5), which has the combination with any one of (1) to (5) One of the antigen binding units described has the same or similar activity.
在具体的实施方式中，所述的抗原结合单元选自：(1)抗原结合单元，所述的抗原结 合单元的重链可变区具有SEQ ID NO：11、11、21、31、36或40所示的氨基酸序列；(2)抗原结合单元，所述的抗原结合单元的轻链可变区具有SEQ ID NO：6、16、26、34、38或43所示的氨基酸序列；(3)抗原结合单元，包含(1)所述抗原结合单元的重链可变区及(2)所述抗原结合单元的轻链可变区；(4)抗原结合单元，(1)～(3)中任一项所述的抗原结合单元的变体，且具备与(1)～(3)中任一项所述的抗原结合单元相同或相似的活性。In a specific embodiment, the antigen-binding unit is selected from: (1) an antigen-binding unit, the heavy chain variable region of the antigen-binding unit has SEQ ID NO: 11, 11, 21, 31, 36 or The amino acid sequence shown in 40; (2) an antigen binding unit, the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 34, 38 or 43; (3 ) an antigen-binding unit, comprising (1) a heavy chain variable region of the antigen-binding unit and (2) a light chain variable region of the antigen-binding unit; (4) an antigen-binding unit, (1) to (3) A variant of the antigen-binding unit according to any one of the above, which has the same or similar activity as the antigen-binding unit according to any one of (1) to (3).
在具体的实施方式中，所述的抗原结合单元选自：(1)抗原结合单元，所述的抗体的重链可变区具有SEQ ID NO：1所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：6所示的氨基酸序列；(2)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：11所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：16所示的氨基酸序列；(3)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：26所示的氨基酸序列；(4)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：31所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：34所示的氨基酸序列；(5)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：36所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：38所示的氨基酸序列；(6)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：40所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：43所示的氨基酸序列；(7)抗原结合单元，(1)～(6)中任一项所述的抗原结合单元的变体，且具备与(1)～(6)中任一项所述的抗原结合单元相同或相似的活性。In a specific embodiment, the antigen binding unit is selected from: (1) an antigen binding unit, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1 and the antigen binding unit The variable region of the light chain has the amino acid sequence shown in SEQ ID NO: 6; (2) the antigen binding unit, the variable region of the heavy chain of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 11 and the The light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 16; (3) the antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 21 amino acid sequence and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 26; (4) antigen binding unit, the heavy chain variable region of the antigen binding unit has SEQ ID NO: The amino acid sequence shown in 31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 34; (5) the antigen binding unit, the heavy chain variable region of the antigen binding unit has The amino acid sequence shown in SEQ ID NO: 36 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 38; (6) an antigen binding unit, the heavy chain of the antigen binding unit The variable region has the amino acid sequence shown in SEQ ID NO: 40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 43; (7) antigen binding unit, (1)～( 6) A variant of the antigen-binding unit of any one of (1) to (6), which has the same or similar activity as the antigen-binding unit of any one of (1) to (6).
在一个方面，本申请提供一种抗原结合单元，其与本申请所述的抗原结合单元识别相同的抗原决定部位；或结合CS1蛋白的Ig-like V-type结构域；或结合CS1蛋白的Ig-like C2-type结构域。In one aspect, the application provides an antigen-binding unit that recognizes the same epitope as the antigen-binding unit described herein; or an Ig-like V-type domain that binds to CS1 protein; or an Ig that binds to CS1 protein -like C2-type domain.
在具体的实施方式中，所述的抗原结合单元是人源化抗体、嵌合抗体或全人抗体；或者所述抗原结合单元是单克隆抗体；或者所述抗原结合单元是scFv、Fv、Fab或(Fab) 2。 In a specific embodiment, the antigen binding unit is a humanized antibody, chimeric antibody or fully human antibody; or the antigen binding unit is a monoclonal antibody; or the antigen binding unit is scFv, Fv, Fab or (Fab) 2 .
在具体的实施方式中，所述的抗原结合单元是人源化的抗体，选自：(1)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：31所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：34所示的氨基酸序列；(2)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：36所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：38所示的氨基酸序列；(3)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：40所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：43所示的氨基酸序列In a specific embodiment, the antigen-binding unit is a humanized antibody selected from: (1) an antigen-binding unit, and the variable region of the heavy chain of the antigen-binding unit is shown in SEQ ID NO: 31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 34; (2) the antigen binding unit, the heavy chain variable region of the antigen binding unit has SEQ ID NO. : the amino acid sequence shown in 36 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 38; (3) the antigen binding unit, the heavy chain variable region of the antigen binding unit Has the amino acid sequence shown in SEQ ID NO: 40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 43
在一个方面，本申请提供编码本申请所述的抗体的核酸。In one aspect, the application provides nucleic acids encoding the antibodies described herein.
在一个方面，本申请提供一种表达载体，其包含本申请所述的核酸。In one aspect, the application provides an expression vector comprising the nucleic acid described herein.
在一个方面，本申请提供一种宿主细胞，其包含本申请所述的表达载体或基因组中整合有本申请所述的核酸。In one aspect, the present application provides a host cell comprising the expression vector described herein or the nucleic acid described herein integrated into the genome.
在一个方面，本申请提供本申请所述的抗体的抗原结合单元，用于制备特异性靶向CS1的肿瘤细胞的靶向性药物，抗体药物偶联物或多功能抗体；或In one aspect, the present application provides an antigen-binding unit of the antibody described in the present application, which is used to prepare a targeted drug, an antibody-drug conjugate or a multifunctional antibody that specifically targets CS1 tumor cells; or
用于制备诊断肿瘤的试剂，该肿瘤表达CS1；或用于制备嵌合抗原受体修饰的免疫细胞；较佳地，所述免疫细胞包括：T淋巴细胞、NK细胞或者NKT淋巴细胞。For preparing a reagent for diagnosing tumors, the tumor expresses CS1; or for preparing immune cells modified with chimeric antigen receptors; preferably, the immune cells include: T lymphocytes, NK cells or NKT lymphocytes.
在一个方面，本申请提供一种多功能免疫辍合物，所述的多功能免疫辍合物包括：In one aspect, the application provides a multifunctional immunoconjugate, the multifunctional immunoconjugate includes:
本申请所述的抗原结合单元；以及与之连接的功能性分子；所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。The antigen binding unit described in this application; and the functional molecule connected to it; the functional molecule is selected from the group consisting of: a molecule targeting tumor surface markers, a tumor-inhibiting molecule, and a surface marker targeting immune cells. Molecular or detectable label.
在具体的实施方式中，所述的抑制肿瘤的分子为抗肿瘤的细胞因子或抗肿瘤的毒素，较佳的，所述的细胞因子包括：IL-12、IL-15、I型干扰素、TNF-alpha。In a specific embodiment, the tumor-inhibiting molecules are anti-tumor cytokines or anti-tumor toxins, preferably, the cytokines include: IL-12, IL-15, type I interferon, TNF-alpha.
在具体的实施方式中，所述的靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志物的抗体或配体；较佳地，所述的免疫细胞表面标志物包括：CD3，CD16，CD28，4-1BB，更佳的，所述的结合免疫细胞表面标志物的抗体是抗CD3抗体。In a specific embodiment, the molecules targeting immune cell surface markers are antibodies or ligands that bind immune cell surface markers; preferably, the immune cell surface markers include: CD3, CD16 , CD28, 4-1BB, more preferably, the antibody that binds to immune cell surface markers is an anti-CD3 antibody.
在具体的实施方式中，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体。In a specific embodiment, the molecule targeting the surface marker of immune cells is an antibody that binds to the surface marker of T cells.
在一个方面，本申请提供编码本申请所述的多功能免疫辍合物的核酸。In one aspect, the application provides nucleic acids encoding the multifunctional immunoconjugates described herein.
在一个方面，本申请提供本申请所述的多功能免疫辍合物的用途，用于制备抗肿瘤药物，或用于制备诊断肿瘤的试剂，该肿瘤表达CS1；或用于制备嵌合抗原受体修饰的免疫细胞；较佳地，所述免疫细胞包括：T淋巴细胞、NK细胞或者NKT淋巴细胞。In one aspect, the present application provides the use of the multifunctional immunoconjugate described in the present application, for the preparation of anti-tumor drugs, or for the preparation of reagents for diagnosing tumors, which express CS1; or for the preparation of chimeric antigen receptors body-modified immune cells; preferably, the immune cells include: T lymphocytes, NK cells or NKT lymphocytes.
在一个面，本申请提供一种嵌合抗原受体，包含胞外域、跨膜域及胞内信号域，所述胞外域包含本申请所述的抗体，该抗体优选单链抗体或结构域抗体。In one aspect, the present application provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular domain comprises the antibody described in the present application, and the antibody is preferably a single-chain antibody or a domain antibody .
在具体的实施方式中，所述胞内信号域包含一个或多个共刺激信号域和/或初级信号域。In specific embodiments, the intracellular signaling domain comprises one or more costimulatory signaling domains and/or primary signaling domains.
在具体的实施方式中，所述嵌合抗原受体还包括铰链域。In specific embodiments, the chimeric antigen receptor further comprises a hinge domain.
在具体的实施方式中，所述的跨膜域选自TCR的alpha、beta、zeta链，CD3ε，CD3ζ，CD4，CD5,CD8α，CD9，CD16，CD22，CD27，CD28，CD33，CD37，CD45，CD64，CD80，CD86，CD134，CD137，CD152，CD154，和PD1的跨膜区；和/或所述共刺激信号域选自CARD11，CD2，CD7，CD27，CD28，CD30，CD40，CD54，CD83，OX40，CD137，CD134，CD150，CD152，CD223，CD270，PD-L2，PD-L1，CD278，DAP10， LAT，NKD2C SLP76，TRIM，FcεRIγ，MyD88，和41BBL的胞内信号区；和/或所述初级信号域选自TCRξ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”)和CD66d，和CD3ζ，更佳的，所述的跨膜域选自CD8α，CD4，CD45，PD1，CD154和CD28的跨膜域；和/或所述共刺激信号域选自CD137，CD134，CD28和OX40；和/或所述初级信号域选自CD3ζ，最优的，所述的跨膜域选自CD8α或CD28，所述共刺激信号域选自CD137或CD28的胞内信号域，所述初级信号域选自CD3ζ。In a specific embodiment, the transmembrane domain is selected from the alpha, beta, zeta chains of TCR, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the transmembrane region of PD1; and/or the costimulatory signaling domain is selected from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, Intracellular signaling regions of OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C SLP76, TRIM, FcεRIγ, MyD88, and 41BBL; and/or the The primary signaling domain is selected from the group consisting of TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS") and CD66d, and CD3ζ, more preferably, the transmembrane domain Transmembrane domains selected from CD8α, CD4, CD45, PD1, CD154 and CD28; and/or said co-stimulatory signaling domains selected from CD137, CD134, CD28 and OX40; and/or said primary signaling domains selected from CD3ζ, most Preferably, the transmembrane domain is selected from CD8α or CD28, the costimulatory signal domain is selected from the intracellular signal domain of CD137 or CD28, and the primary signal domain is selected from CD3ζ.
在具体的实施方式中，所述的嵌合抗原受体包括如下的顺序连接的抗原结合单元，跨膜区和胞内信号区：本申请所述的抗原结合单元、CD8的跨膜区和CD3ζ；本申请所述的抗原结合单元、CD8的跨膜区、CD137的胞内信号区和CD3ζ；本申请所述的抗原结合单元、CD28的跨膜区、CD28的胞内信号区和CD3ζ；或本申请所述的抗原结合单元、CD28的跨膜区、CD28的胞内信号区、CD137和CD3ζ。In a specific embodiment, the chimeric antigen receptor comprises the following sequentially linked antigen binding units, transmembrane regions and intracellular signaling regions: the antigen binding units described in this application, the CD8 transmembrane region and CD3ζ ; the antigen binding unit, the transmembrane region of CD8, the intracellular signal region of CD137 and CD3ζ described in the present application; the antigen binding unit, the transmembrane region of CD28, the intracellular signal region of CD28 and CD3ζ described in the present application; or The antigen binding unit, the transmembrane region of CD28, the intracellular signal region of CD28, CD137 and CD3ζ described in this application.
在具体的实施方式中，所述的嵌合抗原受体的所述胞外域具有SEQ ID NO:50、51或52所示的氨基酸序列；所述跨膜域选自SEQ ID NO：54所示的CD8跨膜域、SEQID NO：72所示的CD28跨膜域；所述共刺激信号域选自SEQ ID NO：73所示的CD28胞内域、SEQ ID NO：56所示的CD3ζ胞内信号结构域和SEQ ID NO：55所示的CD137的胞内域或其混合。In a specific embodiment, the extracellular domain of the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 50, 51 or 52; the transmembrane domain is selected from the group shown in SEQ ID NO: 54 CD8 transmembrane domain, CD28 transmembrane domain shown in SEQ ID NO: 72; the costimulatory signal domain is selected from CD28 intracellular domain shown in SEQ ID NO: 73, CD3ζ intracellular shown in SEQ ID NO: 56 The signaling domain and the intracellular domain of CD137 set forth in SEQ ID NO: 55 or a mixture thereof.
在具体的实施方式中，所述的嵌合抗原受体选自：嵌合抗原受体一，具有SEQ ID NO:50所示的胞外域、SEQ ID NO：53所示的铰链域、SEQID NO：54所示的跨膜域、SEQ ID NO：55所示的共刺激信号域、以及SEQ ID NO：56所示的初级信号域(hu32A12BBz)；或嵌合抗原受体一，具有SEQ ID NO:51所示的胞外域、SEQ ID NO：53所示的铰链域、SEQID NO：54所示的跨膜域、SEQ ID NO：55所示的共刺激信号域、以及SEQ ID NO：56所示的初级信号域(hu37A3BBz)；或嵌合抗原受体一，具有SEQ ID NO:52所示的胞外域、SEQ ID NO：53所示的铰链域、SEQID NO：54所示的跨膜域、SEQ ID NO：55所示的共刺激信号域、以及SEQ ID NO：56所示的初级信号域(hu48G9BBz)。In a specific embodiment, the chimeric antigen receptor is selected from the group consisting of: chimeric antigen receptor one, having the extracellular domain shown in SEQ ID NO:50, the hinge domain shown in SEQ ID NO:53, : a transmembrane domain shown in 54, a costimulatory signaling domain shown in SEQ ID NO: 55, and a primary signaling domain shown in SEQ ID NO: 56 (hu32A12BBz); or a chimeric antigen receptor one with SEQ ID NO : the extracellular domain shown in 51, the hinge domain shown in SEQ ID NO:53, the transmembrane domain shown in SEQ ID NO:54, the costimulatory signal domain shown in SEQ ID NO:55, and the costimulatory signal domain shown in SEQ ID NO:56 The primary signal domain (hu37A3BBz) shown; or the chimeric antigen receptor one, with the extracellular domain shown in SEQ ID NO: 52, the hinge domain shown in SEQ ID NO: 53, and the transmembrane domain shown in SEQ ID NO: 54 , the co-stimulatory signaling domain shown in SEQ ID NO: 55, and the primary signaling domain shown in SEQ ID NO: 56 (hu48G9BBz).
在一个方面，本申请提供编码本申请所述的嵌合抗原受体的核酸。In one aspect, the application provides nucleic acids encoding the chimeric antigen receptors described herein.
在一个方面，本申请提供一种表达载体，其包含本申请所述的核酸。In one aspect, the application provides an expression vector comprising the nucleic acid described herein.
在一个方面，本申请提供一种病毒，所述的病毒包含本申请所述的载体。In one aspect, the present application provides a virus comprising the vector described herein.
在优选的实施方式中，所述病毒是慢病毒。In a preferred embodiment, the virus is a lentivirus.
在一个方面，本申请提供本申请所述的嵌合抗原受体、或本申请所述的核酸、或本申请所述的表达载体、或本申请所述的病毒的用途，用于制备靶向表达CS1的肿瘤细胞的基因修饰的免疫细胞，In one aspect, the present application provides the use of the chimeric antigen receptor described in the present application, or the nucleic acid described in the present application, or the expression vector described in the present application, or the virus described in the present application, for preparing targeted Genetically modified immune cells from tumor cells expressing CS1,
在优选的实施方式中，所述表达CS1的肿瘤是多发性骨髓瘤。In a preferred embodiment, the CS1-expressing tumor is multiple myeloma.
在一个方面，本申请提供一种基因修饰的免疫细胞，其转导有本申请所述的核酸，或本申请所述的表达载体或本申请所述的病毒；或其表达有本申请所述的嵌合抗原受体，In one aspect, the present application provides a genetically modified immune cell transduced with the nucleic acid described in the present application, or the expression vector described in the present application or the virus described in the present application; chimeric antigen receptor,
所述的免疫细胞优选自T淋巴细胞，NK细胞或NKT细胞。Said immune cells are preferably selected from T lymphocytes, NK cells or NKT cells.
在具体的实施方式中，所述基因修饰的免疫细胞还表达有除本申请所述的嵌合抗原受体之外的其他序列，所述其他序列包括细胞因子、或另一种嵌合抗原受体、或趋化因子受体、或降低PD-1表达的siRNA、或阻断PD-L1的蛋白、或TCR、或安全开关；较佳地，所述的细胞因子包括IL-12、IL-15、IL-21、或I型干扰素；较佳地，所述趋化因子受体包括CCR2、CCR5、CXCR2、或CXCR4；较佳地，所述安全开关包括iCaspase-9、Truancated EGFR或RQR8。In a specific embodiment, the genetically modified immune cells also express other sequences other than the chimeric antigen receptors described in the present application, the other sequences include cytokines, or another chimeric antigen receptor body, or chemokine receptor, or siRNA that reduces PD-1 expression, or protein that blocks PD-L1, or TCR, or safety switch; preferably, the cytokines include IL-12, IL- 15. IL-21, or type I interferon; preferably, the chemokine receptor includes CCR2, CCR5, CXCR2, or CXCR4; preferably, the safety switch includes iCaspase-9, Truancated EGFR or RQR8 .
在一个方面，本申请提供本申请所述的基因修饰的免疫细胞的用途，其特征在于，用于制备抑制肿瘤的药物，所述的肿瘤是表达CS1的肿瘤，在优选的实施方式中，所述表达CS1的肿瘤是多发性骨髓瘤。In one aspect, the present application provides the use of the gene-modified immune cells described in the present application, which is characterized in that it is used to prepare a drug for inhibiting tumors, and the tumor is a tumor expressing CS1. In a preferred embodiment, the The CS1-expressing tumor described above is multiple myeloma.
在一个方面，本申请提供一种药物组合物，其包括：本申请所述的抗体或编码该抗体的核酸；或本申请所述的免疫辍合物或编码该辍合物的核酸；或本申请所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或本申请所述的基因修饰的免疫细胞。In one aspect, the present application provides a pharmaceutical composition comprising: the antibody described in the present application or a nucleic acid encoding the antibody; or the immunoconjugate described in the present application or a nucleic acid encoding the conjugate; or the present The chimeric antigen receptor described in the application or the nucleic acid encoding the chimeric antigen receptor; or the genetically modified immune cell described in the application.
在一个方面，本申请提供一种药盒，其包括：容器，以及位于容器中的本申请的药物组合物；或容器，以及位于容器中的本申请的抗体或编码该抗体的核酸；或本申请的免疫辍合物或编码该辍合物的核酸；或本申请的嵌合抗原受体或编码该嵌合抗原受体的核酸；或本申请所述的基因修饰的免疫细胞。In one aspect, the present application provides a kit comprising: a container, and a pharmaceutical composition of the present application in the container; or a container, and an antibody of the present application or a nucleic acid encoding the antibody in the container; or the present The immunoconjugate of the application or the nucleic acid encoding the conjugate; or the chimeric antigen receptor of the application or the nucleic acid encoding the chimeric antigen receptor; or the genetically modified immune cell described in the application.
应理解，在本申请范围内中，本申请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。It should be understood that within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to constitute new or preferred technical solutions. Due to space limitations, it is not repeated here.
附图说明Description of drawings
图1显示了ELISA法测定的CS1杂交瘤抗体32A12MAb、37A3MAb、48G9MAb与重组蛋白hSLAMF7-avi-His的结合情况。Figure 1 shows the binding of CS1 hybridoma antibodies 32A12MAb, 37A3MAb and 48G9MAb to recombinant protein hSLAMF7-avi-His determined by ELISA.
图2显示了CS1杂交瘤抗体与多发性骨髓瘤细胞系MM.1S的结合情况。Figure 2 shows the binding of CS1 hybridoma antibody to the multiple myeloma cell line MM.1S.
图3显示了CS1杂交瘤抗体分别与人、鼠、猴3个种属SLAMF7重组蛋白的结合情况。Figure 3 shows the binding of CS1 hybridoma antibody to SLAMF7 recombinant proteins of human, murine and monkey species, respectively.
图4显示了CS1杂交瘤抗体与MM.1S、NCI H929、RPMI 8226及WI38、HEK293细胞的结合情况。Figure 4 shows the binding of CS1 hybridoma antibody to MM.1S, NCI H929, RPMI 8226 and WI38, HEK293 cells.
图5显示了人源化CS1抗体hu32A12、hu37A3、hu48G9与重组蛋白hSLAMF7-avi-His 的结合情况。Figure 5 shows the binding of humanized CS1 antibodies hu32A12, hu37A3 and hu48G9 to recombinant protein hSLAMF7-avi-His.
图6显示了人源化CS1抗体与多发性骨髓瘤细胞系的结合情况。Figure 6 shows the binding of humanized CS1 antibody to multiple myeloma cell lines.
图7显示了人源化CS1抗体分别与人、鼠、猴3个种属SLAMF7重组蛋白的结合情况。Figure 7 shows the binding of humanized CS1 antibody to SLAMF7 recombinant proteins of human, murine and monkey species, respectively.
图8显示了人源化CS1抗体与MM.1S、NCI H929及WI38、HEK293细胞的结合情况。Figure 8 shows the binding of humanized CS1 antibody to MM.1S, NCI H929 and WI38, HEK293 cells.
图9显示了人源化CS1抗体hu37A3与人SLAMF7的亲和力测定情况。Figure 9 shows the affinity determination of humanized CS1 antibody hu37A3 with human SLAMF7.
图10显示了人源化CS1抗体hu48G9与人SLAMF7的亲和力测定情况。Figure 10 shows the affinity determination of humanized CS1 antibody hu48G9 with human SLAMF7.
图11显示了人源化CS1抗体huLuc63与人SLAMF7的亲和力测定情况。Figure 11 shows the affinity determination of humanized CS1 antibody huLuc63 with human SLAMF7.
图12显示了CS1抗体Luc90与人SLAMF7的亲和力测定情况。Figure 12 shows the affinity determination of CS1 antibody Luc90 with human SLAMF7.
图13显示了人源化CS1抗体hu37A3与猴SLAMF7的亲和力测定情况。Figure 13 shows the affinity determination of humanized CS1 antibody hu37A3 with monkey SLAMF7.
图14显示了人源化CS1抗体hu48G9与猴SLAMF7的亲和力测定情况。Figure 14 shows the affinity determination of humanized CS1 antibody hu48G9 with monkey SLAMF7.
图15显示了人源化CS1抗体hu32A12与人SLAMF7的亲和力测定情况。Figure 15 shows the affinity determination of humanized CS1 antibody hu32A12 with human SLAMF7.
图16显示了SEC测定的人源化CS1抗体hu37A3的聚集结果。Figure 16 shows the aggregation results of humanized CS1 antibody hu37A3 as determined by SEC.
图17显示了SEC测定的人源化CS1抗体hu32A12的聚集结果。Figure 17 shows the aggregation results of humanized CS1 antibody hu32A12 as determined by SEC.
图18显示了SEC测定的人源化CS1抗体hu48G9的聚集结果。Figure 18 shows the aggregation results of the humanized CS1 antibody hu48G9 as determined by SEC.
图19显示了CS1 CAR T细胞CAR的阳性率。Figure 19 shows the positive rate of CS1 CAR T cell CAR.
图20显示了不同时间点的CS 1 CAR T细胞CAR的阳性率。Figure 20 shows the positive rate of CS 1 CAR T cell CAR at different time points.
图21显示了CS1 CAR T在不同效靶比下对CS1表达阳性及阴性细胞的体外杀伤结果。Figure 21 shows the in vitro killing results of CS1 CAR T on CS1 positive and negative cells under different effector-target ratios.
图22显示了CS1 CAR T与CS1表达阳性及阴性靶细胞共孵育后，IFN-γ的分泌情况。Figure 22 shows the secretion of IFN-γ after CS1 CAR T co-incubated with CS1-expressing positive and negative target cells.
图23显示了CS1 CAR T与CS1表达阳性及阴性靶细胞共孵育后，TNF-α的分泌情况。Figure 23 shows the secretion of TNF-α after CS1 CAR T co-incubated with CS1-expressing positive and negative target cells.
图24显示了CS1 CAR T与CS1表达阳性及阴性靶细胞共孵育后，IL-2的分泌情况。Figure 24 shows the secretion of IL-2 after CS1 CAR T co-incubated with CS1-expressing positive and negative target cells.
图25显示了可溶性CS1对CS1 CAR T细胞体外杀伤的影响。Figure 25 shows the effect of soluble CS1 on CS1 CAR T cell killing in vitro.
图26显示了经CS1阳性多发性骨髓瘤细胞刺激后，CS1 CAR T细胞PD-1的表达情况。Figure 26 shows the expression of PD-1 in CS1 CAR T cells after stimulation with CS1-positive multiple myeloma cells.
图27显示了经CS1阳性多发性骨髓瘤细胞刺激后，CS1 CAR T细胞Tim-3的表达情况。Figure 27 shows the expression of Tim-3 in CS1 CAR T cells after stimulation with CS1-positive multiple myeloma cells.
图28显示了经CS1阳性多发性骨髓瘤细胞刺激后，CS1 CAR T细胞LAG-3的表达情况。Figure 28 shows the expression of LAG-3 in CS1 CAR T cells after stimulation with CS1-positive multiple myeloma cells.
图29显示了CS1 CAR T细胞磷酸化CD3-ζ(CAR)的western blot结果。Figure 29 shows western blot results of CS1 CAR T cells phosphorylated CD3-ζ (CAR).
图30显示了CS1 CAR T细胞与单核细胞以及CS1阳性多发性骨髓瘤细胞共孵育后，IL-6的分泌情况。Figure 30 shows the secretion of IL-6 after CS1 CAR T cells co-incubated with monocytes and CS1-positive multiple myeloma cells.
图31显示了经靶细胞刺激后，CS1 CAR T细胞的体外扩增情况。Figure 31 shows the in vitro expansion of CS1 CAR T cells following target cell stimulation.
图32显示了经靶细胞刺激后，CS1 CAR T细胞在不同时间点的活率情况。Figure 32 shows the viability of CS1 CAR T cells at different time points after stimulation with target cells.
图33显示了经靶细胞刺激后，UTD的体外扩增及活率情况。Figure 33 shows the in vitro expansion and viability of UTD after stimulation of target cells.
图34显示了在IL-2刺激下CS1 CAR T细胞的体外扩增及活率情况。Figure 34 shows the in vitro expansion and viability of CS1 CAR T cells under IL-2 stimulation.
图35显示了CS1 CAR T细胞体内治疗多发性骨髓瘤NPG小鼠皮下移植瘤模型中随时间对肿瘤体积的影响及肿瘤照片对比图。Figure 35 shows the effect of CS1 CAR T cells on tumor volume over time in the in vivo treatment of multiple myeloma NPG mouse subcutaneous xenograft tumor model and the comparison of tumor photos.
图36显示了CS1 CAR T细胞对人多发性骨髓瘤细胞RPMI-8226-CS1的NPG小鼠皮下抑制瘤抗肿瘤效果。Figure 36 shows the subcutaneous anti-tumor and anti-tumor effect of CS1 CAR T cells on human multiple myeloma cells RPMI-8226-CS1 in NPG mice.
图37显示了CS1-UCAR-T细胞、CS1-UCAR-CS1-/-T细胞对人多发性骨髓瘤细胞RPMI 8226-CS1的NPG小鼠皮下移植瘤抗肿瘤效果。Figure 37 shows the anti-tumor effect of CS1-UCAR-T cells and CS1-UCAR-CS1-/-T cells on the subcutaneous transplanted tumors of human multiple myeloma cells RPMI 8226-CS1 in NPG mice.
具体实施方式Detailed ways
发明人经过广泛而深入的研究，出乎意料地发现特异性结合CS1的抗原结合单元，这些抗原结合单元可以应用于制备各种靶向性抗肿瘤药物以及诊断肿瘤的药物。在此基础上完成了本申请。After extensive and in-depth research, the inventors unexpectedly found antigen-binding units that specifically bind to CS1, and these antigen-binding units can be used to prepare various targeted antitumor drugs and drugs for tumor diagnosis. This application is completed on this basis.
术语the term
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申请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。Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, where suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, unless otherwise specified, the materials, methods, and examples are illustrative only and not intended to be limiting.
除非另有说明，本申请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)。Unless otherwise indicated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are fully explained in the literature. See, e.g., Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: 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 and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wuetal. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London , 1987); Hand book Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
公开内容中，请求保护的主题的各个方面均以范围形式呈现。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，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述的或中间的值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。所述较小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，除非明确地排除所述范围的上下限。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。Throughout the disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that range. For example, where a range of values is provided, it should be understood that every intervening value between the upper and lower limit of the range, as well as any other stated or intervening value in that range, is included in the claims Within the subject matter, the upper and lower limits of the range also belong to the scope of the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also within the scope of the claimed subject matter, unless the upper and lower limits of the stated ranges are expressly excluded. Where the stated range includes one or both of the limits, the claimed subject matter also includes ranges excluding either or both of the limits. This applies regardless of the width of the range.
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(并描述)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。例如，“约”或“包含”可意指按照在该领域中的实际的标准偏差在1以内或多于1。或者“约”或“包含”可意指至多10％(即±10％)的范围。例如，约5μM可包括在4.5μM与5.5μM之间的任何数目。The term "about" as used herein refers to the usual error range for each value readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself. For example, description of "about X" includes description of "X". For example, "about" or "comprising" may mean within 1 or more than 1 according to the actual standard deviation in the field. Alternatively "about" or "comprising" may mean a range of up to 10% (ie, ±10%). For example, about 5 μM can include any number between 4.5 μM and 5.5 μM.
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。Unless otherwise indicated, 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).
为便于更好地理解本申请，对相关术语定义如下：For a better understanding of this application, the relevant terms are defined as follows:
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、CD8+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是I型跨膜蛋白，胞外段由第23位丝氨酸(S)-第226位甲硫氨酸(M)组成，其包含两个结构域Ig-like V区(23丝氨酸S-124缬氨酸V，远膜端)以及Ig-like C区(131脯氨酸P-206丝氨酸S，近膜端)。示例性，人SLAMF7胞外段的氨基酸序列如SEQ ID NO：57所示，鼠SLAMF7胞外段的氨基酸序列如SEQ ID NO：58所示，食蟹猴SLAMF7胞外段的氨基酸序列如SEQ ID NO：59所示。CS1分子在多发性骨髓瘤 (multiple myeloma,MM)细胞中高表达，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。The term "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+ T cells, activated B cells and mononuclear dendritic cells are basically not expressed in hematopoietic lineage progenitor cells and other human tissues. CS1 is a type I transmembrane protein with an extracellular segment consisting of serine 23 (S) - methionine (M) 226, which contains two domains Ig-like V region (23 serine S-124 Val amino acid V, distal membrane end) and Ig-like C region (131 proline P-206 serine S, proximal membrane end). Exemplarily, the amino acid sequence of the extracellular segment of human SLAMF7 is shown in SEQ ID NO: 57, the amino acid sequence of the extracellular segment of mouse SLAMF7 is shown in SEQ ID NO: 58, and the amino acid sequence of the extracellular segment of cynomolgus monkey SLAMF7 is shown in SEQ ID NO: 58. NO: 59 shown. CS1 molecule is highly expressed in multiple myeloma (MM) cells, and its monoclonal antibody Elotuzumab (huLuc63) has good efficacy in combination with immunomodulators and proteasome inhibitors in the treatment of relapsed or refractory MM patients. , has been approved by the FDA for the treatment of MM.
本申请提供的识别CS1的杂交瘤抗体及其改造后的人源化抗体可用于治疗多发性骨髓瘤。包含本申请制备的CS1抗体的CAR-T细胞在体内外均能显著杀伤多发性骨髓瘤细胞。由于CS1在NK细胞上表达，包含本申请识别CS1抗体的CAR-T细胞能抵抗宿主体内NK细胞对自体或同种异体T细胞的杀伤，从而增加自体或同种异体免疫细胞(示例性的，CS1-CAR-T细胞、CS1-UCAR-T细胞、CS1-UCAR-CS1-/-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。包含本申请制备的CS1抗体，和识别T细胞抗体构成的双特异性抗体，能抵抗宿主体内NK细胞对自体或同种异体T细胞的杀伤，从而增加自体或同种异体免疫细胞(示例性的，CS1-CAR-T细胞、CS1-UCAR-T细胞、CS1-UCAR-CS1-/-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。The hybridoma antibody recognizing CS1 and its modified humanized antibody provided in the present application can be used for the treatment of multiple myeloma. The CAR-T cells containing the CS1 antibody prepared in this application can significantly kill multiple myeloma cells in vitro and in vivo. Since CS1 is expressed on NK cells, CAR-T cells comprising antibodies recognizing CS1 of the present application can resist the killing of autologous or allogeneic T cells by NK cells in the host, thereby increasing autologous or allogeneic immune cells (exemplary, Persistence and/or transplantation survival of CS1-CAR-T cells, CS1-UCAR-T cells, CS1-UCAR-CS1-/-T cells) in the presence of host immune cells. The bispecific antibody comprising the CS1 antibody prepared in the present application and the antibody recognizing T cells can resist the killing of autologous or allogeneic T cells by NK cells in the host, thereby increasing the autologous or allogeneic immune cells (exemplary). , CS1-CAR-T cells, CS1-UCAR-T cells, CS1-UCAR-CS1-/-T cells) persistence and/or transplantation survival in the presence of host immune cells.
术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换使用，指任何长度的氨基酸的聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and refer to polymers of amino acids of any length. The polymer may be linear, cyclic or branched, it may contain modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers such as those that have been processed by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, elimination Amino acid polymers modified by spination, selenylation, transfer-RNA mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation to labeling components. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed from the specified nucleic acid sequence.
术语“抗原结合单元”是指免疫球蛋白分子(或称为“抗体”)和免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。术语“抗原结合单元”中还包括各种物种来源的免疫球蛋白分子，包括无脊椎动物和脊椎动物。结构上，最简单的天然存在的抗体(例如IgG)包含四条多肽链，通过二硫键相互连接的两条重(H)链和两条轻链(L)链。免疫球蛋白代表包括几种类型的分子的一大家族的分子，如IgD、IgG、IgA、IgM和IgE。术语“免疫球蛋白分子”包括例如杂交抗体或改变的抗体及其片段。已经显示抗体的抗原结合功能可以通过天然存在的抗体的片段进行。这些片段统称为“抗原结合单元”。术语“抗原结合单元”中还包括具有与表位吻合并识别表位的特定形状的任何含多肽链的分子结构，其中一个或多个非共价结合相互作用稳定分子结构和表位之间的复合物。所述抗原结合单元的实例包括Fab片段，由VL、VH、CL和CH1结构域组成的单价片段，包含通过铰链区上的二硫桥连接的两个Fab片段的双价片段(F(ab)2片段)；由VH和CH1 结构域组成的Fd片段，由抗体的单臂的VL和VH结构域组成的Fv片段；由VH结构域组成的dAb片段(Ward等人，Nature,341:544-546,1989)；和分离的互补决定区(CDR)或包含这样的抗原结合单元的任何融合蛋白。The term "antigen-binding unit" refers to immunoglobulin molecules (or "antibodies") and immunologically active portions of immunological molecules, ie, molecules that contain an antigen-binding site that specifically binds ("immunoreactively") an antigen. Also included within the term "antigen binding unit" are immunoglobulin molecules derived from various species, including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibodies (eg, IgG) comprise four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Immunoglobulins represent a large family of molecules including several types of molecules, such as IgD, IgG, IgA, IgM and IgE. The term "immunoglobulin molecule" includes, for example, hybrid antibodies or altered antibodies and fragments thereof. It has been shown that the antigen-binding function of antibodies can be carried out by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen binding units". Also included in the term "antigen-binding unit" is any polypeptide chain-containing molecular structure having a specific shape that conforms to and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the relationship between the molecular structure and the epitope. Complex. Examples of such antigen binding units include Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains, bivalent fragments (F(ab) comprising two Fab fragments linked by a disulfide bridge on the hinge region 2 fragments); Fd fragments composed of VH and CH1 domains, Fv fragments composed of the VL and VH domains of the one-armed antibody; dAb fragments composed of VH domains (Ward et al., Nature, 341:544- 546, 1989); and an isolated complementarity determining region (CDR) or any fusion protein comprising such an antigen binding unit.
术语“抗体”在本文中以最广义使用并且包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要其显示所需的抗原结合活性即可。本文中的术语“抗体”指免疫系统的抗原结合蛋白。如本文提到的术语“抗体”包括具有抗原结合区域的完整的全长抗体及其中“抗原结合部分”或“抗原结合区域”保留的其任何片段、或其单链例如单链可变片段(scFv)。“天然抗体”是指天然存在的具有多种结构的免疫球蛋白分子，指包含通过二硫键互联的至少两条重(H)链和两条轻(L)链或其抗原结合片段的糖蛋白。术语“抗体”还包括抗体(特别是本文所述抗体)的所有重组形式，例如在原核细胞中表达的抗体，未糖基化的抗体以及与抗原结合的抗体片段和下文所述的衍生物。每条重链由重链可变区(本文缩写为VH)和重链恒定区组成。每条轻链由轻链可变区(本文缩写为VL)和轻链恒定区组成。术语“可变区或可变结构域”是指参与抗体抗原结合的抗体重链或轻链的结构域。VH和VL可进一步细分为称为互补性决定区(CDR)的高变区，他们散布在称为构架区(FR)的更保守区域中。每条VH和VL由三个CDR和四个FR组成，从氨基端至羧基端通常按以下顺序排列：FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(如效应细胞)和经典补体系统的第一成分(C1q)。单个VH或VL结构域可足以给予抗原结合特异性。此外，结合特定抗原的抗体可以分别使用来自与所述抗原结合的抗体的VH或VL结构域筛选互补VL或VH结构域的文库来分离。参见，例如，Portolano等,J.Immunol.150:880-887(1993)；Clarkson等,Nature352:624-628(1991)。The term "antibody" is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the indicated desired antigen-binding activity. The term "antibody" as used herein refers to an antigen binding protein of the immune system. The term "antibody" as referred to herein includes an intact full-length antibody having an antigen-binding region and any fragment thereof in which the "antigen-binding portion" or "antigen-binding region" remains, or a single chain thereof, such as a single-chain variable fragment ( scFv). "Native antibody" refers to a naturally occurring immunoglobulin molecule with various structures, and refers to a carbohydrate comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding fragments thereof, interconnected by disulfide bonds protein. The term "antibody" also includes all recombinant forms of antibodies, particularly those described herein, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and antigen-binding antibody fragments and derivatives described below. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The term "variable region or variable domain" refers to a domain of an antibody heavy or light chain that is involved in antibody antigen binding. VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, usually arranged in the following order from amino-terminus to carboxy-terminus: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening a library of complementary VL or VH domains using the VH or VL domains, respectively, from antibodies that bind to the antigen. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
术语“高变区”或“互补决定区”或“CDR”是指抗体可变结构域中序列高变和/或形成结构确定的环(“高变环”)和/或含有与抗原接触的残基(“抗原触点”)的各区域。通常，抗体包含六个CDR：VH中的三个(HCDR1，HCDR2，HCDR3)和VL中的三个(LCDR1，LCDR2，LCDR3)。The term "hypervariable region" or "complementarity determining region" or "CDR" refers to an antibody variable domain that is hypervariable in sequence and/or forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting regions of residues ("antigen contacts"). Typically, an antibody contains six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).
抗体片段包括但不限于：(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段(Ward等，1989，Nature 341：544-546)；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点(Bird等，1988，Science 242：423-426；Huston等，1988，Proc.Natl.Acad.Sci. U.S.A 85:5879-5883)；(vii)双特异性单链Fv二聚体(PCT/US92/09965)；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段(Tomlinson等，2000，Methods Enzymol.326:461-479；WO94/13804；Holliger等，1993,Proc.Natl.Acad.Sci.U.S.A 90:6444-6448)；和(ix)与相同或不同抗体遗传融合的scFv(Coloma&Morrison,1997,Nature Biotechnology 15,159-163)。Antibody fragments include, but are not limited to: (i) Fab fragments composed of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments composed of VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) F(ab')2 fragments , a bivalent fragment comprising two linked Fab fragments; (vi) an antigen-binding site for a single-chain Fv molecule (Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc.Natl.Acad.Sci. U.S.A 85:5879-5883); (vii) bispecific single-chain Fv dimers (PCT/US92/09965); (viii) "dimers" or "trimers", multivalent or multimeric constructs constructed by gene fusion specific fragments (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A 90:6444-6448); and (ix) with the same or scFv genetically fused to different antibodies (Coloma & Morrison, 1997, Nature Biotechnology 15, 159-163).
抗体的“分类”是指其重链所具有的恒定结构域或恒定区的类型。主要有五类抗体：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可以被进一步划分成亚类(同种异型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同的类型的免疫球蛋白的重链恒定结构域被分别称为α，δ，ε，γ，和μ。The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (alotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
术语“Fc”或“Fc区”被用于限定含有恒定区的至少一部分的免疫球蛋白重链的C-端区域。该术语包括天然序列Fc区和变体Fc区。The terms "Fc" or "Fc region" are used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.
除非另外指出，在本文中，CDR残基和可变结构域中的其他残基(例如，FR残基)根据以上Kabat等编号。Unless otherwise indicated, herein, CDR residues and other residues in the variable domains (eg, FR residues) are numbered according to Kabat et al. above.
术语“全抗”、“全长抗体”、“完整抗体”可交换使用，是指具有与天然抗体结构基本类似的结构或具有含有如本文中所限定的Fc区的重链或包括具有抗原结合区域的完整的全长抗体。在具体实施例中，本申请提供全长抗体，其重链和轻链可以是全长(例如，抗体可以包括至少一条，优选地两条，完整重链，和至少一条，优选地两条，完整轻链)或可以包括抗原结合部分(Fab、F(ab’)2、Fv或scFv)。在其他实施方案中，抗体重链恒定区选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE。抗体类型的选择将取决于所设计的抗体欲引发的免疫效应子功能。在构建重组免疫球蛋白时，各种免疫球蛋白同种型的恒定区的适宜氨基酸序列和用于产生广泛种类抗体的方法是本领域技术人员已知的。The terms "whole antibody", "full length antibody", "whole antibody" are used interchangeably and refer to having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region as defined herein or including having antigen binding region of the complete full-length antibody. In specific embodiments, the application provides full-length antibodies, the heavy and light chains of which can be full-length (eg, an antibody can include at least one, preferably two, full heavy chains, and at least one, preferably two, whole light chain) or may include an antigen binding portion (Fab, F(ab')2, Fv or scFv). In other embodiments, the antibody heavy chain constant region is selected from, eg, IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE. The choice of antibody type will depend on the immune effector function the antibody is designed to elicit. In the construction of recombinant immunoglobulins, appropriate amino acid sequences for the constant regions of the various immunoglobulin isotypes and methods for generating a wide variety of antibodies are known to those skilled in the art.
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，否则如正如本文中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。在一些实施方案中，针对抗体的VH和VL片段，引入柔性氨基酸(G4S) 3作为接头，构成单链可变片段(scFv)，所述scFv的氨基酸序列赋予分子针对CS1的特异性并且形成本申请的全部抗原结合单元的基础。因此，所述scFv可以用来设计一系列不同“抗体”分子，包括例如全长抗体、其片段如Fab和F(ab’)2、scFv、融合蛋白(包括scFv_Fc)、多价抗体，即具有针对相同抗原或不同抗原的多于一种特异性的抗 体，例如，双特异性T细胞结合抗体(BiTE)、三抗体等(Cuesta等，Multivalent antibodies:when design surpasses evolution,Trends in Biotechnology 28:355-362,2010)。在一些实施方案中，本申请包括具有scFv序列的抗体，所述scFv序列与一个或多个重链恒定区域融合以形成具有人免疫球蛋白Fc区或鼠免疫球蛋白Fc区的抗体以产生双价蛋白，从而增加抗体的总体亲和力和稳定性。此外，Fc部分允许将其他分子(包括但不限于荧光染料、细胞毒素、放射性同位素等)与例如用于抗原定量研究中的抗体直接缀合，以便固定抗体用于亲和力测量、用于定向递送治疗药、使用免疫效应细胞测试Fc介导的细胞毒性和许多其它应用。 The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous ( For example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, a scFv may have the VL and VH variable regions described in any order (eg, with respect to the N-terminus and C-terminus of the polypeptide), the scFv may include a VL-linker-VH or VH-linker-VL can be included. In some embodiments, flexible amino acids (G4S) 3 are introduced as linkers for the VH and VL fragments of the antibody to form a single-chain variable fragment (scFv) whose amino acid sequence confers specificity to the molecule against CS1 and forms the present The basis for all of the application's antigen-binding units. Thus, the scFv can be used to design a range of different "antibody" molecules, including, for example, full-length antibodies, fragments thereof such as Fab and F(ab')2, scFvs, fusion proteins (including scFv-Fc), multivalent antibodies, i.e. with Antibodies with more than one specificity against the same antigen or different antigens, e.g., bispecific T-cell binding antibodies (BiTE), tri-antibodies, etc. (Cuesta et al., Multivalent antibodies: when design surpasses evolution, Trends in Biotechnology 28:355 -362, 2010). In some embodiments, the application includes antibodies having scFv sequences fused to one or more heavy chain constant regions to form antibodies having human immunoglobulin Fc regions or murine immunoglobulin Fc regions to generate dual valent protein, thereby increasing the overall affinity and stability of the antibody. In addition, the Fc portion allows for the direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to antibodies, eg, used in antigen quantification studies, to immobilize antibodies for affinity measurements, for targeted delivery of therapeutics drug, the use of immune effector cells to test Fc-mediated cytotoxicity and many other applications.
术语“单域抗体(Single domain antibody,sdAb)”是指缺失抗体轻链而只有重链可变区的一类抗体，因其分子量小，也被称为纳米抗体(Nanobody)。The term "single domain antibody (sdAb)" refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also called nanobody (Nanobody).
术语“单结构域抗体”是指包含抗体的全部或部分的重链可变结构域或全部或部分的轻链可变结构域。在某些实施方案中，单结构域抗体是人单结构域抗体(Domantis,Inc.,Waltham,MA；参加，例如，美国专利号6248516)。The term "single domain antibody" refers to an antibody comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Pat. No. 6,248,516).
术语“单克隆抗体”、“单抗”是指获自基本上同源的抗体的群体的抗体，即，包括所述群体的个体抗体是相同的和/或结合相同的表位，除可能的变体抗体以外，例如，含天然存在的突变或在单克隆抗体制剂的制备过程中产生，此种变体通常少量存在。对比于多克隆抗体制剂(通常包括针对不同的决定簇(表位)的不同抗体)，单克隆抗体制剂中的各单克隆抗体是针对抗原上的单个决定簇。因此，定于“单克隆”指示抗体的性质为获得自基本上同源的抗体群体，并且不视为要求通过任何特定的方法制备所述抗体。例如，可以通过多种技术制备，包括但不限于杂交瘤方法、重组DNA法、噬菌体展示法，以及利用含有所有或部分的人免疫球蛋白基因座的转基因动物的方法。The terms "monoclonal antibody", "monoclonal antibody" refer to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical and/or bind the same epitope, except where possible In addition to variant antibodies, eg, containing naturally-occurring mutations or produced during the preparation of monoclonal antibody preparations, such variants are usually present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Accordingly, reference to "monoclonal" indicates that the antibody is of the nature obtained from a substantially homogeneous population of antibodies, and is not considered to require that the antibody be prepared by any particular method. For example, it can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals that contain all or a portion of human immunoglobulin loci.
示例性，本申请的单克隆抗体可以由杂交瘤方法生产，杂交瘤可通过分离出受刺激的免疫细胞(诸如来自被接种动物的脾脏的那些)形成。然后可以将这些细胞(诸如骨髓瘤细胞或经转化细胞)与永生化细胞融合，它们能够在细胞培养物中无限复制，由此生产出永生的、分泌免疫球蛋白的细胞系。所利用的永生细胞系被选出(是否缺乏对利用某些营养物所必需的酶)。许多这样的细胞系(诸如骨髓瘤)是本领域技术人员已知的，并且包括例如：胸苷激酶(TK)或次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HGPRT)。这些缺陷允许了：根据融合细胞在例如次黄嘌呤氨基喋呤胸腺嘧啶核苷培养基(HAT)上生长的能力选择出融合细胞。示例性，本申请筛选到3个结合hSLAMF7的杂交瘤抗体32A12、37A3、48G9。Illustratively, the monoclonal antibodies of the present application can be produced by hybridoma methods, which can be formed by isolating stimulated immune cells, such as those from the spleen of a vaccinated animal. These cells, such as myeloma cells or transformed cells, can then be fused with immortalized cells capable of replicating indefinitely in cell culture, thereby producing immortal, immunoglobulin-secreting cell lines. The immortal cell lines utilized are selected (for lack of enzymes necessary to utilize certain nutrients). Many such cell lines, such as myeloma, are known to those of skill in the art and include, for example: thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyltransferase (HGPRT). These deficiencies allow fused cells to be selected on the basis of their ability to grow, for example, on hypoxanthine aminopterin thymidine medium (HAT). Exemplarily, the present application screened 3 hybridoma antibodies 32A12, 37A3, and 48G9 that bind hSLAMF7.
术语“嵌合抗体”是指抗体重链和/或轻链的一部分来源于特定来源或物种，而重链和/或轻链的剩余部分来源于不同的来源或物种的抗体。在某些实施方案中，嵌合抗体包含非人可变区(例如，来源于小鼠、大鼠、仓鼠、兔或非人灵长类动物如猴的可变区)和人恒定区。在另外的实施方案中，嵌合抗体是“类型转换”抗体，其中类型或亚类已经由亲本抗体 的类型或亚类改变。嵌合抗体包括其抗原结合片段。在某些实施方案中，嵌合抗体是“人源化抗体”。The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain of an antibody is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. In certain embodiments, chimeric antibodies comprise non-human variable regions (eg, variable regions derived from mouse, rat, hamster, rabbit, or non-human primates such as monkeys) and human constant regions. In additional embodiments, the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, a chimeric antibody is a "humanized antibody."
术语“人源化”用于非人抗体，例如啮齿动物或灵长类动物等，是含有来源于非人免疫球蛋白的最小序列的杂合免疫球蛋白，免疫球蛋白链或其片段。“人源化抗体”是指一种嵌合抗体，其包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基。在某些实施例中，人源化抗体将包含基本上全部的至少一个(一般为两个)可变结构域，其中所有或基本上所有的CDR对应于非人抗体的CDR，并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。在一些实施方案中，“人源化抗体”可包括突变，例如通过体外随机或定点诱变或通过体内体细胞突变引入的突变。示例性，本申请通过CDR移植的方法对杂交瘤抗体32A12、37A3、48G9进行人源化改造，得到hu32A12、hu37A3、hu48G9。The term "humanized" is used for non-human antibodies, such as rodents or primates, etc., to be hybrid immunoglobulins, immunoglobulin chains or fragments thereof containing minimal sequence derived from non-human immunoglobulins. "Humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one (generally two) variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of the non-human antibody, and all or substantially all of the CDRs correspond to those of the non-human antibody All the FRs above correspond to the FRs of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, "humanized antibodies" may include mutations, such as those introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. Exemplarily, in the present application, the hybridoma antibodies 32A12, 37A3, and 48G9 were humanized by the method of CDR transplantation to obtain hu32A12, hu37A3, and hu48G9.
术语“亲本抗体”或“亲本免疫球蛋白”包括未修饰的抗体，所述抗体之后经修饰产生变体。所述亲本抗体可以使天然存在的抗体，或者天然存在的抗体的变体或改造版本。亲本抗体可以指抗体本身，包含所述亲本抗体的组合物，或其编码氨基酸序列。本文中使用的术语“亲本抗体”或“亲本免疫球蛋白”包括之后经修饰产生人源化抗体的鼠抗体或嵌合抗体。The term "parent antibody" or "parent immunoglobulin" includes unmodified antibodies that have been subsequently modified to produce variants. The parent antibody can be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody. A parent antibody may refer to the antibody itself, a composition comprising said parent antibody, or its encoded amino acid sequence. As used herein, the term "parent antibody" or "parent immunoglobulin" includes murine or chimeric antibodies that are subsequently modified to produce humanized antibodies.
术语“变体抗体”或“抗体变体”包括由于相比亲本的至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。本文中的变体抗体序列优选的具有与亲本抗体序列至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％％、91％、92％、93％、94％、95％％、96％、97％、98％、99％的氨基酸序列同一性。抗体变体可以指抗体本身，包含所述亲本抗体的组合物，或编码其地氨基酸序列。抗体的氨基酸序列变体可以通过向编码所述抗体的核苷酸序列引入合适的修饰或通过肽合成来制备。The terms "variant antibody" or "antibody variant" include antibody sequences that differ from the parent antibody sequence due to at least one amino acid modification compared to the parent. Variant antibody sequences herein preferably have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity. An antibody variant may refer to the antibody itself, a composition comprising the parent antibody, or the amino acid sequence encoding it. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis.
术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”、“氨基酸置换”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸。文中使用的“氨基酸缺失”或“缺失”意指去除亲本多肽序列中特定位置上的氨基酸。可以进行缺失、插入和取代的任意组合以获得最终的构建体，前提是最终的构建体具有所需的特征，例如：结合抗原。The term "amino acid modification" includes amino acid substitutions, additions and/or deletions, "amino acid substitution", "amino acid substitution" means replacing an amino acid at a particular position in the parent polypeptide sequence with another amino acid. "Amino acid insertion" means the addition of an amino acid at a specific position in the parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" means the removal of an amino acid at a particular position in the parent polypeptide sequence. Any combination of deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, eg: binding to antigen.
术语“修饰”一词是指本申请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。The term "modification" refers to a change in the state or structure of a protein or polypeptide of the present application. Modifications can be chemical, structural and functional.
术语“保守修饰”或“保守序列修饰”意指不显著影响或改变含有所述氨基酸序列的抗体的结合特征的氨基酸修饰。此类保守修饰包括氨基酸取代、插入和缺失。可通过本领域已知的标准技术将修饰导入本申请的抗体中，例如定点诱变和PCR介导的诱变。保守的 氨基酸取代是用具有相似侧链的氨基酸残基替换氨基酸残基的取代。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括含碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电的急性侧链(例如，甘氨酸、天冬酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因而，可以用其他相同侧链家族的氨基酸残基替换本申请抗体的CDR区中或框架区中的一个或多个氨基酸残基，并可以测试所改变的抗体(变体抗体)保留的功能。The term "conservative modification" or "conservative sequence modification" means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged acute side chains (eg , glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine) acid, proline, phenylalanine, methionine), beta branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, benzene alanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions or in the framework regions of the antibodies of the present application can be replaced with other amino acid residues of the same side chain family, and the altered antibodies (variant antibodies) can be tested for retained function.
本申请中讨论的所有免疫球蛋白重链恒定区位置，都根据Kabat的EU索引编号(Kabat等，1991，sequences of proteins of immunological interest，第5版，United States Public Health Service,National Institutes of Health,Bethesda，通过引用全文整合)。“Kabat的EU索引”指人IgG1 EU抗体的残基编号，如Edelman等人，1969，Biochemistry 63:78-85所述。All immunoglobulin heavy chain constant region positions discussed in this application are numbered according to the EU index of Kabat (Kabat et al., 1991, sequences of proteins of immunological interest, 5th edition, United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference in its entirety). "Kabat's EU index" refers to the residue numbering of human IgGl EU antibodies, as described by Edelman et al., 1969, Biochemistry 63:78-85.
术语“抗CS1抗体”、“结合CS1的抗体”、“CS1抗体”、“识别CS1的抗体”是指能够以足够的亲和力结合CS1的抗体，所述抗体可用作用于靶向CS1的诊断剂和/或治疗剂。在一个实施方案中，抗CS1抗体与不相关的、非CS1蛋白的结合程度小于所述抗体与CS1的约10％，如通过酶联免疫吸附实验(ELISA)测定。在一些实施例中，本申请靶向CS1的抗原结合单元结合于hCS1胞外结构域的远膜端的Ig-like V-type结构域、或胞外结构域的近膜端Ig-like C2-type结构域。The terms "anti-CS1 antibody", "antibody that binds CS1", "CS1 antibody", "antibody recognizing CS1" refer to an antibody capable of binding CS1 with sufficient affinity for use as a diagnostic agent for targeting CS1 and /or therapeutic agent. In one embodiment, the anti-CS1 antibody binds to an unrelated, non-CS1 protein to less than about 10% of the extent of the antibody to CS1, as determined by an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the antigen binding unit targeting CS1 of the present application binds to the Ig-like V-type domain at the distal membrane end of the extracellular domain of hCS1, or the proximal Ig-like C2-type domain of the extracellular domain. domain.
在本申请中，描述了利用本领域常规杂交瘤抗体制备技术，通过免疫原hSLAMF7-avi-His重组蛋白免疫小鼠获得杂交瘤抗体。还通过CDR移植的方法对杂交瘤抗体进行人源化改造，得到人源化抗体。这些分子展示特异性。具体实施例中，所述抗体识别CS1蛋白。示例性，所述CS1抗体识别表达CS1的细胞，例如肿瘤细胞MM.1S细胞、RPMI 8226细胞、NCI-H929细胞、NK细胞。具体实施例中，所述CS1抗体不识别CS1阴性细胞，例如HEK293、WI38细胞。本申请中如果没有特别说明，本文CS1指人的CS1、鼠的CS1或猴的CS1。In this application, the hybridoma antibody obtained by immunizing mice with the immunogen hSLAMF7-avi-His recombinant protein using conventional hybridoma antibody preparation technology in the art is described. The hybridoma antibody is also humanized by the method of CDR transplantation to obtain a humanized antibody. These molecules exhibit specificity. In a specific embodiment, the antibody recognizes CS1 protein. Exemplarily, the CS1 antibody recognizes cells expressing CS1, such as tumor cells MM.1S cells, RPMI 8226 cells, NCI-H929 cells, NK cells. In a specific embodiment, the CS1 antibody does not recognize CS1 negative cells, such as HEK293, WI38 cells. Unless otherwise specified in this application, CS1 herein refers to human CS1, murine CS1 or monkey CS1.
本文提供的结果突出本申请抗体在靶向CS1时的特异性、灵敏性和效用。The results presented herein highlight the specificity, sensitivity, and utility of the antibodies of the present application in targeting CS1.
本申请提供了识别CS1的抗原结合单元，其包括包含SEQ ID NO：3，13，23任一氨基酸序列的重链CDR1，和/或包含SEQ ID NO：4，14，24，32任一氨基酸序列的重链CDR2，和/或包含SEQ ID NO：5，15，25，41任一氨基酸序列的重链CDR3。在另一个方面，本申请提供了结合CS1的抗原结合单元或其片段，其包括包含SEQ ID NO：8，18，28的氨基酸序列的轻链CDR1，和/或包含SEQ ID NO：9，19，29的氨基酸序列的轻链 CDR2，和/或包含SEQ ID NO：10，20，30任一氨基酸序列的轻链CDR3。在另一方面，本申请提供了结合CS1的抗原结合单元或其片段，其包括包含SEQ ID NO：3，13，23任一氨基酸序列的重链CDR1，和/或包含SEQ ID NO：4，14，24，32任一氨基酸序列的重链CDR2，和/或包含SEQ ID NO：5，15，25，41任一的氨基酸序列的重链CDR3，和/或包含SEQ ID NO：8，18，28的氨基酸序列的轻链CDR1，和/或包含SEQ ID NO：9，19，29的氨基酸序列的轻链CDR2，和/或包含SEQ ID NO：10，20，30任一的氨基酸序列的轻链CDR3。优选的，所述结合CS1的抗体或其片段包括包含SEQ ID NO：3，13，23任一氨基酸序列的重链CDR1，和包含SEQ ID NO：4，14，24，32任一氨基酸序列的重链CDR2，和包含SEQ ID NO：5，15，25，41任一的氨基酸序列的重链CDR3，和/或包含SEQ ID NO：8，18，28的氨基酸序列的轻链CDR1，和包含SEQ ID NO：9，19，29的氨基酸序列的轻链CDR2，和包含SEQ ID NO：10，20，30任一的氨基酸序列的轻链CDR3。更优选的，所述结合CS1的抗体或其片段包括包含SEQ ID NO：3，13，23的氨基酸序列的重链CDR1，和包含SEQ ID NO：4，14，24，32的氨基酸序列的重链CDR2，和包含SEQ ID NO：5，15，25，41任一的氨基酸序列的重链CDR3，和包含SEQ ID NO：8，18，28的氨基酸序列的轻链CDR1，和包含SEQ ID NO：9，19，29的氨基酸序列的轻链CDR2，和包含SEQ ID NO：10，20，30任一的氨基酸序列的轻链CDR3。The present application provides an antigen-binding unit that recognizes CS1, comprising a heavy chain CDR1 comprising the amino acid sequence of any of SEQ ID NOs: 3, 13, 23, and/or comprising any of the amino acids of SEQ ID NO: 4, 14, 24, 32 The heavy chain CDR2 of the sequence, and/or the heavy chain CDR3 comprising the amino acid sequence of any of SEQ ID NOs: 5, 15, 25, 41. In another aspect, the application provides an antigen-binding unit or fragment thereof that binds CS1, comprising a light chain CDR1 comprising the amino acid sequences of SEQ ID NOs: 8, 18, 28, and/or comprising SEQ ID NOs: 9, 19 , the light chain CDR2 of the amino acid sequence of 29, and/or the light chain CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 10, 20, and 30. In another aspect, the application provides an antigen-binding unit or fragment thereof that binds CS1, comprising a heavy chain CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 3, 13, 23, and/or comprising SEQ ID NO: 4, The heavy chain CDR2 of any amino acid sequence of 14, 24, 32, and/or the heavy chain CDR3 comprising the amino acid sequence of any of SEQ ID NOs: 5, 15, 25, 41, and/or the heavy chain CDR3 comprising SEQ ID NO: 8, 18 , the light chain CDR1 of the amino acid sequence of 28, and/or the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, 19, 29, and/or the light chain CDR2 comprising the amino acid sequence of any of SEQ ID NO: 10, 20, 30 Light chain CDR3. Preferably, the antibody or fragment thereof that binds to CS1 comprises the heavy chain CDR1 comprising any of the amino acid sequences of SEQ ID NOs: 3, 13, 23, and the heavy chain CDR1 comprising any of the amino acid sequences of SEQ ID NOs: 4, 14, 24, and 32 Heavy chain CDR2, and heavy chain CDR3 comprising the amino acid sequence of any of SEQ ID NOs: 5, 15, 25, 41, and/or light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, 18, 28, and comprising The light chain CDR2 of the amino acid sequence of SEQ ID NO: 9, 19, 29, and the light chain CDR3 comprising the amino acid sequence of any of SEQ ID NO: 10, 20, 30. More preferably, the antibody or fragment thereof that binds to CS1 comprises the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 3, 13, 23, and the heavy chain comprising the amino acid sequence of SEQ ID NO: 4, 14, 24, 32. Chain CDR2, and heavy chain CDR3 comprising the amino acid sequence of any of SEQ ID NOs: 5, 15, 25, 41, and light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, 18, 28, and comprising SEQ ID NO : the light chain CDR2 of the amino acid sequence of 9, 19, 29, and the light chain CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 10, 20, 30.
在另一方面，本申请提供了识别CS1的抗原结合单元，其包括选自SEQ ID NO：1，11，21，31，36或40的重链可变区序列。In another aspect, the application provides an antigen binding unit recognizing CS1 comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21, 31, 36 or 40.
在另一方面，本申请提供了识别CS1的抗原结合单元，其包括选自SEQ ID NO：6，16，26，34，38或43的轻链可变区序列。In another aspect, the application provides an antigen binding unit recognizing CS1 comprising a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 6, 16, 26, 34, 38 or 43.
考虑到这些重链和轻链可变区序列各自可以识别CS1，可以“混合和匹配”重链和轻链可变区序列来产生本申请的抗CS1的结合分子。Given that each of these heavy and light chain variable region sequences can recognize CS1, the heavy and light chain variable region sequences can be "mixed and matched" to generate the anti-CS1 binding molecules of the present application.
在另一方面，本申请提供了识别CS1的抗原结合单元，其包括选自重链可变区具有SEQ ID NO：1所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：6所示的氨基酸序列；重链可变区具有SEQ ID NO：11所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：16所示的氨基酸序列；重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：26所示的氨基酸序列；重链可变区具有SEQ ID NO：31所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：34所示的氨基酸序列；重链可变区具有SEQ ID NO：36所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：38所示的氨基酸序列；重链可变区具有SEQ ID NO：40所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：43所示的氨基酸序列。In another aspect, the application provides an antigen binding unit that recognizes CS1, comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region of the antigen binding unit having SEQ ID The amino acid sequence shown in NO: 6; the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 11 and the light chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 16; the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 11; The chain variable region has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 26; the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 31 The amino acid sequence shown and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 34; the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 36 and the antigen binding The light chain variable region of the unit has the amino acid sequence shown in SEQ ID NO: 38; the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 40 and the light chain variable region of the antigen binding unit has SEQ ID NO: 40 The amino acid sequence shown in NO:43.
在另一个方面，本申请提供了识别CS1的抗原结合单元的变体。因而本申请提供了抗原结合单元，具有与重链或轻链的可变区序列至少80％相同的重链和/或轻链可变区。优选的，重链和/或轻链可变区的氨基酸序列同一性是至少85％，更优选至少90％，最优选至少95％，特别是96％，更特别97％，甚至更特别98％，最特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。变体可以以本申请所述的抗体为母本抗体，通过酵母库筛选、噬菌体库筛选、点突变等方法得到。In another aspect, the application provides variants of antigen binding units that recognize CS1. The application thus provides antigen binding units having heavy and/or light chain variable regions that are at least 80% identical in sequence to the variable region sequences of the heavy or light chains. Preferably, the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98% , most particularly 99%, including e.g. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. Variants can be obtained by using the antibody described in the present application as the parent antibody by yeast library screening, phage library screening, point mutation and other methods.
在另一个方面，本申请提供了与前述的抗CS1的抗原结合单元识别相同的抗原决定部位的抗原结合单元；或者结合CS1的Ig-like V-type结构域；或结合CS1的Ig-like C2-type结构域。本申请的识别CS1的抗原结合单元均能够特异性的结合CS1阳性细胞，不结合CS1阴性细胞，显示其良好的治疗潜能。本申请的部分识别CS1的抗原结合单元的scFv结构稳定，不容易聚集。In another aspect, the present application provides an antigen-binding unit that recognizes the same epitope as the aforementioned anti-CS1 antigen-binding unit; or an Ig-like V-type domain that binds CS1; or an Ig-like C2 that binds CS1 -type field. All the antigen-binding units that recognize CS1 of the present application can specifically bind to CS1-positive cells, but do not bind to CS1-negative cells, showing their good therapeutic potential. Part of the scFv of the present application that recognizes the antigen-binding unit of CS1 is structurally stable and does not easily aggregate.
在另一个方面，本申请提供了识别CS1的抗原结合单元，所述抗原结合单元是杂交瘤抗体、人源化抗体、嵌合抗体或全人抗体；或者所述抗原结合单元是单克隆抗体；或者所述抗原结合单元是全抗、scFv、单域抗体、Fv片段、Fab片段、Fab’片段、(Fab’)2片段、dAb片段或多功能抗体。In another aspect, the application provides an antigen binding unit that recognizes CS1, the antigen binding unit is a hybridoma antibody, a humanized antibody, a chimeric antibody or a fully human antibody; or the antigen binding unit is a monoclonal antibody; Alternatively the antigen binding unit is a whole antibody, scFv, single domain antibody, Fv fragment, Fab fragment, Fab' fragment, (Fab')2 fragment, dAb fragment or multifunctional antibody.
通过本领域已知的多种测定可以鉴定、筛选本文中提供的抗CS1抗体或表征其物理/化学性质和/或生物学活性。包括例如ELISA、biacore、酶标仪和流式细胞仪分析。合适的测定详细描述在实施例中。The anti-CS1 antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art. These include, for example, ELISA, biacore, microplate reader and flow cytometric analysis. Suitable assays are described in detail in the Examples.
术语“抗原”是指被抗原结合单元识别并特异性结合的物质。抗原可以包括肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合。非限制性示例性抗原包括肿瘤抗原或病原体抗原。“抗原”也可以指引发免疫反应的分子。这种免疫反应可能涉及抗体产生或特定免疫活性细胞(immunologically-competent cells)的活化，或两者兼有。本领域技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽，都可以作为抗原。本申请使用的抗原包括人、小鼠、猴的SLAMF7重组蛋白。The term "antigen" refers to a substance that is recognized and specifically bound by an antigen-binding unit. Antigens can include peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof. Non-limiting exemplary antigens include tumor antigens or pathogen antigens. "Antigen" can also refer to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunologically-competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Antigens used in this application include human, mouse and monkey SLAMF7 recombinant proteins.
术语“亲和力”是指分子(例如：抗体)的单个结合位点及其结合配体(例如：抗原)之间非共价相互作用的力量总和。除非另外指出，如本文中使用的“结合亲和力”是指固有结合亲和力，其反应结合对的成员(例如：抗体和抗原)之间1：1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(Kd)代表。亲和力可以通过本领域中已知的常规方法测量，所述方法包括本文中所述的利用Biacore测定抗体的亲和力。本文中的抗体对CS1的“亲和力”以抗体的KD表示。抗体的KD是指抗体——抗原相互作用的平衡解离常数。抗体结合其抗原的KD值越大，其对所述具体抗原的结合亲和力越弱。示例性，本申请测定 了CS1抗体与不同种属(示例性，人、小鼠、猴的)SLAMF7重组蛋白的亲和力，KD值范围为940pM-107nM。The term "affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the use of Biacore to determine the affinity of an antibody as described herein. The "affinity" of an antibody for CS1 herein is expressed as the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The greater the KD value of an antibody for binding to its antigen, the weaker its binding affinity for that particular antigen. Exemplarily, the present application determined the affinity of CS1 antibodies to SLAMF7 recombinant proteins of different species (exemplarily, human, mouse, monkey), with KD values ranging from 940pM to 107nM.
术语“EC50”，半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。本申请用ELISA法检测了CS1抗体与重组蛋白hSLAMF7-avi-His的EC50值，范围为0.01-0.058μg/ml；用流式细胞仪检测了CS1抗体与CS1阳性细胞MM.1S细胞的EC50值，范围为0.05-1.76μg/ml。The term "EC50", concentration for 50% of maximal effect (EC50), refers to the concentration that elicits 50% of the maximal effect. In this application, the EC50 values of CS1 antibody and recombinant protein hSLAMF7-avi-His were detected by ELISA, in the range of 0.01-0.058 μg/ml; the EC50 values of CS1 antibody and CS1 positive cells MM.1S cells were detected by flow cytometry , in the range of 0.05-1.76 μg/ml.
在一些实施例中，本申请还用酶标仪检测了CS1抗体与不同种属(示例性，人、小鼠、猴的)CS1的结合情况，抗体均与人CS1结合，不与小鼠CS1结合，抗体37A3和48G9还与猴CS1结合。In some embodiments, the present application also uses a microplate reader to detect the binding of CS1 antibodies to CS1 of different species (exemplarily, human, mouse, monkey), and the antibodies all bind to human CS1, but not to mouse CS1 Binding, antibodies 37A3 and 48G9 also bound to monkey CS1.
术语“抗原决定部位”又称“抗原表位”，或“表位”或“抗原决定簇”，包括任何能够被抗体结合的决定簇或区域。抗原表位是抗原中被靶向所述抗原的抗体结合的区域，包括与抗体直接接触的特定氨基酸。示例性，抗原表位可以由CS1蛋白序列的连续序列组成，也可以由CS1蛋白序列不连续的三维结构组成。示例性，本文中使用的抗原是人、鼠或猴的CS1。本申请对CS1抗体与人CS1结合表位进行了分析，得到结合表位在人CS1的Ig-like V-type结构域，远膜端；或在人CS1的Ig-like C2-type结构域，近膜端。The term "antigenic epitope", also known as "antigenic epitope", or "epitope" or "antigenic determinant", includes any determinant or region capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody targeting the antigen and includes specific amino acids that are in direct contact with the antibody. Exemplarily, the epitope may consist of a contiguous sequence of CS1 protein sequences, or it may consist of a three-dimensional structure in which CS1 protein sequences are discontinuous. Exemplarily, the antigen used herein is human, murine or monkey CS1. In this application, the binding epitope of CS1 antibody and human CS1 is analyzed, and the binding epitope is obtained in the Ig-like V-type domain of human CS1, at the far membrane end; or in the Ig-like C2-type domain of human CS1, proximal end.
本申请还提供了免疫缀合物，所述的免疫辍合物包括本文所述的抗体，以及与之连接的功能性分子。所述抗体与所述功能性分子可以通过共价连接、偶联、附着、交联等方式构成缀合物。The application also provides immunoconjugates comprising the antibodies described herein, and functional molecules linked thereto. The antibody and the functional molecule can form a conjugate by covalent connection, coupling, attachment, cross-linking and the like.
所述功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。在一些实施方案中，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗原结合单元(例如抗体)，其与本申请所述的抗原结合单元形成T细胞参与的双功能抗原结合单元(例如双功能抗体)。The functional molecule is selected from the group consisting of: a molecule targeting a tumor surface marker, a tumor-inhibiting molecule, a molecule targeting an immune cell surface marker, or a detectable marker. In some embodiments, the molecule targeting the surface marker of an immune cell is an antigen-binding unit (eg, an antibody) that binds to a T-cell surface marker, which forms a T-cell participation with the antigen-binding unit described herein. Bifunctional antigen binding units (eg, diabodies).
术语“连接”或“融合”在本文中可互换使用。这些术语是指通过包括化学缀合或重组方法的任何手段将两个以上化学元件或组件连接在一起。“框内融合”是指以维持原始开放阅读框(ORF)的正确阅读框的方式连接两个或更多个ORF以形式连续的较长的ORF。因此，所得到的重组融合蛋白是含有两个或更多个片段的单一蛋白质，这些片段对应于由原始ORF编码的多肽(这些片段在自然状态通常不是如此连接)。尽管阅读框因此在整个融合片段中是连续的，但这些片段可以在物理上或空间上通过例如框内连接序列(例如“flexon”)分开。The terms "connected" or "fused" are used interchangeably herein. These terms refer to the joining together of two or more chemical elements or components by any means including chemical conjugation or recombinant methods. An "in-frame fusion" refers to a longer ORF that joins two or more ORFs to form contiguous in a manner that maintains the correct reading frame of the original open reading frame (ORF). Thus, the resulting recombinant fusion protein is a single protein containing two or more fragments corresponding to the polypeptide encoded by the original ORF (the fragments are not normally so linked in their natural state). Although the reading frame is thus contiguous throughout the fusion fragment, the fragments may be physically or spatially separated by, for example, in-frame linking sequences (eg "flexon").
本申请另一方面提供了编码本申请的至少一种抗体、其功能变体或免疫缀合物的核酸分子。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。Another aspect of the present application provides nucleic acid molecules encoding at least one antibody, functional variant or immunoconjugate thereof of the present application. Once the relevant sequences have been obtained, recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
本申请还提供了编码前述抗体的核酸分子，优选的，本申请核酸分子是选自编码重链可变区的SEQ ID NO:2、12、22、33、37或42，和/或选自编码轻链可变区的SEQ ID NO:7、17、27、35、39或44。更优选的，是这样的核酸分子，所述核酸分子包含SEQ ID NO:2的重链可变区序列，和包含SEQ ID NO:7的轻链可变区序列；或者包含SEQ ID NO:12的重链可变区序列，和包含SEQ ID NO:17的轻链可变区序列；或者包含SEQ ID NO:22的重链可变区序列，和包含SEQ ID NO:27的轻链可变区序列；或者包含SEQ ID NO:33的重链可变区序列，和包含SEQ ID NO:35的轻链可变区序列；或者包含SEQ ID NO:37的重链可变区序列，和包含SEQ ID NO:39的轻链可变区序列；或者包含SEQ ID NO:42的重链可变区序列，和包含SEQ ID NO:44的轻链可变区序列。The present application also provides nucleic acid molecules encoding the aforementioned antibodies, preferably, the nucleic acid molecules of the present application are selected from SEQ ID NO: 2, 12, 22, 33, 37 or 42 encoding the variable region of the heavy chain, and/or selected from SEQ ID NO: 7, 17, 27, 35, 39 or 44 encoding the light chain variable region. More preferably, it is such a nucleic acid molecule comprising the heavy chain variable region sequence of SEQ ID NO:2, and the light chain variable region sequence comprising SEQ ID NO:7; or comprising SEQ ID NO:12 The heavy chain variable region sequence, and the light chain variable region sequence comprising SEQ ID NO:17; or the heavy chain variable region sequence comprising SEQ ID NO:22, and the light chain variable region comprising SEQ ID NO:27 region sequence; or the heavy chain variable region sequence comprising SEQ ID NO:33, and the light chain variable region sequence comprising SEQ ID NO:35; or the heavy chain variable region sequence comprising SEQ ID NO:37, and comprising The light chain variable region sequence of SEQ ID NO:39; or the heavy chain variable region sequence comprising SEQ ID NO:42, and the light chain variable region sequence comprising SEQ ID NO:44.
在一个实施方案中，提供一种或多种包含上述核酸的载体(例如，表达载体)。In one embodiment, one or more vectors (eg, expression vectors) comprising the above-described nucleic acids are provided.
本申请还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。The present application also relates to vectors comprising the appropriate DNA sequences described above together with appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins. Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
术语“细胞”指人或非人的、来源于动物的细胞。在一实施例中，所述工程细胞或工程化细胞指表达CS1-CAR的T细胞。The term "cell" refers to cells of animal origin, human or non-human. In one embodiment, the engineered cells or engineered cells refer to T cells expressing CS1-CAR.
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。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“患者”是患有疾病、病症或病况或者有患疾病、病症或病况的风险或者在其他方面需要本文提供的组合物和方法的受试者。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. In some embodiments, 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. In some embodiments, the subject is or may be suffering from an immune disease, such as an autoimmune disease, or following treatment with a transplant. The term "patient" is a subject suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
术语“宿主细胞”指被引入外源核酸的细胞，包括此种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括转化的原代细胞及来源于其的后代(不考虑传代次数)。后代的核酸内容可以与亲本细胞不完全相同，并且可以含有突变。本文中包括具有与对于原始转化的细胞中筛选或选择的相同的功能或生物学活性的突变体后代。The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include transformed primary cells and progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be identical to the parental cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.
在一个实施例中，提供制备抗CS1抗体的方法，其中所述方法包括在适合于表达如上所述的抗体的条件下培养包含编码所述抗体的核酸的宿主细胞，和任选地从宿主细胞(或宿主细胞培养基)回收抗体。In one embodiment, a method of making an anti-CS1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expressing the antibody as described above, and optionally extracting the antibody from the host cell (or host cell culture medium) to recover the antibody.
在一实施方案中，提供包含编码上述抗体的核酸的宿主细胞。宿主细胞包含(例如，转导有)：(1)载体，所述载体包含核酸，所述核酸编码包含抗体VL的氨基酸序列和包含 抗体VH的氨基酸序列，或(2)包含编码包含抗体VL的氨基酸序列的核酸的第一载体，和包含编码包含抗体VH的氨基酸序列的核酸的第二载体。在一个实施方案中，宿主细胞是真核细胞，例如，293T细胞。In one embodiment, host cells comprising nucleic acids encoding the above-described antibodies are provided. The host cell comprises (e.g., transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of the antibody, or (2) comprising a nucleic acid encoding a VL comprising the antibody A first vector of nucleic acid comprising an amino acid sequence, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is a eukaryotic cell, eg, a 293T cell.
在另一实施方案中，所述宿主细胞还可以表达趋化因子受体。In another embodiment, the host cell may also express a chemokine receptor.
在另一实施方案中，所述宿主细胞还可以表达安全开关。In another embodiment, the host cell may also express a safety switch.
在一优选例中，所述宿主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；和/或所述宿主细胞与改善其相关的一种或多种副作用的药剂联合施用；和/或所述宿主细胞与表达靶向CS1之外的嵌合抗原受体的宿主细胞联合施用。In a preferred embodiment, the host cell is administered in combination with an agent that enhances its function, preferably, in combination with a chemotherapeutic drug; and/or the host cell is administered in combination with an agent that improves one or more side effects associated therewith and/or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor targeting a chimeric antigen other than CS1.
在一些实施方案中，所述宿主细胞是免疫效应细胞。In some embodiments, the host cell is an immune effector cell.
术语“免疫效应细胞”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，所述的免疫效应细胞为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细胞可以是自体NK细胞或同种异体NK细胞。术语“CIK细胞“，即细胞因子诱导的杀伤细胞(Cytokine-Induced Killer，CIK)是一种新型的免疫活性细胞，CIK增殖能力强，细胞毒作用强，具有一定的免疫特性。由于该细胞同时表达CD3和CD56两种膜蛋白分子，故又称为NK细胞(自然杀伤细胞)样T淋巴细胞，兼具有T淋巴细胞强大的抗瘤活性，和NK细胞的非MHC限制性杀瘤优点。The term "immune effector cell" refers to a cell involved in an immune response that produces an immune effector, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages , mast cells, etc. In some embodiments, the immune effector cells are T cells, NK cells, NKT cells. In some embodiments, the T cells can be autologous T cells, xenogeneic T cells, allogeneic T cells. In some embodiments, the NK cells may be autologous NK cells or allogeneic NK cells. The term "CIK cells", that is, cytokine-induced killer cells (Cytokine-Induced Killer, CIK) is a new type of immune active cells, CIK has strong proliferation ability, strong cytotoxicity, and has certain immune characteristics. Because this cell expresses both CD3 and CD56 membrane protein molecules, it is also called NK cell (natural killer cell)-like T lymphocyte, which has both strong antitumor activity of T lymphocyte and non-MHC restriction of NK cell. Tumor-killing advantages.
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。The term "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. For example, 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.
本文所述的T细胞可以从许多来源获得，包括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、脾组织和肿瘤的组织获取的天然的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αβT细胞、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，从个体收集的血液获得T细胞。T细胞可以是任何类型的T细胞，可以是任何发育阶段，包括但不限于CD4+/CD8+双阳性T细胞、CD4+辅助T细胞，例如Th1和Th2细胞、CD8+T细胞(例如细胞毒T细胞)、肿瘤浸润细 胞、记忆T细胞、天真T细胞等。T细胞可能是CD8+T细胞或CD4+T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。在一个实施方案中，可以从健康供体，或来自诊断患有癌症个体的衍生细胞。The T cells described herein can be obtained from a number of sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and naive T cells obtained from tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors, and also It can be a cell population with specific phenotypic characteristics obtained by sorting, etc., or a mixed cell population with different phenotypic characteristics, such as "T cells" can be cells that contain at least one T cell subset: memory stem cell-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). In some instances, a "T cell" may be a specific subtype of T cell, such as αβ T cells, γδ T cells. In some cases, 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 Ficoll™ separation. T cells can be of any type and of any developmental stage, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells such as Th1 and Th2 cells, CD8+ T cells (e.g. cytotoxic T cells) , tumor infiltrating cells, memory T cells, naive T cells, etc. The T cells may be CD8+ T cells or CD4+ T cells. In one embodiment, 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. In one embodiment, cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps. In one embodiment, cells can be derived from a healthy donor, or from an individual diagnosed with cancer.
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。The terms "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. For example, the term "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. "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. Using CD3/CD28 magnetic beads, antigen stimulation in vitro or in vivo will affect the degree and duration of T cell activation. In one embodiment, the engineered T cells are co-incubated with tumor cells containing a specific target antigen or activated after viral infection.
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。The term "peripheral blood mononuclear cells" (PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, and the like.
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。The term "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). As used herein, 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. In one embodiment, 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 inducing the expression of certain regulatory genes or by exogenously applying certain proteins.
“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。"Pluripotent stem cell characteristics" refers to cell characteristics that distinguish pluripotent stem cells from other cells. For example, 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.
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。在一个实施例中，所述工程化是指核酸的一个或多个改变，例如生物体基因组内的核酸。在一个实施例中，所述工程化是指基因的改变、添加和/或缺失。在一个实施例中，所述工程细胞或所述工程化细胞还可以指具有加入、缺失和/或改变的基因的细胞。The term "engineering" 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. A comprehensive science and technology of products. In one embodiment, the engineering refers to one or more alterations of nucleic acids, such as nucleic acids within the genome of an organism. In one embodiment, the engineering refers to changes, additions and/or deletions of genes. In one embodiment, the engineered cell or the engineered cell may also refer to a cell with added, deleted and/or altered genes.
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。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 noncoding 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. In some embodiments, 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 chimeric antigen receptors (CARs) or T cell receptors (TCRs), which can be integrated into the cell genome.
所述“TCR沉默”是指内源性的TCR不表达或低表达。The "TCR silencing" refers to no or low expression of endogenous TCR.
所述“MHC沉默”是指内源性的MHC不表达或低表达。The "MHC silencing" refers to no or low expression of endogenous MHC.
本申请所述“低表达”是工程化细胞中的目标基因表达的蛋白质和/或RNA水平低于细胞工程化处理之前的表达水平。具体实施例中，B2M或TCR或CS1的低表达是指细胞中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％。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或TCR或CS1的特异性抗体测定细胞中蛋白的表达或含量。"Low expression" as used herein means that the protein and/or RNA level of the target gene expressed in the engineered cell is lower than the expression level before the cell engineering treatment. In specific embodiments, low expression of B2M or TCR or CS1 refers to a decrease in 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%. 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 or TCR or CS1.
术语“B2M”为β-2微球蛋白，也称为B2M，是MHC I类分子的轻链。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。The term "B2M" is beta-2 microglobulin, also known as B2M, the light chain of an MHC class I molecule. Playing an important role in the transplant response, rejection is mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.
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。TCR分为两类：TCR1和TCR2；TCR1由γ和δ两条链组成，TCR2由α和β两条链组成。术语“TRAC”是指TCRα链的恒定区。The term "T cell receptor (TCR)" mediates T cell recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens, including classical TCR receptors and optimized TCR receptors. body. TCRs are divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, γ and δ, and TCR2 is composed of two chains, α and β. The term "TRAC" refers to the constant region of the TCRα chain.
术语“基因编辑”，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，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。The term "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 to alter DNA sequences. This technique is sometimes called "gene clipping" or "genome engineering." Gene editing can be used to achieve precise and efficient gene knockout or gene knock-in.
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、Base Editor技术、引导编辑技术和/或归巢核酸内切酶技术。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. In a preferred embodiment, 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, Base Editor technology, guide editing technology and/or homing endonuclease technology.
本申请一个实施例采用CRISPR/Cas9技术制备得到UCAR-T细胞。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。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杂交促进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。一般而言，指导序列(gRNA)是与靶多核苷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苷酸序列。本申请凡涉及gRNA的序列时，其可以为靶向的DNA序列，亦可以为所述DNA对应的核糖核苷酸与crRNA、TracrRNA形成的完整Cas9引导序列。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。术语“sgRNA”指短小的gRNA。An example of this application uses CRISPR/Cas9 technology to prepare UCAR-T cells. The term "CRISPR (Clustered regularly interspaced short palindromicrepeats)" refers to clustered regularly interspaced short palindromic repeats. The term "Cas9 (CRISPR associated nuclease)" is a CRISPR-associated nuclease, an RNA-guided technology that uses Cas9 nuclease to edit targeted genes. In the context of CRISPR complex formation, "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. After the CRISPR complex is formed, under the action of the cas9 enzyme, it can cut specific sites in the genome to introduce gene mutations; it can also regulate gene expression, such as activation or inhibition. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In general, a guide sequence (gRNA) is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. When the application involves the sequence of gRNA, it can be a targeted DNA sequence, or it can also be a complete Cas9 guide sequence formed by the ribonucleotides corresponding to the DNA, crRNA and TracrRNA. In some embodiments, 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. The term "sgRNA" refers to short gRNAs.
CRISPR/Cas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。CRISPR/Cas transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.
本申请通过敲除基因TRAC、B2M获得通用型T细胞或通用型CAR-T细胞。在一实施例中，分别对在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。在一个实施例中，敲除TCR使用的gRNA选自SEQ ID NO:76、77、78、79、80、81、82和/或83所示的序列。在一实施例中，采用CRISPR/Cas9技术敲除所述工程化T细胞B2M基因，使用的gRNA选自SEQ ID NO:84、85、86和/或87所示的序列。This application obtains universal T cells or universal CAR-T cells by knocking out the genes TRAC and B2M. In one embodiment, the exons of the corresponding genes encoding the constant regions of one or both of the alpha and beta chains of B2M, TCR are knocked out using CRISPR/Cas technology, respectively. In one embodiment, the gRNA used for knocking out TCR is selected from the sequences shown in SEQ ID NO: 76, 77, 78, 79, 80, 81, 82 and/or 83. In one embodiment, the engineered T cell B2M gene is knocked out using CRISPR/Cas9 technology, and the gRNA used is selected from the sequences shown in SEQ ID NOs: 84, 85, 86 and/or 87.
在一个实施例中，敲除细胞CS1基因，减少CS1-CAR-T细胞对自身的杀伤。示例性，采用CRISPR/Cas9技术敲除CS1基因，使用的gRNA选自SEQ ID NO:88、89、90、91、92、93、94和/或95所示的序列。In one embodiment, knocking out the cellular CS1 gene reduces self-killing by CS1-CAR-T cells. Exemplarily, the CS1 gene is knocked out using CRISPR/Cas9 technology, and the gRNA used is selected from the sequences shown in SEQ ID NOs: 88, 89, 90, 91, 92, 93, 94 and/or 95.
“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％。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或TCR或CS1的特异性抗体测定细胞中蛋白的表达或含量。"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%. 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 or TCR or CS1.
本申请在一实施例中，先构建表达特异性CAR-T细胞，再利用CRISPER/Cas9技术 敲除CAR-T细胞内源性TRAC、B2M和/或CS1构建对应的UCAR-T。在一个实施例中，先利用CRISPER/Cas9技术敲除内源性TRAC、B2M和/或CS1构建通用型T细胞，再表达特异性CAR构建UCAR-T细胞。在一个实施例中，CRISPER/Cas9技术敲除内源性TRAC,B2M和/或CS1和表达特异性CAR同时操作构建UCAR-T细胞。In one embodiment of the present application, specific CAR-T cells are constructed first, and then CRISPER/Cas9 technology is used to knock out the endogenous TRAC, B2M and/or CS1 of the CAR-T cells to construct the corresponding UCAR-T. In one embodiment, CRISPER/Cas9 technology is used to knock out endogenous TRAC, B2M and/or CS1 to construct universal T cells, and then express specific CAR to construct UCAR-T cells. In one embodiment, CRISPER/Cas9 technology knocks out endogenous TRAC, B2M and/or CS1 and expresses specific CAR simultaneously to construct UCAR-T cells.
术语“转染”是指外源性核酸通过其转移或引入到宿主细胞中的过程。“转染的”或“转化的”或“转导的”细胞是已经用外源性核酸转染、转化或转导的细胞。所述细胞包括原发性受试者细胞及其后代。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)。The term "transfection" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cells include primary subject cells and their progeny. 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.
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。The terms "nucleic acid molecule encoding", "encoding DNA sequence" and "encoding DNA" refer 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.
当用于指核苷酸序列或多核苷酸序列时，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。As used herein, the term "sequence" when used in reference to a nucleotide sequence or a polynucleotide sequence may include DNA or RNA, and may be single-stranded or double-stranded.
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。The term sequence "identity" as used herein 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.
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苷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)。为了表达蛋白质，可以将编码本申请抗原结合单元的核酸整合到表达载体中。用于本申请的表达载体包括但不限于使蛋白质能够在哺乳动物细胞、细菌、昆虫细胞、酵母和体外系统中的表达载体。The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleotide 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). To express the protein, the nucleic acid encoding the antigen binding unit of the present application can be integrated into an expression vector. Expression vectors for use in this application include, but are not limited to, expression vectors that enable proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.
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。The term "vector" as used herein 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. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "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.
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。The term "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.
术语“内源”是指一个核酸分子或多肽等来自生物体自身。The term "endogenous" refers to a nucleic acid molecule or polypeptide or the like that is derived from the organism itself.
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或多肽片段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)。The term "chimeric receptor" refers to a fusion molecule formed by linking DNA fragments or corresponding cDNA or polypeptide fragments of proteins 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).
术语“嵌合T细胞受体”，由一个TCR亚基与一个抗原结合结构域(如抗体结构域)结合组成，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。在具体实施例中，所述嵌合T细胞受体的抗原结合结构域包括本申请所述的CS1抗原识别单元。The term "chimeric T cell receptor" consists of a TCR subunit combined with an antigen binding domain (such as an antibody domain), wherein the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, TCR The stimulatory domain of the intracellular signaling domain; the TCR subunit is operably linked to the antibody domain. In specific embodiments, the antigen binding domain of the chimeric T cell receptor comprises the CS1 antigen recognition unit described herein.
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。具体实施例中，所述TAC抗原结合结构域包括本申请所述的CS1抗原识别单元。The term "T cell antigen coupler (TAC)" 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 bringing the TAC receptor close to the TCR receptor; (3) the transmembrane domain and the intracellular CD4 co-receptor Area. In a specific embodiment, the TAC antigen binding domain includes the CS1 antigen recognition unit described in this application.
本申请提供了一种能显著杀伤靶细胞的T细胞。在具体实施例中，本申请提供了包含所述CS1抗原识别单元的CAR-T细胞，例如hu32A12 CAR-T细胞、hu37A3 CAR-T细胞、hu48G9 CAR-T细胞。所述CS1-CAR-T细胞具有以下任一优点：The present application provides a T cell that can significantly kill target cells. In specific embodiments, the application provides CAR-T cells comprising the CS1 antigen recognition unit, such as hu32A12 CAR-T cells, hu37A3 CAR-T cells, hu48G9 CAR-T cells. The CS1-CAR-T cells have any of the following advantages:
(1)在体内外实验中，所述CS1-CAR-T细胞与CS1阳性靶细胞共孵育后，所述CS1-CAR-T细胞会产生显著的特异性增殖。(1) In in vitro and in vivo experiments, after the CS1-CAR-T cells were co-incubated with CS1-positive target cells, the CS1-CAR-T cells would produce significant specific proliferation.
(2)所述CS1-CAR-T细胞能特异性杀伤CS1阳性靶细胞。示例性，所述CS1-CAR-T细胞能特异性杀伤CS1阳性肿瘤细胞。具体实施例中，在体内外实验中，所述CS1-CAR-T细胞特异性杀伤CS1阳性多发性骨髓瘤。由于CS1是淋巴细胞激活分子家族成员，参与NK细胞活化功能，故CS1在NK细胞上表达；示例性，在体内外实验中，所述CS1-CAR-T细胞特异性杀伤宿主NK细胞、或者所述CS1-CAR-T细胞能抵抗宿主NK细胞对自体或同种异体免疫细胞(例如T细胞、CAR-T细胞)的杀伤、提高自体或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。示例性，在体内外实验中，可溶性CS1不影响所述CS1-CAR-T细胞对CS1阳性靶细胞的杀伤作用。(2) The CS1-CAR-T cells can specifically kill CS1-positive target cells. Exemplarily, the CS1-CAR-T cells can specifically kill CS1-positive tumor cells. In a specific embodiment, in vitro and in vivo experiments, the CS1-CAR-T cells specifically kill CS1-positive multiple myeloma. Since CS1 is a member of the lymphocyte activating molecule family and participates in the activation function of NK cells, CS1 is expressed on NK cells; for example, in in vitro and in vivo experiments, the CS1-CAR-T cells specifically kill host NK cells, or all The CS1-CAR-T cells can resist the killing of autologous or allogeneic immune cells (such as T cells, CAR-T cells) by host NK cells, and improve the persistence of autologous or allogeneic immune cells in the presence of host immune cells. Sex and/or transplant survival. Exemplarily, in vitro and in vivo experiments, soluble CS1 did not affect the killing effect of CS1-CAR-T cells on CS1-positive target cells.
(2)细胞因子分泌：在体内外实验中，所述CS1-CAR-T细胞与CS1阳性靶细胞共孵育后可显著激活进而分泌细胞因子TNF-α、IL-2或IFN-γ；在体内外实验中，所述CS1-CAR-T细胞与CS1阴性细胞共孵育后不产生显著的非特异细胞因子TNF-α、 IL-2或IFN-γ分泌；在体内外实验中，所述CS1-CAR-T细胞与单核细胞和CS1阳性靶细胞共同孵育后，细胞因子IL-6分泌水平较低。(2) Cytokine secretion: In in vitro and in vivo experiments, the CS1-CAR-T cells can be significantly activated after co-incubation with CS1 positive target cells to secrete cytokines TNF-α, IL-2 or IFN-γ; in vivo In vitro and in vivo experiments, the CS1-CAR-T cells did not produce significant non-specific cytokine TNF-α, IL-2 or IFN-γ secretion after co-incubation with CS1-negative cells; After co-incubation of CAR-T cells with monocytes and CS1-positive target cells, the secretion of cytokine IL-6 was lower.
(3)耗竭程度检测：在体内外实验中，所述CS1-CAR-T细胞与CS1阳性靶细胞共孵育后，所述CS1-CAR-T细胞耗竭标志物PD-1、TIM-3、LAG-3表达较低；在无靶细胞刺激的体外培养过程中，所述CS1-CAR-T细胞的CD3-ζ磷酸化程度较低。(3) Depletion degree detection: In in vitro and in vivo experiments, after the CS1-CAR-T cells were co-incubated with CS1 positive target cells, the CS1-CAR-T cell exhaustion markers PD-1, TIM-3, LAG -3 expression was low; the CS1-CAR-T cells had a low degree of CD3-ζ phosphorylation during in vitro culture without target cell stimulation.
在具体的实施例中，本申请提供了包含所述CS1-CAR，且内源性TCR和B2M双敲除的通用型T细胞(CS1-UCAR-T)，和内源性CS1、TCR和B2M三敲除的CS1-UCAR-T细胞。所述双敲除或三敲除的CS1-UCAR-T细胞具有以下任一优点：特异性杀伤CS1阳性靶细胞。示例性，所述CS1-UCAR-T细胞能特异性杀伤CS1阳性肿瘤细胞。具体实施例中，在体内外实验中，所述CS1-UCAR-T细胞特异性杀伤CS1阳性多发性骨髓瘤细胞。示例性，在体内外实验中，所述CS1-UCAR-T细胞特异性杀伤宿主NK细胞、或者所述CS1-UCAR-T细胞能抵抗宿主NK细胞对自体或同种异体免疫细胞(例如T细胞、CAR-T细胞、CS1-UCAR-T)的杀伤、提高自体或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。In specific embodiments, the present application provides universal T cells comprising the CS1-CAR and double knockout of endogenous TCR and B2M (CS1-UCAR-T), and endogenous CS1, TCR and B2M Triple knockout CS1-UCAR-T cells. The double-knockout or triple-knockout CS1-UCAR-T cells have any of the following advantages: specifically kill CS1-positive target cells. Exemplarily, the CS1-UCAR-T cells can specifically kill CS1-positive tumor cells. In a specific embodiment, in vitro and in vivo experiments, the CS1-UCAR-T cells specifically kill CS1-positive multiple myeloma cells. Exemplarily, in vitro and in vivo experiments, the CS1-UCAR-T cells specifically kill host NK cells, or the CS1-UCAR-T cells can resist host NK cells against autologous or allogeneic immune cells (such as T cells). , CAR-T cells, CS1-UCAR-T) killing, improving the persistence and/or transplantation survival rate of autologous or allogeneic immune cells in the presence of host immune cells.
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。胞外抗原结合结构域包括本申请识别CS1的抗原结合单元、或包括衍生自本申请识别CS1的抗原结合单元的Fv、Fab、Fab'、Fab'-SH、F(ab')2、scFv或多特异性抗体。The term "chimeric antigen receptor" (CAR) includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain includes an antigen-binding unit of the present application that recognizes CS1, or includes an Fv, Fab, Fab', Fab'-SH, F(ab')2, scFv, or derived from an antigen-binding unit of the present application that recognizes CS1. multispecific antibodies.
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；或包括刺激性分子和/或共刺激性分子的全部细胞内部分、或全部天然胞内信号传导结构域、或其功能片段或衍生物。在一个方面，刺激性分子包括与T细胞受体复合体结合的CD3ζ链；在一个方面，胞内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，多肽组彼此连接。示例性，靶向CS1的CAR包含SEQ ID NO：50、51或52所示的抗原结合域，示例性，靶向CS1的CAR包含SEQ ID NO：45、46或47所示的序列。上述SEQ ID NO：45、46、47的跨膜域和胞内域本领域技术人员可以选择常规的跨膜域和胞内域进行替换，且均落入本申请的保护范围。Intracellular signaling domains include functional signaling domains of stimulatory and/or costimulatory molecules; or entire intracellular portions, or entire native intracellular signaling structures including stimulatory and/or costimulatory molecules domain, or a functional fragment or derivative thereof. In one aspect, the stimulatory molecule comprises a CD3ζ chain bound to a T cell receptor complex; in one aspect, the intracellular signaling domain further comprises a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (ie CD137), CD27 and/or CD28. In certain embodiments, groups of polypeptides are linked to each other. Exemplarily, a CAR targeting CS1 comprises the antigen binding domain set forth in SEQ ID NO: 50, 51 or 52, and exemplary, a CAR targeting CS1 comprises the sequence set forth in SEQ ID NO: 45, 46 or 47. The transmembrane domains and intracellular domains of the above-mentioned SEQ ID NOs: 45, 46, and 47 can be replaced by those skilled in the art by selecting conventional transmembrane domains and intracellular domains, and all fall within the protection scope of the present application.
刺激性分子的功能信号传导结构域，也称为术语“初级信号域”，以刺激性方式调节TCR复合物的初始活化。一方面，初级信号域由例如TCR/CD3复合物与加载了肽的MHC分子的结合而引发，由此介导T细胞反应(包括但不限于，增殖、活化、分化等)。以刺激性方式起作用的初级信号域可以包含免疫受体酪氨酸激活基序或ITAM的信号传导基序。在本申请中尤其有用的包含ITAM的初级信号域的例子包括但不限于，源自TCRξ、CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”) 和CD66d的序列。在特例的本申请CAR中，在任何一个或多个本申请CAR中胞内信号传导结构域包含胞内信号传导序列，例如CD3ξ的初级信号域。The functional signaling domain of the stimulatory molecule, also referred to by the term "primary signaling domain", modulates the initial activation of the TCR complex in a stimulatory manner. In one aspect, the primary signaling domain is initiated by, for example, binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.). Primary signaling domains that act in a stimulatory manner may contain immunoreceptor tyrosine activation motifs or signaling motifs of ITAMs. Examples of ITAM-containing primary signaling domains that are particularly useful in this application include, but are not limited to, those derived from TCRξ, CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "" ICOS") and CD66d sequences. In a special case of the CAR of the present application, the intracellular signaling domain in any one or more of the CARs of the present application comprises an intracellular signaling sequence, such as the primary signaling domain of CD3ξ.
术语“共刺激信号域”指“共刺激分子”，指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。为T细胞上的关联结合性配偶体，其特异性结合共刺激配体，由此介导T细胞的共刺激反应，包括但不限于细胞增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。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)。The term "costimulatory signaling domain" refers to "costimulatory molecule" and refers to a signal that binds to a cell stimulatory signaling molecule, such as TCR/CD3, in combination resulting in T cell proliferation and/or up- or down-regulation of key molecules. is an cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, including but not limited to cell proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response. 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 ).
示例性，靶向CS1的CAR的信号传导结构域包括CD3ζ。示例性，靶向CS1的CAR的信号传导结构域包括CD28胞内域、CD137胞内域。在具体实施例中，CD28胞内域包含SEQ ID NO:73所示序列，CD137胞内域包含SEQ ID NO:55所示序列。在本申请中“CD3ζ”与“CD3z”及“CD3Z”可互换使用。在具体实施例中，CD3ζ为人CD3ζ分子，包含SEQ ID NO:56所示序列。Exemplarily, the signaling domain of a CAR targeting CS1 includes CD3ζ. Exemplarily, the signaling domains of a CAR targeting CS1 include CD28 intracellular domain, CD137 intracellular domain. In a specific embodiment, the CD28 intracellular domain comprises the sequence shown in SEQ ID NO:73, and the CD137 intracellular domain comprises the sequence shown in SEQ ID NO:55. "CD3ζ" is used interchangeably with "CD3z" and "CD3Z" in this application. In a specific embodiment, CD3ζ is a human CD3ζ molecule comprising the sequence shown in SEQ ID NO:56.
具体实施例中，CAR包含可选的前导序列。一方面，CAR在胞外抗原识别结构域的N端还包含前导序列，其中前导序列任选地在CAR的细胞加工和定位至细胞膜的过程中从抗原识别结构域(例如scFv)切下。在一实施例中，前导序列包含SEQ ID NO:71所示序列。In specific embodiments, the CAR includes an optional leader sequence. In one aspect, the CAR further comprises a leader sequence N-terminal to the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane. In one embodiment, the leader sequence comprises the sequence shown in SEQ ID NO:71.
在一些实施例中，CAR的胞外抗原结合区包括本申请识别CS1的抗原结合单元和连接片段(也称为铰链、间隔区或连接体)。连接片段可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。连接片段可以是任何长度的。示例性，在一个实施例中，CAR包括一个铰链域，是CD8α铰链，优选地，CD8α铰链域包含SEQ ID NO:53所示的氨基酸序列。In some embodiments, the extracellular antigen-binding region of the CAR includes an antigen-binding unit and a linker fragment (also referred to as a hinge, spacer, or linker) of the present application that recognizes CS1. Linked fragments can be considered as part of a CAR used to provide flexibility to the extracellular antigen binding region. Linked fragments can be of any length. Exemplarily, in one embodiment, the CAR includes a hinge domain, which is a CD8α hinge, preferably, the CD8α hinge domain comprises the amino acid sequence set forth in SEQ ID NO:53.
CAR的跨膜(TM)结构域(或称为结构区)可以将CAR锚定在细胞的质膜上。示例性，TM是CD8或CD28跨膜结构域。在优选实施例中，TM是人CD8跨膜结构域或人CD28跨膜结构域。优选地，CD8跨膜结构域包含SEQ ID NO:54的氨基酸，CD28跨膜结构域包含SEQ ID NO:72的氨基酸。The transmembrane (TM) domain (or domain) of a CAR can anchor the CAR to the plasma membrane of cells. Exemplarily, a TM is a CD8 or CD28 transmembrane domain. In preferred embodiments, the TM is a human CD8 transmembrane domain or a human CD28 transmembrane domain. Preferably, the CD8 transmembrane domain comprises the amino acids of SEQ ID NO:54 and the CD28 transmembrane domain comprises the amino acids of SEQ ID NO:72.
术语“干扰素”是免疫系统产生的细胞因子，主要分为α、β、γ三种类型，具有抗病毒、抗肿瘤及免疫调节作用。The term "interferon" is a cytokine produced by the immune system, mainly divided into three types: α, β, and γ, and has anti-viral, anti-tumor and immunomodulatory effects.
术语“白细胞介素-2(白介素2、IL-2)”是趋化因子家族的一种细胞因子，对机体的免疫应答和抗病毒感染等有重要作用。The term "interleukin-2 (interleukin-2, IL-2)" is a cytokine in the chemokine family, which plays an important role in the body's immune response and antiviral infection.
术语“白细胞介素6(白介素6、IL-6)”是一种功能广泛的多效性细胞因子。IL-6是细胞因子风暴的一个生物标识物。The term "interleukin-6 (interleukin-6, IL-6)" is a broadly functional pleiotropic cytokine. IL-6 is a biomarker of cytokine storm.
术语“T细胞耗竭”，是一种T细胞功能失常的状态，其表现为进展性的功能丧失、基因表达谱的变化、和抑制性细胞因子的持续分泌。T细胞耗竭的生物标识物包括PD-1、TIM-3、LAG-3等。The term "T cell exhaustion" is a state of T cell dysfunction manifested by progressive loss of function, changes in gene expression profiles, and sustained secretion of inhibitory cytokines. Biomarkers of T cell exhaustion include PD-1, TIM-3, LAG-3, etc.
本申请的抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述抗体、免疫辍合物、嵌合受体、核酸或宿主细胞，还可包含药学上可接受的载体。术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。药学上可接受的载体可以是常规使用的载体之一，并且仅受化学物理考虑的限制，例如溶解度和不与活性剂反应，以及给药途径。本文描述的医药上可接受载体，例如佐剂、辅料和稀释剂，是本领域技术人员熟知的，并且易于公众使用。优选的，药物上可接受的载体是在使用条件下无害、无毒副作用的载体。本申请的药物组合物有多种合适的剂型。制备可给药(例如，肠外给药)组合物的方法对于本领域技术人员是已知的或显而易见的。The antibodies, immunoconjugates, chimeric receptors, and host cells comprising the antibodies of the present application can be applied to the preparation of pharmaceutical compositions or diagnostic reagents. In addition to comprising an effective amount of the antibody, immunoconjugate, chimeric receptor, nucleic acid or host cell, the composition may also comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans. The pharmaceutically acceptable carrier can be one of those conventionally used and is limited only by chemical physical considerations, such as solubility and inactivity with the active agent, and the route of administration. The pharmaceutically acceptable carriers described herein, such as adjuvants, excipients and diluents, are well known to those skilled in the art and are readily available to the public. Preferably, a pharmaceutically acceptable carrier is one that is innocuous under the conditions of use and has no toxic side effects. There are a variety of suitable dosage forms for the pharmaceutical compositions of the present application. Methods of preparing administrable (eg, parenterally) compositions are known or apparent to those skilled in the art.
在一些实施方案中，所述组合物包含化疗剂。In some embodiments, the composition comprises a chemotherapeutic agent.
本申请的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。The composition of the present application can be prepared into various dosage forms as required, and the physician can determine the dosage beneficial to the patient according to factors such as the type, age, weight and general disease state of the patient, and the mode of administration. The mode of administration can be, for example, parenteral administration (eg, injection) or other therapeutic modes.
免疫原性组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。"Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
本申请的工程化细胞可以任何适当的方式给予受试者。优选地，通过注射(例如，皮下、静脉内、肿瘤内、动脉内、肌肉内、皮内、腹膜间或鞘内)施用本申请的抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞。优选地，本申请的抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞经静脉给予。用于本申请注射用抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞的合适的医药上可接受的载体可包括任何等渗载体，例如，生理盐水(水中约0.90％w/v NaCl，水中约300mOsm/L NaCl，或每升水约9.0g NaCl)，常温或电解质溶液。在一个实施例中，医药上可接受的载体补充有人血清蛋白。The engineered cells of the present application can be administered to a subject in any suitable manner. Preferably, the antibodies of the present application, immunoconjugates comprising the antibodies, chimeric receptors, host cell. Preferably, the antibodies of the present application, immunoconjugates comprising the antibodies, chimeric receptors, host cells are administered intravenously. Suitable pharmaceutically acceptable carriers for injectable antibodies of the present application, immunoconjugates comprising the antibodies, chimeric receptors, host cells may include any isotonic carrier, for example, physiological saline (about 0.90% w in water). /v NaCl, about 300mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), normal temperature or electrolyte solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum protein.
“有效量”或“治疗有效量”是指足以预防或治疗个体疾病(癌症)的剂量。治疗性或预防性使用的有效剂量取决于所治疗疾病的阶段和严重程度、受试者的年龄、体重和一般健康状况以及处方医生的判断。剂量的大小还取决于所选择的活性物质、给药方法、给药时间和频率、可能伴随特定活性物给药的不良副作用的存在、性质和程度以及所需的生理效应。根据处方医生或本领域技术人员判断可能需要一轮或多轮、或多次给与本申请抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞。An "effective amount" or "therapeutically effective amount" refers to a dose sufficient to prevent or treat a disease (cancer) in a subject. Effective doses for therapeutic or prophylactic use depend on the stage and severity of the disease being treated, the age, weight and general health of the subject, and the judgment of the prescribing physician. The size of the dose will also depend on the active substance selected, the method of administration, the timing and frequency of administration, the presence, nature and extent of adverse side effects that may accompany administration of the particular active substance, and the desired physiological effect. According to the judgment of the prescribing physician or those skilled in the art, one or more rounds, or multiple administrations of the antibody of the present application, the immunoconjugate comprising the antibody, the chimeric receptor, and the host cell may be required.
本申请的实施例还包括在施用本申请的抗体、包含该抗体的免疫辍合物、嵌合受体、 宿主细胞之前去除哺乳动物的淋巴细胞，包括但不限于非清髓性淋巴耗竭化疗、清髓性淋巴耗竭化疗、全身照射等。Embodiments of the present application also include depletion of mammalian lymphocytes prior to administration of the antibodies of the present application, immunoconjugates comprising the antibodies, chimeric receptors, host cells, including but not limited to non-myeloablative lymphoid depletion chemotherapy, Myeloablative lymphatic depletion chemotherapy, total body irradiation, etc.
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。在具体实施例中，本申请提供的工程化T细胞能抑制肿瘤细胞增殖、和/或体内抑制肿瘤细胞增殖、肿瘤体积增加。The term "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. In a specific embodiment, the engineered T cells provided in the present application can inhibit tumor cell proliferation, and/or inhibit tumor cell proliferation and increase tumor volume in vivo.
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。The term "prevention" refers to interventions that are attempted prior to the development of a disease such as rejection of a cell transplant.
本申请提供了用于治疗或预防哺乳动物肿瘤的本申请的抗体、CAR、核酸、重组表达载体、宿主细胞、细胞集合或药物组合物。The application provides the antibodies, CARs, nucleic acids, recombinant expression vectors, host cells, cell collections or pharmaceutical compositions of the present application for use in the treatment or prevention of mammalian tumors.
本申请的所提供的工程化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。本申请所提供的工程化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。The engineered T cells provided in this application 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 . The engineered T cells provided herein 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) ), 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, for example, idiopathic nephrotic syndrome or minimal change nephropathy), multiple myeloma, other types of tumors, skin and Inflammatory diseases of the cornea, myositis, loosening of bone implants, metabolic disorders (such as obesity, atherosclerosis and other cardiovascular diseases including dilated cardiomyopathy, myocarditis, type 2 diabetes and dyslipidemia) and autoimmunity Sexual thyroid disease (including Hashimoto's thyroiditis), primary vasculitis of small and medium vessels, large vessel vasculitis including giant cell arteritis, hidradenitis suppurativa, neuromyelitis optica, Sjogren's syndrome, Behçet's Disease, atopic and contact dermatitis, bronchiolitis, inflammatory muscle disease, autoimmune peripheral neuropathy, immune kidney, liver and thyroid disease, inflammation and atherosclerosis, autoinflammatory fever syndrome, immunohematology Disorders and bullous diseases of the skin and mucous membranes.
本申请所提供的工程化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。The engineered T cells provided herein can be used to treat, prevent or ameliorate asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema, and other obstructive or inflammatory diseases of the airways.
可将本申请的工程化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。The engineered T cells of the present application 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 anticancer agents (eg, as adjuvants thereof or in combination with them). ), for example, to treat or prevent diseases associated with immune disorders.
本申请所述肿瘤可以是任何肿瘤，包括急性淋巴细胞癌、急性髓系白血病、肺泡横纹肌肉瘤、膀胱癌、骨癌、脑癌(例如髓母细胞瘤)、乳腺癌、肛门癌、肛管癌或肛肠癌、眼癌，肝内胆管癌、关节癌、颈癌、胆囊癌或胸膜癌、鼻癌、鼻腔癌或中耳癌、口腔癌、外阴癌、慢性淋巴细胞白血病(CLL)、慢性髓细胞癌、结肠癌、食管癌、宫颈癌、纤维肉瘤、胃肠道类癌、头颈部癌(如头颈部鳞状细胞癌)、霍奇金淋巴瘤、下咽癌、肾癌、喉癌、白血病、肝癌、肺癌(如非小细胞肺癌)、淋巴瘤、恶性间皮瘤、肥大细胞瘤、黑色素瘤、多发性骨髓瘤、鼻咽癌、非霍奇金淋巴瘤、B-慢性淋巴细胞白血病、B-前体急性淋巴细胞白血病(B-ALL)、前B细胞前体急性淋巴细胞白血病(BCP-ALL)、B细胞淋巴瘤、急性淋巴细胞白血病(ALL)，伯基特淋巴瘤、卵巢癌、胰腺癌、咽癌、前列腺癌、直肠癌、肾癌、皮肤癌、小肠癌、软组织癌、胃癌、睾丸癌、甲状腺癌、输尿管癌。优选地，所述肿瘤以CS1表达为特征，并且更优选地是以CS1表达为特征的多发性骨髓瘤。The tumor described herein can be any tumor, including acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (eg, medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal, eye, intrahepatic bile duct, joint, neck, gallbladder or pleura, nose, nasal cavity or middle ear, oral cavity, vulvar, chronic lymphocytic leukemia (CLL), chronic myeloid Cell cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid, head and neck cancer (such as head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer Cancer, leukemia, liver cancer, lung cancer (eg, non-small cell lung cancer), lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin lymphoma, B-chronic lymphoma B-cell leukemia, B-precursor acute lymphoblastic leukemia (B-ALL), B-cell precursor acute lymphoblastic leukemia (BCP-ALL), B-cell lymphoma, acute lymphoblastic leukemia (ALL), Burkitt lymphoma , ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer. Preferably, the tumor is characterized by CS1 expression, and more preferably is a multiple myeloma characterized by CS1 expression.
“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。在某些方面，本申请的过度增生性病症是指癌症。"Tumor antigen" refers to an antigen that is new or overexpressed during the development, progression of a hyperproliferative disease. In certain aspects, the hyperproliferative disorder of the present application refers to cancer.
本申请提供了一种T细胞被基因工程化表达CS1-CAR用于显著杀伤靶细胞；提供了所述基因工程化T细胞的制备方法。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR。本申请还提供了一种不仅能显著杀伤肿瘤细胞，还能抵抗自体或同种异体NK细胞杀伤的T细胞。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR，且利用基因编辑技术敲除内源性CS1。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR,且利用基因编辑技术敲除内源性B2M和TCR。在一些实施方案中，工程化T细胞被基因工程化表达CS1-CAR,且利用基因编辑技术敲除内源性CS1、B2M和TCR。The application provides a T cell that is genetically engineered to express CS1-CAR for significantly killing target cells; and a method for preparing the genetically engineered T cell. In some embodiments, the engineered T cells are genetically engineered to express CS1-CAR. The present application also provides a T cell that can not only kill tumor cells significantly, but also resist the killing of autologous or allogeneic NK cells. In some embodiments, the engineered T cells are genetically engineered to express CS1-CAR, and gene editing techniques are used to knock out endogenous CS1. In some embodiments, the engineered T cells are genetically engineered to express CS1-CAR, and gene editing techniques are used to knock out endogenous B2M and TCR. In some embodiments, the engineered T cells are genetically engineered to express CS1-CAR, and gene editing techniques are used to knock out endogenous CS1, B2M, and TCR.
本申请包括，例如中国申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN 108884459A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN 109908176A、CN109880803A、CN 110055275A、CN110123837A、CN 110438082A、CN 110468105A国际申请公开号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、WO2019/210863、WO2019/219029中公开的那些CAR-T细胞及其制备方法。本申请包括，例如中国申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN 108884459A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、 CN 109908176A、CN109880803A、CN 110055275A、CN110123837A、CN 110438082A、CN 110468105A国际申请公开号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, WO2019/210863, WO2019/210863, those CAR-CAR-19 cells disclosed in WO2019/149279 Preparation.
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。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。The present application will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.
实施例1、CS1重组蛋白的制备Example 1. Preparation of CS1 recombinant protein
(1)hSLAMF7-avi-His、hSLAMF7-huFc、mSLAMF7-huFc，cySLAMF7-huFc，h1m2D-SLAMF7-huFc，m1h2D-SLAMF7-huFc表达质粒的构建(1) Construction of hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7-huFc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc expression plasmids
根据Genbank中human SLAMF7(Gene ID:57823)、mouse SLAMF7(Gene ID:75345)、Cynomolgus Monkey SLAMF7(Gene ID:102133710)序列信息设计引物，通过分子克隆技术将human SLAMF7胞外区域(SEQ ID NO:57)与avi标签和His标签连接，构建hSLAMF7-avi-His表达质粒；将human SLAMF7胞外区域(SEQ ID NO:57)与人Fc片段(SEQ ID NO:60)融合构建hSLAMF7-huFc表达质粒；将mouse SLAMF7胞外区域(SEQ ID NO:58)与人Fc片段(SEQ ID NO:60)融合构建mSLAMF7-huFc表达质粒；将Cynomolgus Monkey SLAMF7胞外区域(SEQ ID NO:59)与人Fc片段(SEQ ID NO:60)融合构建cySLAMF7-huFc表达质粒；将人Ig like-V结构域和鼠Ig like-C结构域融合，即将human SLAMF7氨基酸序列第23-130位(SEQ ID NO:67)与mouse SLAMF7氨基酸序列第128-224位(SEQ ID NO:68)融合得到序列(SEQ ID NO:65)，再与人Fc片段(SEQ ID NO:60)融合构建h1m2D-SLAMF7-huFc表达质粒；将鼠Ig like-V结构域和人Ig like-C结构域融合，即将mouse SLAMF7氨基酸序列第23-127位(SEQ ID NO:69)与human SLAMF7氨基酸序列第131-226位(SEQ ID NO:70)融合得到序列(SEQ ID NO:66)，再与人Fc片段(SEQ ID NO:60)融合构建m1h2D-SLAMF7-huFc表达质粒。Primers were designed according to the sequence information of human SLAMF7 (Gene ID: 57823), mouse SLAMF7 (Gene ID: 75345), Cynomolgus Monkey SLAMF7 (Gene ID: 102133710) in Genbank, and the extracellular region of human SLAMF7 (SEQ ID NO: 57) Connect with avi tag and His tag to construct hSLAMF7-avi-His expression plasmid; fuse human SLAMF7 extracellular region (SEQ ID NO:57) with human Fc fragment (SEQ ID NO:60) to construct hSLAMF7-huFc expression plasmid The mouse SLAMF7 extracellular region (SEQ ID NO:58) was fused with human Fc fragment (SEQ ID NO:60) to construct mSLAMF7-huFc expression plasmid; Cynomolgus Monkey SLAMF7 extracellular region (SEQ ID NO:59) was combined with human Fc The fragment (SEQ ID NO:60) was fused to construct the cySLAMF7-huFc expression plasmid; the human Ig like-V domain and the mouse Ig like-C domain were fused, that is, positions 23-130 of the amino acid sequence of human SLAMF7 (SEQ ID NO:67 ) fused with mouse SLAMF7 amino acid sequence 128-224 (SEQ ID NO:68) to obtain sequence (SEQ ID NO:65), and then fused with human Fc fragment (SEQ ID NO:60) to construct h1m2D-SLAMF7-huFc expression plasmid ; The mouse Ig like-V domain and the human Ig like-C domain are fused, that is, the 23-127th position (SEQ ID NO:69) of the mouse SLAMF7 amino acid sequence and the 131-226th position (SEQ ID NO: 69) of the human SLAMF7 amino acid sequence. :70) fused to obtain the sequence (SEQ ID NO:66), and then fused with the human Fc fragment (SEQ ID NO:60) to construct the m1h2D-SLAMF7-huFc expression plasmid.
(2)重组蛋白hSLAMF7-avi-His、hSLAMF7-huFc、mSLAMF7-huFc，cySLAMF7-huFc，h1m2D-SLAMF7-huFc，m1h2D-SLAMF7-huFc的转染表达及纯化(2) Transfection, expression and purification of recombinant proteins hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7-huFc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc
将上述hSLAMF7-avi-His、hSLAMF7-huFc、mSLAMF7-huFc，cySLAMF7-huFc，h1m2D-SLAMF7-huFc，m1h2D-SLAMF7-huFc表达质粒转染生长良好的HEK 293F细胞 (来自上海市肿瘤研究所)，37℃，5％CO 2，125rpm摇床连续培养7天，4000rpm离心10min，去除沉淀，收集上清，并用0.45μm滤膜过滤，将处理好的样品以HisTrap或者Mabselect Sure(购自GE)亲和层析柱进行纯化，最终获得纯化的重组蛋白hSLAMF7-avi-His、hSLAMF7-huFc、mSLAMF7-huFc，cySLAMF7-huFc，h1m2D-SLAMF7-huFc，m1h2D-SLAMF7-huFc。 The above-mentioned hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7-huFc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc expression plasmids were transfected into well-growing HEK 293F cells (from Shanghai Cancer Institute), 37°C, 5% CO 2 , 125 rpm shaker for continuous cultivation for 7 days, centrifugation at 4000 rpm for 10 min, remove the precipitate, collect the supernatant, and filter it with a 0.45 μm filter membrane. The purified recombinant proteins hSLAMF7-avi-His, hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7-huFc, h1m2D-SLAMF7-huFc and m1h2D-SLAMF7-huFc were finally obtained by purifying with a chromatography column.
实施例2、抗CS1杂交瘤抗体的产生Example 2. Production of anti-CS1 hybridoma antibodies
(1)免疫(1) Immunity
选用6-8周龄的雌性Balb/c小鼠，将免疫原hSLAMF7-avi-His重组蛋白和弗氏完全佐剂1：1(体积比)混匀，经皮下多点注射进行初次免疫，免疫计量为100μg/只。然后将免疫原hSLAMF7-avi-His重组蛋白和弗氏不完全佐剂1：1(体积比)混匀，经皮下多点注射进行后续免疫，每隔两周免疫一次，共免疫2次，免疫计量为100μg/只。在融合采脾前3天，采用腹腔注射100μg/只hSLAMF7-avi-His重组蛋白的方式进行加强免疫。Female Balb/c mice aged 6-8 weeks were selected, the immunogen hSLAMF7-avi-His recombinant protein and Freund's complete adjuvant were mixed at 1:1 (volume ratio), and the primary immunization was performed by subcutaneous injection at multiple points. The dosage is 100μg/only. Then, the immunogen hSLAMF7-avi-His recombinant protein and incomplete Freund's adjuvant were mixed at 1:1 (volume ratio), followed by subcutaneous injection at multiple points for subsequent immunization, once every two weeks, for a total of 2 immunizations. The dosage is 100μg/only. Three days before fusion and spleen harvesting, booster immunization was performed by intraperitoneal injection of 100 μg/hSLAMF7-avi-His recombinant protein.
(2)杂交瘤制备(2) Hybridoma preparation
分离免疫小鼠的脾细胞，与小鼠骨髓瘤细胞SP2/0(ATCC,CRL 1581)按2:1的比例混合，使用50％PEG(Sigma)将分离的脾细胞与SP2/0按公开的标准方案(Kohler、Milstein，1975)进行融合。经HAT培养基(sigma，H0262)及ELISA、流式细胞术筛选后得到3个结合hSLAMF7的杂交瘤抗体32A12MAb(IgG1/k)、37A3MAb(IgG1/k)、48G9MAb(IgG2b/k)。Splenocytes from immunized mice were isolated and mixed with mouse myeloma cells SP2/0 (ATCC, CRL 1581) at a ratio of 2:1. The isolated splenocytes were mixed with SP2/0 using 50% PEG (Sigma) as published Fusion was performed using standard protocols (Kohler, Milstein, 1975). After screening in HAT medium (sigma, H0262), ELISA and flow cytometry, three hybridoma antibodies 32A12MAb (IgG1/k), 37A3MAb (IgG1/k) and 48G9MAb (IgG2b/k) that bind hSLAMF7 were obtained.
对照抗体Luc90和huLuc63的序列来自专利US9175081B2，Luc90重链可变区序列(SEQ ID NO:61)，Luc90轻链可变区序列(SEQ ID NO:62)，huLuc63的重链可变区序列(SEQ ID NO:63)，huLuc63的轻链可变区序列(SEQ ID NO:64)；通过常规的重组表达技术，制备对照抗体用于后续检测。The sequences of control antibodies Luc90 and huLuc63 are from patent US9175081B2, Luc90 heavy chain variable region sequence (SEQ ID NO:61), Luc90 light chain variable region sequence (SEQ ID NO:62), heavy chain variable region sequence of huLuc63 ( SEQ ID NO: 63), the light chain variable region sequence of huLuc63 (SEQ ID NO: 64); by conventional recombinant expression technology, a control antibody was prepared for subsequent detection.
表1 杂交瘤抗体序列Table 1 Hybridoma antibody sequences
   氨基酸序列amino acid sequence 核苷酸序列Nucleotide sequence
32A12 VH32A12 VH SEQ ID NO:1SEQ ID NO: 1 SEQ ID NO:2SEQ ID NO: 2
32A12 VL32A12 VL SEQ ID NO:6SEQ ID NO: 6 SEQ ID NO:7SEQ ID NO: 7
37A3 VH37A3 VH SEQ ID NO:11SEQ ID NO: 11 SEQ ID NO:12SEQ ID NO: 12
37A3 VL37A3 VL SEQ ID NO:16SEQ ID NO: 16 SEQ ID NO:17SEQ ID NO: 17
48G9 VH48G9 VH SEQ ID NO:21SEQ ID NO: 21 SEQ ID NO:22SEQ ID NO: 22
48G9 VL48G9 VL SEQ ID NO:26SEQ ID NO: 26 SEQ ID NO:27SEQ ID NO: 27
实施例3、利用ELISA法测定抗CS1杂交瘤抗体与重组蛋白hSLAMF7-avi-His的 结合活性Example 3. Determination of the binding activity of anti-CS1 hybridoma antibody and recombinant protein hSLAMF7-avi-His by ELISA
包被hSLAMF7-avi-his，1.5μg/ml，100μL/孔，4℃过夜；5％脱脂奶粉PBS室温封闭2h；封闭后用PBST清洗，之后分别加入纯化抗体32A12MAb、37A3MAb、48G9MAb从100μg/mL开始5倍梯度稀释11次，100μL/孔，室温孵育1小时；PBST清洗后，每孔加入100μL 4000倍稀释的HRP-Goat-anti-Mouse IgG(Fc specific)(sigma，CAT A0168，LOT 077M4820V)，室温孵育1小时，清洗后加入TMB显色。用酶标仪(Spectra
Figure PCTCN2022084732-appb-000001
340PC 384)测定OD450nM和OD650nM吸光值，并以OD450-OD650作为最后检测结果，使用GraphPad Prism8.0作图。 Coated with hSLAMF7-avi-his, 1.5μg/ml, 100μL/well, overnight at 4°C; blocked with 5% nonfat dry milk at room temperature for 2h; washed with PBST after blocking, and then added purified antibodies 32A12MAb, 37A3MAb, 48G9MAb from 100μg/mL 11 times of 5-fold serial dilution, 100 μL/well, incubated at room temperature for 1 hour; after washing with PBST, 100 μL of 4000-fold diluted HRP-Goat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M4820V) was added to each well. , incubate for 1 hour at room temperature, and add TMB to develop color after washing. Using a microplate reader (Spectra
Figure PCTCN2022084732-appb-000001
340PC 384) to measure the absorbance values of OD450nM and OD650nM, and take OD450-OD650 as the final detection result, and use GraphPad Prism8.0 to make a graph.
结果如图1所示，3个杂交瘤抗体32A12MAb、37A3MAb、48G9MAb及对照抗体huLuc63与hSLAMF7-avi-his蛋白结合，且呈现浓度梯度依赖性，EC50值接近(范围为0.034μg/ml-0.058μg/ml)。The results are shown in Figure 1. The three hybridoma antibodies 32A12MAb, 37A3MAb, 48G9MAb and the control antibody huLuc63 bind to hSLAMF7-avi-his protein in a concentration gradient-dependent manner, and the EC50 values are close (range 0.034μg/ml-0.058μg /ml).
实施例4、抗CS1杂交瘤抗体与人多发性骨髓瘤细胞结合活性测定Example 4. Determination of the binding activity of anti-CS1 hybridoma antibody to human multiple myeloma cells
取CS1阳性的MM.1S细胞(人多发性骨髓瘤细胞，来自中国科学院典型培养物保藏委员会细胞库)2×10 5个细胞/孔于96孔圆底培养板中，清洗后分别加入纯化抗体32A12MAb、37A3MAb、48G9MAb 20μg/mL开始5倍梯度稀释7次，100μL，4℃45min；500g离心5min，弃上清后用含1％FBS的PBS清洗2次，之后加入Goat-anti-Mouse FITC(KANFCHEN，CAT:KC-MM-095，1:200使用)，50μL/孔，4℃孵育45min；500g离心5min，弃上清后用含1％FBS的PBS清洗2次，之后加入200μL含1％FBS的PBS重悬细胞用流式细胞仪(Guava easyCyte 8HT)进行检测，结果用FlowJo7.6.1统计及GraphPad Prism8.0作图。 Take CS1-positive MM.1S cells (human multiple myeloma cells, from the cell bank of the Type Culture Collection, Chinese Academy of Sciences) 2 × 10 5 cells/well in a 96-well round-bottom culture plate, and add purified antibodies after washing. 32A12MAb, 37A3MAb, 48G9MAb 20μg/mL start 5-fold serial dilution 7 times, 100μL, 4 ℃ 45min; 500g centrifugation for 5min, discard the supernatant, wash twice with PBS containing 1% FBS, then add Goat-anti-Mouse FITC ( KANFCHEN, CAT:KC-MM-095, used at 1:200), 50 μL/well, incubated at 4°C for 45 min; centrifuged at 500 g for 5 min, discarded the supernatant, washed twice with PBS containing 1% FBS, and then added 200 μL containing 1% The cells resuspended in FBS in PBS were detected by a flow cytometer (Guava easyCyte 8HT), and the results were plotted using FlowJo7.6.1 statistics and GraphPad Prism8.0.
结果(图2)显示杂交瘤抗体37A3MAb和48G9MAb与MM.1S细胞结合能力较强，EC50值分别为0.05μg/ml和0.18μg/ml，杂交瘤抗体32A12MAb与MM.1S细胞结合能力较弱，EC50值为1.76μg/ml。The results (Fig. 2) showed that the hybridoma antibodies 37A3MAb and 48G9MAb had strong binding ability to MM.1S cells, with EC50 values of 0.05 μg/ml and 0.18 μg/ml, respectively, while the hybridoma antibody 32A12MAb had a weaker binding ability to MM.1S cells. The EC50 value was 1.76 μg/ml.
实施例5、利用ELISA法检测抗CS1杂交瘤抗体与不同种属SLAMF7重组蛋白的结合Example 5. Detecting the binding of anti-CS1 hybridoma antibodies to SLAMF7 recombinant proteins of different species by ELISA
包被hSLAMF7-huFc，mSLAMF7-huFc，cySLAMF7-huFc，hu-Fc，5μg/mL，100μL/孔，4℃过夜；5％脱脂奶粉PBS室温封闭2h；封闭后用PBST清洗，之后分别加入纯化抗体32A12MAb、37A3MAb、48G9MAb 5μg/mL，100μL/孔，室温孵育1小时；PBST清洗后，每孔加入100μL 4000倍稀释的HRP-Goat-anti-Mouse IgG(Fc specific)(sigma，CAT A0168，LOT 077M4820V)，室温孵育1小时，清洗后加入TMB显色。用酶标仪测定OD450nM和OD650nM吸光值，并以OD450-OD650作为最后检测结果。Coated with hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7-huFc, hu-Fc, 5 μg/mL, 100 μL/well, overnight at 4°C; blocked with 5% nonfat milk powder at room temperature for 2 h; washed with PBST after blocking, and then added purified antibodies 32A12MAb, 37A3MAb, 48G9MAb 5μg/mL, 100μL/well, incubated at room temperature for 1 hour; after washing with PBST, 100μL of 4000-fold diluted HRP-Goat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M4820V) was added to each well ), incubated at room temperature for 1 hour, and added TMB to develop color after washing. The absorbance values of OD450nM and OD650nM were measured with a microplate reader, and OD450-OD650 was used as the final detection result.
结果(图3)显示3个杂交瘤抗体32A12MAb、37A3MAb、48G9MAb均与人 SLAMF7-huFc结合，不与小鼠SLAMF7-huFc结合，抗体37A3MAb和48G9MAb与猴SLAMF7-huFc结合，抗体32A12MAb不与猴SLAMF7-huFc结合。The results (Fig. 3) showed that the three hybridoma antibodies 32A12MAb, 37A3MAb and 48G9MAb all bound to human SLAMF7-huFc, but not to mouse SLAMF7-huFc. Antibodies 37A3MAb and 48G9MAb bound to monkey SLAMF7-huFc, and antibody 32A12MAb did not bind to monkey SLAMF7. -huFc binding.
实施例6、检测抗CS1杂交瘤抗体与hSLAMF7的结合表位Example 6. Detection of the binding epitope of anti-CS1 hybridoma antibody to hSLAMF7
包被hSLAMF7-huFc，hu-Fc，h1m2D-SLAMF7-huFc，m1h2D-SLAMF7-huFc，5μg/mL，100μL/孔，4℃过夜；室温封闭2h；清洗后分别加入纯化抗体32A12MAb、37A3MAb、48G9MAb 5μg/mL，100μL/孔，室温孵育1小时；清洗后，每孔加入100μL4000倍稀释的HRP-Goat-anti-Mouse IgG(Fc specific)(sigma，CAT A0168，LOT 077M4820V)，室温孵育1小时，清洗后加入TMB显色。用酶标仪测定OD450nM和OD650nM吸光值，并以OD450-OD650作为最后检测结果。Coated with hSLAMF7-huFc, hu-Fc, h1m2D-SLAMF7-huFc, m1h2D-SLAMF7-huFc, 5 μg/mL, 100 μL/well, overnight at 4°C; blocked at room temperature for 2 hours; after washing, 5 μg of purified antibodies 32A12MAb, 37A3MAb and 48G9MAb were added respectively /mL, 100μL/well, incubate at room temperature for 1 hour; after washing, add 100μL of 4000-fold diluted HRP-Goat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M4820V) to each well, incubate at room temperature for 1 hour, wash Then add TMB to develop color. The absorbance values of OD450nM and OD650nM were measured with a microplate reader, and OD450-OD650 was used as the final detection result.
结果(表2)显示抗体37A3MAb、48G9MAb以及对照抗体Luc90与hCS1结合表位在hCS1的Ig-like V-type结构域，远膜端；抗体32A12MAb以及对照抗体huLuc63与hCS1结合表位在hCS1的Ig-like C2-type结构域，近膜端。The results (Table 2) show that the binding epitope of antibodies 37A3MAb, 48G9MAb and control antibody Luc90 to hCS1 is in the Ig-like V-type domain of hCS1, at the far membrane end; the binding epitope of antibody 32A12MAb and control antibody huLuc63 to hCS1 is in the Ig of hCS1 -like C2-type domain, membrane proximal.
表2 CS1抗体与hSLAMF7-huFc结合表位分析结果Table 2 Analysis results of CS1 antibody and hSLAMF7-huFc binding epitope
抗体Antibody hSLAMF7-huFchSLAMF7-huFc h1m2D-SLAMF7-huFch1m2D-SLAMF7-huFc m1h2D-SLAMF7-huFcm1h2D-SLAMF7-huFc hu-Fchu-Fc
32A12MAb32A12MAb ++ -- ++ --
37A3MAb37A3MAb ++ ++ -- --
48G9MAb48G9MAb ++ ++ -- --
huLuc63huLuc63 ++ -- ++ --
Luc90Luc90 ++ ++ -- --
实施例7、检测抗CS1杂交瘤抗体与细胞的结合Example 7. Detection of binding of anti-CS1 hybridoma antibody to cells
分别取CS1阳性的人骨髓瘤细胞MM.1S(中国科学院典型培养物保藏委员会细胞库)、NCI H929(中国科学院典型培养物保藏委员会细胞库)、RPMI 8226(ATCC)；及CS1阴性的人胚胎肺成纤维细胞WI38(中国科学院典型培养物保藏委员会细胞库)、人胚胎肾细胞HEK293(中国典型培养物保藏中心)，2×10 5个细胞/孔96孔圆底培养板中，离心弃上清后清洗2次，之后分别加入纯化抗体32A12MAb、37A3MAb、48G9MAb10μg/mL，100μL，4℃45min；离心弃上清后清洗2次，之后加入Goat-anti-Mouse FITC(1:200使用)，50μL/孔，4℃孵育45min；离心弃上清后清洗2次，之后重悬细胞用流式细胞仪(Guava easyCyte 8HT)进行检测，结果用FlowJo7.6.1统计作图。 Take CS1-positive human myeloma cells MM.1S (Cell Bank of Type Culture Collection, Chinese Academy of Sciences), NCI H929 (Cell Bank of Type Culture Collection, Chinese Academy of Sciences), RPMI 8226 (ATCC); and CS1-negative human embryos Lung fibroblasts WI38 (Cell Bank of Type Culture Collection, Chinese Academy of Sciences), human embryonic kidney cells HEK293 (China Type Culture Collection), 2×10 5 cells/well in a 96-well round-bottom culture plate, centrifuged and discarded After clearing, wash twice, then add purified antibodies 32A12MAb, 37A3MAb, 48G9MAb at 10 μg/mL, 100 μL, 45min at 4°C; centrifuge and discard the supernatant, wash twice, then add Goat-anti-Mouse FITC (used at 1:200), 50 μL /well, incubated at 4°C for 45 min; centrifuged to discard the supernatant, washed twice, and then resuspended the cells for detection by a flow cytometer (Guava easyCyte 8HT), and the results were plotted using FlowJo7.6.1 statistics.
结果(图4)显示3个CS1杂交瘤抗体32A12MAb、37A3MAb、48G9MAb特异性的结合CS1阳性细胞(MM.1S、RPMI8226、NCI-H929)，不结合CS1阴性细胞(HEK293、WI38)。The results ( FIG. 4 ) showed that the three CS1 hybridoma antibodies 32A12MAb, 37A3MAb, and 48G9MAb specifically bound to CS1-positive cells (MM.1S, RPMI8226, NCI-H929), but did not bind to CS1-negative cells (HEK293, WI38).
实施例8、人源化抗体的制备Example 8. Preparation of humanized antibodies
通过CDR移植的方法对杂交瘤抗体32A12、37A3、48G9进行人源化改造(文献Jones et al.1986)。经过序列相似性比对，选取相似度最高的抗体种系作为抗体模板。The hybridoma antibodies 32A12, 37A3 and 48G9 were humanized by the method of CDR transplantation (document Jones et al. 1986). After sequence similarity comparison, the antibody germline with the highest similarity was selected as the antibody template.
选取来自IMGT数据库的germline序列IGHV3-7*01作为32A12重链的抗体模板，选取来自IMGT数据库的germline序列IGKV1-9*01作为32A12轻链的抗体模板。将32A12抗体的CDR区替换掉抗体模板的CDR区，同时，将重链第61位的苏氨酸突变成丙氨酸以去掉N糖基化位点，从而形成人源化抗体hu32A12的轻链(SEQ ID NO:34)和重链(SEQ ID NO:31)。The germline sequence IGHV3-7*01 from the IMGT database was selected as the antibody template of the 32A12 heavy chain, and the germline sequence IGKV1-9*01 from the IMGT database was selected as the antibody template of the 32A12 light chain. The CDR region of the 32A12 antibody was replaced with the CDR region of the antibody template, and at the same time, the threonine at position 61 of the heavy chain was mutated to alanine to remove the N-glycosylation site, thereby forming the light of the humanized antibody hu32A12. chain (SEQ ID NO:34) and heavy chain (SEQ ID NO:31).
选取来自IMGT数据库的germline序列IGHV1-69*02作为37A3重链的抗体模板，选取来自IMGT数据库的germline序列IGKV1-33*01作为37A3轻链的抗体模板。将37A3抗体的CDR区替换掉抗体模板的CDR区，形成人源化抗体hu37A3的轻链(SEQ ID NO:38)和重链(SEQ ID NO:36)。The germline sequence IGHV1-69*02 from the IMGT database was selected as the antibody template of the 37A3 heavy chain, and the germline sequence IGKV1-33*01 from the IMGT database was selected as the antibody template of the 37A3 light chain. The CDR regions of the 37A3 antibody were replaced with the CDR regions of the antibody template to form the light chain (SEQ ID NO:38) and heavy chain (SEQ ID NO:36) of the humanized antibody hu37A3.
选取来自IMGT数据库的germline序列IGHV1-2*02作为48G9重链的抗体模板，选取来自IMGT数据库的germline序列IGKV1-33*01作为48G9轻链的抗体模板。将48G9抗体的CDR区替换掉抗体模板的CDR区，同时，将重链第102位的丝氨酸突变成丙氨酸以去掉N糖基化位点，从而形成人源化抗体hu48G9的轻链(SEQ ID NO:43)和重链(SEQ ID NO:40)。The germline sequence IGHV1-2*02 from the IMGT database was selected as the antibody template of the 48G9 heavy chain, and the germline sequence IGKV1-33*01 from the IMGT database was selected as the antibody template of the 48G9 light chain. The CDR region of the 48G9 antibody was replaced with the CDR region of the antibody template, and at the same time, the serine at position 102 of the heavy chain was mutated to alanine to remove the N-glycosylation site, thereby forming the light chain of the humanized antibody hu48G9 ( SEQ ID NO:43) and heavy chain (SEQ ID NO:40).
通过常规的重组表达技术，制备人源化抗体hu32A12、hu37A3、hu48G9用于后续检测。Through conventional recombinant expression technology, humanized antibodies hu32A12, hu37A3, and hu48G9 were prepared for subsequent detection.
表3 人源化抗体的序列Table 3 Sequences of humanized antibodies
   氨基酸序列amino acid sequence 核苷酸序列Nucleotide sequence
hu32A12 VHhu32A12 VH SEQ ID NO:31SEQ ID NO: 31 SEQ ID NO:33SEQ ID NO: 33
hu32A12 VLhu32A12 VL SEQ ID NO:34SEQ ID NO: 34 SEQ ID NO:35SEQ ID NO: 35
hu37A3 VHhu37A3 VH SEQ ID NO:36SEQ ID NO: 36 SEQ ID NO:37SEQ ID NO: 37
hu37A3 VLhu37A3 VL SEQ ID NO:38SEQ ID NO: 38 SEQ ID NO:39SEQ ID NO: 39
hu48G9 VHhu48G9 VH SEQ ID NO:40SEQ ID NO: 40 SEQ ID NO:42SEQ ID NO: 42
hu48G9 VLhu48G9 VL SEQ ID NO:43SEQ ID NO: 43 SEQ ID NO:44SEQ ID NO: 44
实施例9、利用ELISA测定CS1人源化抗体与重组蛋白hSLAMF7-avi-His的结合活性Example 9. Determination of the binding activity of CS1 humanized antibody to recombinant protein hSLAMF7-avi-His by ELISA
包被hSLAMF7-avi-his，1.5μg/ml，100μL/孔，4℃过夜；室温封闭2h；封闭后清洗，之后分别加入人源化抗体hu32A12、hu37A3、hu48G9从100μg/mL开始5倍梯度稀释11次，100μL/孔，室温孵育1小时；清洗后，每孔加入100μL 4000倍稀释的 HRP-Goat-anti-Mouse IgG(Fc specific)(sigma，CAT A0168，LOT 077M4820V)，室温孵育1小时，清洗后加入TMB显色。用酶标仪(Spectra
Figure PCTCN2022084732-appb-000002
340PC 384)测定OD450nM和OD650nM吸光值，并以OD450-OD650作为最后检测结果，使用GraphPad Prism8.0作图。 Coated with hSLAMF7-avi-his, 1.5μg/ml, 100μL/well, overnight at 4°C; blocked at room temperature for 2 hours; washed after blocking, and then humanized antibodies hu32A12, hu37A3, and hu48G9 were added to 5-fold gradient dilution from 100 μg/mL 11 times, 100 μL/well, incubate at room temperature for 1 hour; after washing, add 100 μL of 4000-fold diluted HRP-Goat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M4820V) to each well, incubate at room temperature for 1 hour, After washing, add TMB to develop color. Using a microplate reader (Spectra
Figure PCTCN2022084732-appb-000002
340PC 384) to measure the absorbance values of OD450nM and OD650nM, and take OD450-OD650 as the final detection result, and use GraphPad Prism8.0 to make a graph.
结果(图5)显示3个人源化抗体hu32A12、hu37A3、hu48G9与hSLAMF7-avi-his蛋白结合EC50在同一水平，略好于对照抗体Luc90、huLuc63，且好于对应的鼠抗32A12MAb、37A3MAb、48G9MAb。The results (Fig. 5) showed that the three humanized antibodies hu32A12, hu37A3, and hu48G9 bound to the hSLAMF7-avi-his protein at the same EC50 level, slightly better than the control antibodies Luc90, huLuc63, and better than the corresponding mouse anti-32A12MAb, 37A3MAb, 48G9MAb .
表4 鼠抗及人源化抗体与重组蛋白hSLAMF7-avi-His的结合情况对比Table 4 Comparison of binding between mouse anti- and humanized antibodies and recombinant protein hSLAMF7-avi-His
EC50(μg/mL)EC50(μg/mL) 32A1232A12 37A337A3 48G948G9 Luc90Luc90 huLuc63huLuc63
鼠抗mouse anti 0.05840.0584 0.034240.03424 0.034960.03496 0.021170.02117   
人源化抗体humanized antibody 0.011260.01126 0.017260.01726 0.015760.01576    0.0220.022
实施例10、测定CS1人源化抗体与人多发性骨髓瘤细胞的结合活性Example 10. Determination of the binding activity of CS1 humanized antibody to human multiple myeloma cells
取MM.1S细胞2×10 5个细胞/孔于96孔圆底培养板中，离心弃上清后清洗2次，之后分别加入人源化抗体hu32A12、hu37A3、hu48G9 40μg/mL开始5倍梯度稀释7次，100μL，4℃45min；离心弃上清后清洗2次，之后加入Goat-anti-Mouse FITC(KANFCHEN，CAT:KC-MM-095，1:200使用)，50μL/孔，4℃孵育45min；离心弃上清后清洗2次，之后重悬细胞用流式细胞仪(Guava easyCyte 8HT)进行检测，结果用FlowJo7.6.1统计及GraphPad Prism8.0作图。 Take 2×10 5 cells/well of MM.1S cells in a 96-well round-bottom culture plate, centrifuge and discard the supernatant, wash twice, and then add humanized antibodies hu32A12, hu37A3, and hu48G9 at 40 μg/mL to start a 5-fold gradient Diluted 7 times, 100 μL, 45min at 4°C; centrifuged and discarded the supernatant, washed twice, then added Goat-anti-Mouse FITC (KANFCHEN, CAT:KC-MM-095, used at 1:200), 50 μL/well, 4°C Incubate for 45 min; centrifuge to discard the supernatant, wash twice, and then resuspend the cells for detection by flow cytometer (Guava easyCyte 8HT).
结果(图6)显示人源化抗体hu37A3、hu48G9与MM.1S结合能力较强，和对照抗体Luc90-mFc相似；人源化抗体hu32A12与MM.1S结合能力较弱，和对照抗体huLuc63-mFc相似。The results (Fig. 6) showed that the humanized antibodies hu37A3 and hu48G9 had strong binding ability to MM.1S, similar to the control antibody Luc90-mFc; the humanized antibody hu32A12 had weaker binding ability to MM.1S, and the control antibody huLuc63-mFc resemblance.
表5 鼠抗及人源化抗体与MM.1S结合能力对比Table 5 Comparison of the binding ability of mouse anti- and humanized antibodies to MM.1S
EC50(μg/mL)EC50(μg/mL) 32A1232A12 37A337A3 48G948G9 Luc90Luc90 huLuc63huLuc63
鼠抗mouse anti 1.7581.758 0.053860.05386 0.18450.1845 0.082350.08235   
人源化抗体humanized antibody 96.0196.01 0.067660.06766 0.094780.09478    ～7644~7644
实施例11、检测CS1人源化抗体与不同种属SLAMF7重组蛋白的结合Example 11. Detection of binding of CS1 humanized antibody to SLAMF7 recombinant proteins of different species
包被hSLAMF7-huFc，mSLAMF7-huFc，cySLAMF7，hu-Fc，5μg/mL，100μL/孔，4℃过夜；室温封闭2h；清洗，之后分别加入人源化抗体hu32A12、hu37A3、hu48G95μg/mL，100μL/孔，室温孵育1小时；清洗后，每孔加入100μL 4000倍稀释的HRP-Goat-anti-Mouse IgG(Fc specific)(sigma，CAT A0168，LOT 077M4820V)，室温 孵育1小时，清洗后加入TMB显色。用酶标仪测定OD450nM和OD650nM吸光值，并以OD450-OD650作为最后检测结果。Coated with hSLAMF7-huFc, mSLAMF7-huFc, cySLAMF7, hu-Fc, 5 μg/mL, 100 μL/well, overnight at 4°C; blocked at room temperature for 2 h; washed, and then added humanized antibodies hu32A12, hu37A3, hu48G 95 μg/mL, 100 μL /well, incubate at room temperature for 1 hour; after washing, add 100 μL of 4000-fold diluted HRP-Goat-anti-Mouse IgG (Fc specific) (sigma, CAT A0168, LOT 077M4820V) to each well, incubate at room temperature for 1 hour, and add TMB after washing Color rendering. The absorbance values of OD450nM and OD650nM were measured with a microplate reader, and OD450-OD650 was used as the final detection result.
结果(图7)显示人源化抗体hu37A3、hu48G9均与人SLAMF7和猴SLAMF7结合，人源化抗体hu32A12只与人SLAMF7结合，3个人源化抗体hu37A3、hu48G9、hu32A12都不与鼠SLAMF7结合。The results (Fig. 7) showed that the humanized antibodies hu37A3 and hu48G9 both bound to human SLAMF7 and monkey SLAMF7, the humanized antibody hu32A12 only bound to human SLAMF7, and the three humanized antibodies hu37A3, hu48G9, and hu32A12 did not bind to murine SLAMF7.
实施例12、检测CS1人源化抗体与细胞的结合Example 12. Detection of binding of CS1 humanized antibody to cells
取CS1阳性细胞MM.1S、NCI-H929及CS1阴性细胞WI38、HEK293，2×10 5个细胞/孔96孔圆底培养板中，离心弃上清后清洗2次，之后分别加入人源化抗体hu32A12、hu37A3、hu48G9 5μg/mL，100μL，4℃45min；离心弃上清后清洗2次，之后加入Goat-anti-Mouse FITC(1:200使用)，50μL/孔，4℃孵育45min；离心弃上清后清洗2次，之后重悬细胞用流式细胞仪(Guava easyCyte 8HT)进行检测，结果用FlowJo7.6.1统计作图。 Take CS1-positive cells MM.1S, NCI-H929 and CS1-negative cells WI38, HEK293, 2 × 10 5 cells/well in a 96-well round-bottom culture plate, centrifuge and discard the supernatant, wash twice, and then add humanized cells respectively. Antibodies hu32A12, hu37A3, hu48G9 5μg/mL, 100μL, 4°C for 45min; centrifuge to discard the supernatant, wash twice, then add Goat-anti-Mouse FITC (used at 1:200), 50μL/well, incubate at 4°C for 45min; centrifuge The supernatant was discarded, washed twice, and the cells were resuspended for detection by flow cytometer (Guava easyCyte 8HT), and the results were plotted using FlowJo7.6.1.
结果(图8)显示CS1人源化抗体hu37A3、hu48G9均与CS1阳性细胞(MM.1S、NCI-H929)结合明显，hu32A12与CS1阳性细胞(MM.1S、NCI-H929)结合有微弱结合，三个人源化抗体都不与CS1阴性细胞(HEK293、WI38)结合。The results (Fig. 8) showed that the CS1 humanized antibodies hu37A3 and hu48G9 were obviously bound to CS1-positive cells (MM.1S, NCI-H929), and hu32A12 was weakly bound to CS1-positive cells (MM.1S, NCI-H929). None of the three humanized antibodies bound to CS1 negative cells (HEK293, WI38).
实施例13、测定CS1人源化抗体的亲和力Example 13. Determination of the affinity of CS1 humanized antibody
使用Cytiva提供的Mouse Antibody Capture Kit(cat:BR100838)，根据说明书将3个人源化抗体hu32A12、hu37A3、hu48G9分别通过氨基偶联的方式包被在CM5芯片(cat:BR100012)上。目标抗体hu32A12、hu37A3、hu48G9分别与mFc(SEQ ID NO：75)融合表达得到hu32A12-mFc、hu37A3-mFc、hu48G9-mFc融合蛋白作为配体，重组表达的huSLAMF7-avi-his或cySLAMF7-huFc 500nM起3倍梯度稀释作为流动相。检测完成后使用Evaluation Software拟合得到亲和力结果。Using the Mouse Antibody Capture Kit (cat: BR100838) provided by Cytiva, three humanized antibodies hu32A12, hu37A3, and hu48G9 were coated on a CM5 chip (cat: BR100012) by amino coupling according to the instructions. The target antibodies hu32A12, hu37A3, and hu48G9 were respectively fused and expressed with mFc (SEQ ID NO: 75) to obtain hu32A12-mFc, hu37A3-mFc, hu48G9-mFc fusion proteins as ligands, and recombinantly expressed huSLAMF7-avi-his or cySLAMF7-huFc 500nM A 3-fold serial dilution was used as the mobile phase. After the detection is completed, use the Evaluation Software to fit the affinity results.
结果(表6)显示人源化抗体hu 37A3、hu 48G9和人CS1的亲和力KD分别为3.9nM(图9)、940pM(图10)，亲和力较高，优于对照抗体KD7nM(图11、图12)；人源化抗体hu 37A3、hu 48G9和猴CS1的亲和力KD分别为37nM(图13)和107nM(图14)，亲和力弱。抗体hu32A12与人CS1亲和力为12.1nM(图15)，亲和力较弱。The results (Table 6) show that the affinity KDs of the humanized antibodies hu 37A3, hu 48G9 and human CS1 are 3.9nM (Fig. 9) and 940pM (Fig. 10), respectively, and the affinity is higher than that of the control antibody KD7nM (Fig. 11, Fig. 10). 12); the affinity KDs of the humanized antibodies hu 37A3, hu 48G9 and monkey CS1 are 37nM (Fig. 13) and 107nM (Fig. 14), respectively, and the affinity is weak. The affinity of antibody hu32A12 to human CS1 was 12.1 nM ( FIG. 15 ), which was weak.
表6 CS1人源化抗体的亲和力测定结果Table 6 Affinity determination results of CS1 humanized antibody
Figure PCTCN2022084732-appb-000003
Figure PCTCN2022084732-appb-000003
Figure PCTCN2022084732-appb-000004
Figure PCTCN2022084732-appb-000004
实施例14、CS1人源化抗体-Fc融合蛋白聚集情况分析Example 14. Analysis of the aggregation of CS1 humanized antibody-Fc fusion protein
亲和层析后的人源化单链抗体融合蛋白hu37A3-mFc、hu48G9-mFc、hu32A12-mFc，采用SEC分析其聚集情况。柱型为XK16/70(GE Healthcare)，层析介质为120mL的Superdex200 prep grade，流动相为PBS溶液，1mL/min流速平衡柱子1.5CV以上直至UV280、电导和pH基线平稳。样品使用0.5mL样品环上样，纯化流速为1mL/min，单体峰出峰体积67mL左右。The humanized single chain antibody fusion proteins hu37A3-mFc, hu48G9-mFc and hu32A12-mFc after affinity chromatography were analyzed by SEC for their aggregation. The column type is XK16/70 (GE Healthcare), the chromatography medium is 120 mL of Superdex200 prep grade, the mobile phase is PBS solution, and the flow rate of 1 mL/min balances the column for more than 1.5 CV until the UV280, conductivity and pH baselines are stable. The sample was loaded with a 0.5mL sample loop, the purification flow rate was 1mL/min, and the peak volume of the monomer peak was about 67mL.
结果(图16-图18)显示hu37A3-mFc、hu32A12-mFc、hu48G9-mFc的单体形式的抗体占比分别为82％、83％、93％。从聚集程度来看，3个人源化抗体的单体率都在80％以上，说明这3个抗体的scFv结构稳定，不容易聚集。The results (Fig. 16-Fig. 18) showed that the proportion of antibodies in the monomeric form of hu37A3-mFc, hu32A12-mFc, and hu48G9-mFc was 82%, 83%, and 93%, respectively. In terms of the degree of aggregation, the monomer ratios of the three humanized antibodies are all above 80%, indicating that the scFv structures of the three antibodies are stable and not easy to aggregate.
实施例15：抗CS1嵌合抗原受体CAR-T细胞的制备Example 15: Preparation of anti-CS1 chimeric antigen receptor CAR-T cells
(1)hu32A12 scFv、hu37A3 scFv、hu48G9 scFv的构建(1) Construction of hu32A12 scFv, hu37A3 scFv, and hu48G9 scFv
分别针对人源化抗体hu32A12，hu37A3，hu48G9的VH和VL片段，构成hu32A12 scFv、hu37A3 scFv、hu48G9 scFv。hu 32A12 scFv氨基酸序列(SEO ID NO:50)，hu 37A3 scFv氨基酸序列(SEO ID NO:51)，hu 48G9 scFv氨基酸序列(SEO ID NO:52)。The VH and VL fragments of the humanized antibodies hu32A12, hu37A3, and hu48G9, respectively, constitute hu32A12 scFv, hu37A3 scFv, and hu48G9 scFv. hu 32A12 scFv amino acid sequence (SEO ID NO: 50), hu 37A3 scFv amino acid sequence (SEO ID NO: 51), hu 48G9 scFv amino acid sequence (SEO ID NO: 52).
(2)嵌合抗原受体CAR-T细胞的制备(2) Preparation of chimeric antigen receptor CAR-T cells
以PRRLSIN-cPPT.EF-1α为载体，分别插入表7所示的嵌合抗原受体序列构建了表达人源化抗体hu32A12、hu37A3、hu48G9、huLuc63、Luc90的二代嵌合抗原受体的慢病毒质粒，包括PRRL-hu32A12 BBz，PRRL-hu37A3 BBz，PRRL-hu48G9 BBz，PRRL-huLuc63 BBz，PRR-Luc90 BBz。具体方法如(Berahovich R,2018)文献描述。将上述慢病毒质粒分别转染293T细胞，得到相应慢病毒hu32A12 BBz CAR、hu37A3 BBz CAR、hu48G9 BBz CAR、huLuc63 BBz CAR、Luc90 BBz CAR。Using PRRLSIN-cPPT.EF-1α as a vector, the chimeric antigen receptor sequences shown in Table 7 were inserted to construct the second-generation chimeric antigen receptor expressing humanized antibodies hu32A12, hu37A3, hu48G9, huLuc63, Luc90. Viral plasmids, including PRRL-hu32A12 BBz, PRRL-hu37A3 BBz, PRRL-hu48G9 BBz, PRRL-huLuc63 BBz, PRR-Luc90 BBz. The specific method is described in the literature (Berahovich R, 2018). The above lentiviral plasmids were transfected into 293T cells to obtain the corresponding lentiviruses hu32A12 BBz CAR, hu37A3 BBz CAR, hu48G9 BBz CAR, huLuc63 BBz CAR, and Luc90 BBz CAR.
表7 嵌合抗原受体Table 7 Chimeric Antigen Receptors
Figure PCTCN2022084732-appb-000005
Figure PCTCN2022084732-appb-000005
Figure PCTCN2022084732-appb-000006
Figure PCTCN2022084732-appb-000006
注：CD8α信号肽序列(SEQ ID NO:71)；scFv(hu32A12)序列(SEQ ID NO：50)；scFv(hu37A3)序列(SEQ ID NO：51)；scFv(hu48G9)序列(SEQ ID NO：52)；huLuc63 VH序列(SEQ ID NO：63)、(G4S)3linker(SEQ ID NO：74)、huLuc63 VL序列(SEQ ID NO：64)；Luc90 VH序列(SEQ ID NO：61)、Luc90 VL序列(SEQ ID NO：62)；CD8α铰链区序列(SEQ ID NO:53)；CD8α跨膜结构域序列(SEQ ID NO:54)、CD137胞内信号结构域序列(SEQ ID NO:55)、CD3Zeta胞内信号结构域序列(SEQ ID NO:56)、hu32A12 BBz序列(SEQ ID NO：45)、hu37A3 BBz序列(SEQ ID NO：46)、hu48G9 BBz序列(SEQ ID NO：47)、huLuc63 BBz序列(SEQ ID NO：48)、Luc90 BBz序列(SEQ ID NO：49)。Note: CD8α signal peptide sequence (SEQ ID NO: 71); scFv (hu32A12) sequence (SEQ ID NO: 50); scFv (hu37A3) sequence (SEQ ID NO: 51); scFv (hu48G9) sequence (SEQ ID NO: 51) 52); huLuc63 VH sequence (SEQ ID NO: 63), (G4S)3linker (SEQ ID NO: 74), huLuc63 VL sequence (SEQ ID NO: 64); Luc90 VH sequence (SEQ ID NO: 61), Luc90 VL sequence (SEQ ID NO:62); CD8α hinge region sequence (SEQ ID NO:53); CD8α transmembrane domain sequence (SEQ ID NO:54), CD137 intracellular signal domain sequence (SEQ ID NO:55), CD3Zeta intracellular signaling domain sequence (SEQ ID NO:56), hu32A12 BBz sequence (SEQ ID NO:45), hu37A3 BBz sequence (SEQ ID NO:46), hu48G9 BBz sequence (SEQ ID NO:47), huLuc63 BBz Sequence (SEQ ID NO:48), Luc90 BBz sequence (SEQ ID NO:49).
T淋巴细胞活化：从人外周血中分离出PBMC细胞，以约1×10 6个细胞/mL密度加入淋巴细胞培养基液培养，加入同时包被有抗CD3和CD28抗体的磁珠和终浓度300U/mL的重组人IL-2在37℃、5％CO 2下刺激培养48h； T lymphocyte activation: PBMC cells were isolated from human peripheral blood, cultured in lymphocyte culture medium at a density of about 1×10 6 cells/mL, and magnetic beads coated with both anti-CD3 and CD28 antibodies were added and the final concentration 300U/mL recombinant human IL-2 was stimulated and cultured at 37°C, 5% CO 2 for 48h;
然后分别加入1x10 7个上述制备的hu32A12 BBz CAR、hu37A3 BBz CAR、hu48G9 BBz CAR、huLuc63 BBz CAR、Luc90 BBz CAR慢病毒转导，分别得到hu32A12 BBz CAR-T细胞、hu37A3 BBz CAR-T细胞、hu48G9 BBz CAR-T细胞、huLuc63 BBz CAR-T细胞、Luc90 BBz CAR-T细胞。 Then, 1×10 7 hu32A12 BBz CARs, hu37A3 BBz CARs, hu48G9 BBz CARs, huLuc63 BBz CARs, and Luc90 BBz CARs prepared above were added for lentiviral transduction to obtain hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, and hu48G9 respectively. BBz CAR-T cells, huLuc63 BBz CAR-T cells, Luc90 BBz CAR-T cells.
实施例16：T淋巴细胞嵌合抗原受体表达Example 16: T lymphocyte chimeric antigen receptor expression
将实施例15制备的CAR-T细胞洗涤并悬浮在流式细胞仪缓冲液(含1％胎牛血清(FBS)的磷酸盐缓冲液(PBS))中，细胞密度为1x10 6个细胞/孔。加入用生物素(Biotin)标记的CS1抗原(10μg/mL，用FACS缓冲液稀释)，在4℃下孵育45分钟。用流式细胞仪缓冲液冲洗两次。加入Streptavidin-PE Conjugate抗体(eBiosciences，1:200，用FACS缓冲液稀释)，在4℃，避光下孵育30分钟。用流式细胞仪缓冲液冲洗两次，再用300μl流式细胞仪缓冲液重悬细胞，用流式仪检测。 The CAR-T cells prepared in Example 15 were washed and suspended in flow cytometry buffer (phosphate buffered saline (PBS) containing 1 % fetal bovine serum (FBS)) at a cell density of 1x10 cells/well . CS1 antigen labeled with Biotin (10 μg/mL, diluted with FACS buffer) was added and incubated at 4°C for 45 minutes. Rinse twice with flow cytometry buffer. Streptavidin-PE Conjugate antibody (eBiosciences, 1:200, diluted with FACS buffer) was added and incubated at 4°C for 30 minutes in the dark. Rinse twice with flow cytometry buffer, resuspend cells in 300 μl of flow cytometry buffer, and detect by flow cytometry.
结果显示：抗体hu32A12，hu37A3，hu48G9形成的CAR T细胞CAR阳性率优于对照抗体huluc63(图19)，且十分稳定，经过5-11天的培养阳性率基本保持不变(图 20)。The results showed that the CAR positive rate of CAR T cells formed by antibodies hu32A12, hu37A3 and hu48G9 was better than that of the control antibody huluc63 (Figure 19), and was very stable, and the positive rate remained basically unchanged after 5-11 days of culture (Figure 20).
实施例17：CS1 CAR T细胞的细胞毒性测定Example 17: Cytotoxicity assay of CS1 CAR T cells
通过乳酸脱氢酶(LDH)法检测CS1 CAR-T细胞在体外对肿瘤细胞系的杀伤效果。采用CytoTox
Figure PCTCN2022084732-appb-000007
Non-Radioactive Cytotoxicity Assay试剂盒(Promega，G1780)检测细胞毒性，具体步骤参照试剂盒说明书。 The killing effect of CS1 CAR-T cells on tumor cell lines in vitro was detected by lactate dehydrogenase (LDH) assay. Using CytoTox
Figure PCTCN2022084732-appb-000007
Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780) detects cytotoxicity, and the specific steps refer to the kit instructions.
靶细胞：表达CS1的多发性骨髓瘤细胞MM.1S，NCl-H929、RPMI-8226和不表达CS1的HEK293细胞。Target cells: CS1 expressing multiple myeloma cells MM.1S, NCl-H929, RPMI-8226 and HEK293 cells not expressing CS1.
效应细胞：UTD(未感染病毒的T细胞)、hu32A12 BBz CAR-T细胞、hu37A3 BBz CAR-T细胞、hu48G9 BBz CAR-T细胞、huLuc63 BBz CAR-T细胞、Luc90 BBz CAR-T细胞。Effector cells: UTD (virus-uninfected T cells), hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, Luc90 BBz CAR-T cells.
分别按效靶比3:1,1:1,1:3将效应细胞和靶细胞(10000个细胞/孔)先后铺入96孔板中(每孔200ul 1640+10％FBS体系)，在37℃、5％CO 2下共培养16小时，同时根据说明书设置自发释放孔和最大释放孔。第二天在最大释放孔中加入20μl裂解液，反应30分钟。然后从每孔中取出50μl培养上清，再加入50μl反应底物，室温下避光孵育25分钟，加入50μl终止液终止反应，用酶标仪检测每孔的OD490/650nm吸收峰。 The effector cells and target cells (10,000 cells/well) were successively plated into 96-well plates (200ul 1640+10% FBS system per well) according to the effector-target ratio of 3:1, 1:1, 1:3 respectively. Co-incubate for 16 hours at °C, 5% CO2 , while setting up spontaneous release wells and maximal release wells according to the instructions. The next day, 20 μl of lysate was added to the maximum release well, and the reaction was carried out for 30 minutes. Then remove 50 μl of culture supernatant from each well, add 50 μl of reaction substrate, incubate at room temperature for 25 minutes in the dark, add 50 μl of stop solution to stop the reaction, and use a microplate reader to detect the OD490/650nm absorption peak of each well.
按照下面公式计算杀伤率：Calculate the kill rate according to the following formula:
细胞毒性％＝[实验组LDH释放量(Avg.)–效应细胞自发LDH释放量(Avg.)–靶细胞自发LDH释放量(Avg.)]/[靶细胞最大LDH释放量(Avg.)-靶细胞自发LDH释放量(Avg.)–体积校准(Avg.)]×100％Cytotoxicity %=[The amount of LDH released by experimental group (Avg.) – The amount of spontaneous LDH released by effector cells (Avg.) – The amount of spontaneous LDH released by target cells (Avg.)]/[The maximum amount of LDH released by target cells (Avg.)- Target cell spontaneous LDH release (Avg.) – volume calibration (Avg.)] × 100%
结果显示：hu32A12 BBz CAR-T细胞、hu37A3 BBz CAR-T细胞、hu48G9 BBz CAR-T细胞对于表达CS1的靶细胞的体外杀伤效果明显，且与效靶比成明显的剂量效应关系。对于不表达CS1的HEK293细胞，CS1 CAR T在不同的效靶比下几乎没有杀伤作用(图21)。说明了hu32A12 BBz CAR-T细胞、hu37A3 BBz CAR-T细胞、hu48G9 BBz CAR-T细胞能较好的特异性识别并杀伤CS1阳性肿瘤细胞。The results showed that hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, and hu48G9 BBz CAR-T cells had obvious killing effects on target cells expressing CS1 in vitro, and there was an obvious dose-effect relationship with the effect-target ratio. For HEK293 cells that do not express CS1, CS1 CAR T had almost no killing effect at different effector-target ratios (Figure 21). It shows that hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, and hu48G9 BBz CAR-T cells can better specifically recognize and kill CS1-positive tumor cells.
实施例18：CS1 CAR T细胞体外诱导细胞因子释放实验Example 18: CS1 CAR T cells induce cytokine release experiment in vitro
进一步检查实施例17中hu32A12 BBz CAR-T细胞、hu37A3 BBz CAR-T细胞、hu48G9 BBz CAR-T细胞、huLuc63 BBz CAR-T细胞、Luc90 BBz CAR-T细胞在靶细胞刺激下细胞因子的分泌情况。The cytokine secretion of hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, and Luc90 BBz CAR-T cells under stimulation by target cells in Example 17 was further examined .
收集实施例17中hu32A12 BBz CAR-T细胞、hu37A3 BBz CAR-T细胞、hu48G9 BBz CAR-T细胞、huLuc63 BBz CAR-T细胞、Luc90 BBz CAR-T细胞与肿瘤细胞共孵育的上清液，并按BD TM CBA试剂盒(BD Pharmingen公司)说明书进行细胞因子检测。 The supernatants of the hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, and Luc90 BBz CAR-T cells incubated with tumor cells in Example 17 were collected, and Cytokine detection was performed according to the instructions of BD ™ CBA kit (BD Pharmingen).
结果显示：细胞因子IFN-γ的分泌情况：hu32A12 BBz CAR-T细胞、hu37A3 BBz  CAR-T细胞、hu48G9 BBz CAR-T细胞、huLuc63 BBz CAR-T细胞、Luc90 BBz CAR-T细胞与MM.1S细胞共孵育后，可以激活IFN-γ细胞因子的分泌，且与效靶比呈现一定的剂量效应关系(图22)。The results showed: the secretion of cytokine IFN-γ: hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, Luc90 BBz CAR-T cells and MM.1S After the cells were co-incubated, the secretion of IFN-γ cytokines could be activated, and there was a certain dose-effect relationship with the effector-target ratio (Figure 22).
细胞因子TNF-α的分泌情况：hu32A12 BBz CAR-T细胞、hu37A3 BBz CAR-T细胞、hu48G9 BBz CAR-T细胞、huLuc63 BBz CAR-T细胞、Luc90 BBz CAR-T细胞与MM.1S细胞共孵育后，各CS1 CAR-T TNF-α的分泌明显增强(图23)。与不表达CS1的HEK293细胞共孵育后,各CS1 CAR T都无明显的TNF-α分泌，说明其不产生非特异的TNF-α细胞因子分泌。Cytokine TNF-α secretion: hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, Luc90 BBz CAR-T cells were incubated with MM.1S cells After that, the secretion of each CS1 CAR-T TNF-α was significantly enhanced (Fig. 23). After co-incubation with HEK293 cells that do not express CS1, each CS1 CAR T has no obvious TNF-α secretion, indicating that it does not produce non-specific TNF-α cytokine secretion.
细胞因子IL-2的分泌情况：hu32A12 BBz CAR-T细胞、hu37A3 BBz CAR-T细胞、hu48G9 BBz CAR-T细胞、huLuc63 BBz CAR-T细胞、Luc90 BBz CAR-T细胞与MM.1S细胞共孵育后，IL-2的分泌量与效靶比呈负相关，效靶比越低IL-2的浓度越高，这可能与T细胞增殖消耗IL-2有关(图24)。与不表达CS1的HEK293的细胞共孵育后，各CS1 CAR-T都无明显的IL-2分泌，说明CS1 CAR-T与CS1阴性细胞共孵育后不产生非特异IL-2分泌。Cytokine IL-2 secretion: hu32A12 BBz CAR-T cells, hu37A3 BBz CAR-T cells, hu48G9 BBz CAR-T cells, huLuc63 BBz CAR-T cells, Luc90 BBz CAR-T cells were incubated with MM.1S cells Afterwards, the secretion amount of IL-2 was negatively correlated with the effector-target ratio, and the lower the effector-target ratio was, the higher the IL-2 concentration was, which may be related to the consumption of IL-2 by T cell proliferation (Figure 24). After co-incubation with HEK293 cells that do not express CS1, each CS1 CAR-T had no obvious IL-2 secretion, indicating that CS1 CAR-T did not produce non-specific IL-2 secretion after co-incubation with CS1-negative cells.
实施例19：可溶性CS1蛋白对CS1 CAR T细胞体外细胞毒性杀伤的影响Example 19: Effect of soluble CS1 protein on cytotoxic killing of CS1 CAR T cells in vitro
临床研究中发现，多发性骨髓瘤(MM)病人体内有可溶性的CS1蛋白存在，且有文献(Mariko Ishibashi，2018)指出，MM病人血清中可溶性CS1的浓度大多数在0.091–14.7ng/mL。因此，我们在实施例17LDH法检测CAR-T体外毒性杀伤的基础上，在加入效应细胞和靶细胞后立即加入不同浓度的重组表达的CS1蛋白(0、1、10、100ng/mL)，检测其对CS1 CAR T细胞毒性杀伤的影响。Clinical studies have found that soluble CS1 protein exists in patients with multiple myeloma (MM), and some literature (Mariko Ishibashi, 2018) pointed out that the concentration of soluble CS1 in serum of MM patients is mostly 0.091-14.7ng/mL. Therefore, we added different concentrations of recombinantly expressed CS1 protein (0, 1, 10, 100ng/mL) immediately after adding effector cells and target cells on the basis of the LDH method in Example 17 to detect the toxicity of CAR-T in vitro. Its effect on CS1 CAR T cytotoxic killing.
结果显示，CS1 CAR-T细胞对靶细胞的杀伤效果随可溶性CS1蛋白浓度上升并无明显变化(图25)，说明可溶性CS1不影响CS1 CAR-T体外细胞毒性杀伤。The results showed that the killing effect of CS1 CAR-T cells on target cells did not change significantly with the increase of soluble CS1 protein concentration (Figure 25), indicating that soluble CS1 did not affect the cytotoxic killing of CS1 CAR-T in vitro.
实施例20：CS1 CAR T细胞经靶抗原刺激后耗竭标志物表达情况测定Example 20: Determination of the expression of exhaustion markers in CS1 CAR T cells stimulated by target antigens
通过流式检测CS1 CAR-T在受靶抗原CS1阳性多发性骨髓瘤细胞系NCl-H929刺激后，耗竭标志物PD-1，TIM-3，LAG-3的表达情况。具体过程如下：分别加入CS1阳性多发性骨髓瘤细胞系NCl-H929(50000个细胞/孔，500μl/孔)及各CS1 CAR-T(50000个细胞/孔，500μl/孔)于48孔细胞培养板，并同时设置UTD(未转染CAR的T细胞)为对照。在37℃、5％CO 2培养箱中静置培养48小时。离心48孔板，取细胞。清洗细胞，并转移至96孔板中。分成两组，一组加入50μl/孔PD-1(BD HorizonTM，562516)/TIM-3(BD PharmingenTM，563422)/CD3(Invitrogen，17-0028-42)，另一组加入LAG-3(BD HorizonTM，565720)/CD3(BD，1:100，用FACS缓冲液稀释)抗体混合溶液，4℃避光孵育30分钟。 The expression of depletion markers PD-1, TIM-3 and LAG-3 was detected by flow cytometry after CS1 CAR-T was stimulated by the target antigen CS1-positive multiple myeloma cell line NCl-H929. The specific process is as follows: CS1-positive multiple myeloma cell line NCl-H929 (50,000 cells/well, 500 μl/well) and each CS1 CAR-T (50,000 cells/well, 500 μl/well) were added to 48-well cell culture Plate, and set UTD (untransfected CAR T cells) as a control at the same time. Incubate for 48 hours in a 37°C, 5% CO2 incubator. The 48-well plate was centrifuged to remove the cells. Cells were washed and transferred to 96-well plates. Divided into two groups, one group added 50μl/well PD-1 (BD HorizonTM, 562516)/TIM-3 (BD PharmingenTM, 563422)/CD3 (Invitrogen, 17-0028-42), the other group added LAG-3 (BD HorizonTM, 565720)/CD3 (BD, 1:100, diluted with FACS buffer) antibody mixed solution, incubated at 4°C for 30 minutes in the dark.
清洗细胞，每个样品用300μl FACS缓冲液重悬细胞，用流式细胞仪检测。Cells were washed, resuspended in 300 μl of FACS buffer per sample, and detected by flow cytometry.
结果显示：耗竭标志物PD-1：与UTD相比，经靶细胞刺激后，各CS1 CAR-T PD-1的表达明显上调，其中本申请所提供的CS1-CAR T的PD-1表达水平均低于对照luc90 CAR T的PD-1表达水平(图26)。The results showed: the depletion marker PD-1: Compared with UTD, the expression of each CS1 CAR-T PD-1 was significantly up-regulated after target cell stimulation, and the PD-1 expression level of CS1-CAR T provided in this application All were lower than the PD-1 expression level of the control luc90 CAR T (Figure 26).
耗竭标志物Tim-3:与UTD相比，经靶细胞刺激后，各CS1 CAR-T Tim-3的表达明显上调，其中本申请所提供的CS1-CAR T的Tim-3表达水平均低于对照luc90 CAR T(图27)。Depletion marker Tim-3: Compared with UTD, the expression of each CS1 CAR-T Tim-3 was significantly up-regulated after target cell stimulation, and the Tim-3 expression level of CS1-CAR T provided by the application was lower than Control luc90 CAR T (Figure 27).
耗竭标志物LAG-3：与UTD相比，经靶细胞刺激后，各CS1 CAR-T LAG-3的表达明显上调，其中hu37A3 CAR-T和hu48G9 CAR-T的LAG-3表达水平均低于对照luc90 CAR T(图28)。Depletion marker LAG-3: Compared with UTD, the expression of each CS1 CAR-T LAG-3 was significantly up-regulated after target cell stimulation, and the LAG-3 expression levels of hu37A3 CAR-T and hu48G9 CAR-T were lower than Control luc90 CAR T (Figure 28).
综合以上结果，CS1 CAR-T在经靶抗原CS1阳性多发性骨髓瘤细胞刺激后，hu32A12 CAR-T，hu37A3 CAR-T，hu48G9 CAR-T组耗竭标志物表达较弱。说明hu32A12 CAR-T，hu37A3 CAR-T，hu48G9 CAR-T与肿瘤细胞孵育后更少进入耗竭状态。Based on the above results, after CS1 CAR-T was stimulated by target antigen CS1-positive multiple myeloma cells, the expression of depletion markers in hu32A12 CAR-T, hu37A3 CAR-T, and hu48G9 CAR-T groups was weaker. It shows that hu32A12 CAR-T, hu37A3 CAR-T, and hu48G9 CAR-T are less likely to enter the exhausted state after incubation with tumor cells.
实施例21：CS1 CAR-T CD3-ζ磷酸化自激活(tonic signaling)研究Example 21: CS1 CAR-T CD3-ζ phosphorylation tonic signaling study
通过常规分子生物学技术Western-blot方法检测CS1 CAR-T胞内CD3-ζ磷酸化的比例，判断其抗原非依赖性信号转导通路的激活情况。The ratio of CD3-ζ phosphorylation in CS1 CAR-T cells was detected by Western-blot method using conventional molecular biology techniques, and the activation of antigen-independent signal transduction pathway was judged.
取细胞裂解液进行SDS凝胶电泳、转膜、封闭。抗体染色：用CD3-ζ和p-CD3-ζ抗体溶液与膜孵育过夜清洗后，孵育羊抗鼠-HRP二抗(1：5000)，室温1小时后进行化学发光和曝光检测(图29)。结果(图29)表明，hu32A12 CAR-T细胞，hu37A3 CAR-T细胞，hu48G9 CAR-T细胞在体外常规培养至第9天时，CD3-ζ磷酸化程度明显弱于对照huluc63 CAR T细胞和luc90 CAR T细胞，说明hu32A12 CAR-T细胞，hu37A3 CAR-T细胞，hu48G9 CAR-T细胞在无靶细胞刺激的体外培养过程中存在较少的自激活现象。预示着hu32A12 CAR-T细胞、hu37A3 CAR-T细胞、hu48G9 CAR-T细胞的自身耗竭更少。The cell lysate was taken for SDS gel electrophoresis, membrane transfer, and blocking. Antibody staining: Incubate the membrane with CD3-ζ and p-CD3-ζ antibody solution overnight and wash, incubate with goat anti-mouse-HRP secondary antibody (1:5000), and perform chemiluminescence and exposure detection after 1 hour at room temperature (Figure 29) . The results (Fig. 29) showed that when hu32A12 CAR-T cells, hu37A3 CAR-T cells, and hu48G9 CAR-T cells were routinely cultured in vitro to the 9th day, the phosphorylation of CD3-ζ was significantly weaker than that of control huluc63 CAR T cells and luc90 CAR T cells, indicating that hu32A12 CAR-T cells, hu37A3 CAR-T cells, and hu48G9 CAR-T cells have less self-activation during in vitro culture without target cell stimulation. Indicates less self-depletion of hu32A12 CAR-T cells, hu37A3 CAR-T cells, and hu48G9 CAR-T cells.
实施例22：CS1 CAR T细胞与单核细胞以及肿瘤细胞共孵育后IL-6的分泌情况测定Example 22: Determination of IL-6 secretion after co-incubation of CS1 CAR T cells with monocytes and tumor cells
将CS1 CAR-T与单核细胞以及CS1阳性多发性骨髓瘤细胞NCl-H929按细胞数目比1:1:1的比例共同培养48小时，收取培养上清，用BD TM CBA试剂盒按照说明书进行细胞因子检测。 CS1 CAR-T was co-cultured with monocytes and CS1-positive multiple myeloma cells NCl-H929 at a ratio of 1:1:1 to the number of cells for 48 hours, and the culture supernatant was collected and carried out with the BD TM CBA kit according to the instructions. Cytokine detection.
结果如图30所示，CAR-T与单核细胞和靶细胞共同孵育后分泌细胞因子IL-6。hu32A12 CAR-T，hu37A3 CAR-T，hu48G9 CAR-T细胞分泌的IL-6较低。The results are shown in Figure 30, after CAR-T co-incubated with monocytes and target cells, the cytokine IL-6 was secreted. IL-6 secreted by hu32A12 CAR-T, hu37A3 CAR-T, and hu48G9 CAR-T cells was lower.
实施例23：CS1 CAR T细胞体外特异性扩增试验Example 23: In vitro specific expansion test of CS1 CAR T cells
本实验以转导CS1-CAR的T细胞和未转导CAR的T细胞(UTD)为效应细胞，分别与CS1阳性靶细胞MM.1S、NCl-H929、RPMI-8226细胞，和CS1阴性靶细胞HEK293共孵育，效靶比为2：1。随后每三天对总细胞数进行计数并检测CD3阳性率和PI染色的活率，持续9天。In this experiment, CS1-CAR-transduced T cells and untransduced CAR-transduced T cells (UTD) were used as effector cells, which were associated with CS1-positive target cells MM.1S, NCl-H929, RPMI-8226 cells, and CS1-negative target cells, respectively HEK293 was co-incubated with an effector-target ratio of 2:1. The total number of cells was then counted every three days and the CD3 positivity and PI staining viability were determined every three days for 9 days.
结果显示，培养基中不添加huIL-2时，hu37A3 CAR-T，hu48G9 CAR-T，huluc63 CAR-T，luc90 CAR-T分别与CS1阳性靶细胞共孵育后,CAR-T细胞都出现了明显的特异性增殖(图31)，CAR-T细胞的活率(PI染色鉴定)也呈现显著增加(图32)。与此同时，UTD细胞与所有靶细胞共孵育后均增殖缓慢(图33)。培养基中添加300IU/mL huIL-2而不加靶细胞的阳性对照组中，UTD，hu37A3 CAR-T，hu48G9 CAR-T细胞都有持续的增殖，且活率均保持在90％以上(图34)。The results showed that when huIL-2 was not added to the medium, hu37A3 CAR-T, hu48G9 CAR-T, huluc63 CAR-T, and luc90 CAR-T were co-incubated with CS1-positive target cells, and the CAR-T cells appeared obvious. The specific proliferation of CAR-T cells (Figure 31), the viability of CAR-T cells (identified by PI staining) also showed a significant increase (Figure 32). At the same time, UTD cells proliferated slowly after co-incubation with all target cells (Figure 33). In the positive control group in which 300IU/mL huIL-2 was added to the medium without the addition of target cells, UTD, hu37A3 CAR-T, and hu48G9 CAR-T cells all continued to proliferate, and the viability remained above 90% (Fig. 34).
结果显示，hu37A3 CAR-T，hu48G9 CAR-T细胞与CS1阳性多发性骨髓瘤细胞共孵育后，CAR-T会产生显著的特异性增殖。The results showed that after co-incubation of hu37A3 CAR-T and hu48G9 CAR-T cells with CS1-positive multiple myeloma cells, CAR-T produced significant specific proliferation.
实施例24：CS1 CAR T细胞在小鼠体内的细胞杀伤实验Example 24: Cell killing experiments of CS1 CAR T cells in mice
(1)CS1 CAR T细胞在小鼠体内杀伤骨髓瘤细胞MM.1S实验(1) Experiment of CS1 CAR T cells killing myeloma cells MM.1S in mice
接种3×10 6人多发性骨髓瘤细胞MM.1S于5～6周龄，雌性NPG小鼠(来自北京维通达生物技术有限公司)右侧腋部皮下，肿瘤接种日记为D0。当肿瘤体积长至约180mm 3，随机分为6组，分别为UTD对照组、hu37A3 CAR-T、hu48G9 CAR T细胞治疗组，尾静脉注射相应的CAR T细胞，剂量为4×10 6个细胞/鼠。 Female NPG mice (from Beijing Weitongda Biotechnology Co., Ltd.) were inoculated with 3×10 6 human multiple myeloma cells MM.1S subcutaneously in the right axilla of 5-6 weeks old, and the tumor inoculation diary was D0. When the tumor volume grew to about 180mm 3 , they were randomly divided into 6 groups: UTD control group, hu37A3 CAR-T, and hu48G9 CAR T cell treatment groups, respectively, and the corresponding CAR T cells were injected into the tail vein at a dose of 4×10 6 cells /mouse.
实验结果显示(图35)，D38时，与UTD相比，各组抑瘤率分别为hu37A3 CAR-T：99.9％(5只小鼠4只肿瘤消退)、hu48G9 CAR-T：100％(5只小鼠肿瘤全部消退)。从结果可以看到，hu37A3 CAR T组、hu48G9 CAR T组均能显著抑瘤。The experimental results (Fig. 35) showed that at D38, compared with UTD, the tumor inhibition rates of each group were hu37A3 CAR-T: 99.9% (4 tumors in 5 mice regressed), and hu48G9 CAR-T: 100% (5 mice). tumor regressed in all mice). It can be seen from the results that both the hu37A3 CAR T group and the hu48G9 CAR T group can significantly inhibit tumors.
(2)CS1 CAR T细胞对人多发性骨髓瘤细胞RPMI-8226-CS1的NPG小鼠皮下抑制瘤抗肿瘤效果(2) Subcutaneous antitumor and antitumor effect of CS1 CAR T cells on human multiple myeloma cells RPMI-8226-CS1 in NPG mice
采用常规分子生物学技术，通过慢病毒介导的方式将人SLAMF7胞外段(SEQ ID NO:57)转入RPMI-8226细胞构建成过表达CS1的RPMI-8226稳转细胞株RPMI-8226-CS1。Using conventional molecular biology techniques, the extracellular segment of human SLAMF7 (SEQ ID NO: 57) was transferred into RPMI-8226 cells by lentivirus-mediated method to construct a stable RPMI-8226 cell line RPMI-8226-overexpressing CS1 CS1.
接种5×10 6过表达CS1的人多发性骨髓瘤细胞RPMI-8226-CS1于5～6周龄，雌性NPG小鼠(来自北京维通达生物技术有限公司)右侧腋部皮下，肿瘤接种日记为D0。当肿瘤体积长至约110mm 3，随机分为7组，分别为： 5 × 10 6 human multiple myeloma cells RPMI-8226-CS1 overexpressing CS1 were inoculated subcutaneously in the right axilla of 5-6 week old female NPG mice (from Beijing Weitongda Biotechnology Co., Ltd.), tumor inoculation diary is D0. When the tumor volume grew to about 110mm 3 , they were randomly divided into 7 groups:
Group1：剂量2×10 6UTD组；Group2：剂量0.6×10 6hu37A3 CAR-T组；Group3：剂量1×10 6hu37A3 CAR-T组；Group4：剂量2×10 6hu37A3 CAR-T组；Group5：剂量0.6×10 6hu48G9 CAR-T组；Group6：剂量1×10 6hu48G9 CAR-T组；Group7：剂量2×10 6hu48G9  CAR-T组。 Group1: Dose 2×10 6 UTD group; Group2: Dose 0.6×10 6 hu37A3 CAR-T group; Group3: Dose 1×10 6 hu37A3 CAR-T group; Group4: Dose 2×10 6 hu37A3 CAR-T group; Group5 : Dose 0.6×10 6 hu48G9 CAR-T group; Group6: Dose 1×10 6 hu48G9 CAR-T group; Group 7: Dose 2×10 6 hu48G9 CAR-T group.
尾静脉注射相应的CAR T细胞，注射后，每周2次测量体重(包括分组给药及安乐死当天)，并用游标卡尺测量并记录肿瘤长径、短径，计算肿瘤体积，根据肿瘤体积绘制肿瘤生长曲线，并比较各组间肿瘤生长曲线的差异(肿瘤体积：V＝1/2×长径×短径 2)。 The corresponding CAR T cells were injected into the tail vein. After the injection, the body weight was measured twice a week (including group administration and the day of euthanasia), and the long and short diameters of the tumor were measured and recorded with a vernier caliper, the tumor volume was calculated, and the tumor growth was plotted according to the tumor volume. curve, and compare the difference of tumor growth curve among each group (tumor volume: V=1/2×long diameter×short diameter 2 ).
实验结果显示，低剂量(0.6×10 6和1×10 6)的hu48G9 CAR-T、hu37A3 CAR-T细胞都能显著抑制肿瘤的生长(图36)。 The experimental results showed that low doses (0.6×10 6 and 1×10 6 ) of hu48G9 CAR-T and hu37A3 CAR-T cells could significantly inhibit tumor growth ( FIG. 36 ).
实施例25：CS1-UCAR-T细胞及CS1敲除UCAR-T细胞的制备Example 25: Preparation of CS1-UCAR-T cells and CS1 knockout UCAR-T cells
体外扩增hu48G9 CAR-T细胞48小时后，按照常规分子生物学CRISPR/Cas9技术,分别对hu48G9 CAR-T细胞进行TRAC和B2M双基因敲除、或三基因敲除(敲除TRAC、B2M和CS1)，得到hu48G9-UCAR-T细胞(TRAC和B2M双基因敲除)，hu48G9-UCAR-CS1-/-T细胞(TCR、B2M、CS1三基因敲除)。按照试剂说明书(GeneArt TM Precision gRNA Synthesis Kit，Thermo Tisher)体外合成靶向TRAC、B2M、CS1的gRNA序列，其中，TRAC-gRNA的核酸序列如SEQ ID NO：76所示，B2M-gRNA的核酸序列如SEQ ID NO：84所示，CS1 gRNA的核酸序列如SEQ ID NO：89所示。 After 48 hours of in vitro expansion of hu48G9 CAR-T cells, according to conventional molecular biology CRISPR/Cas9 technology, hu48G9 CAR-T cells were subjected to TRAC and B2M double-gene knockout, or triple-gene knockout (knockout TRAC, B2M and CS1), hu48G9-UCAR-T cells (TRAC and B2M double knockout), hu48G9-UCAR-CS1-/-T cells (TCR, B2M, CS1 triple knockout) were obtained. According to the reagent instructions (GeneArt ™ Precision gRNA Synthesis Kit, Thermo Tisher), the gRNA sequences targeting TRAC, B2M and CS1 were synthesized in vitro, wherein the nucleic acid sequence of TRAC-gRNA was shown in SEQ ID NO: 76, and the nucleic acid sequence of B2M-gRNA was shown in SEQ ID NO: 76. As shown in SEQ ID NO:84, the nucleic acid sequence of CS1 gRNA is shown in SEQ ID NO:89.
实施例26：CS1-UCAR-T细胞、CS1-UCAR-CS1-/-T细胞对人多发性骨髓瘤细胞RPMI 8226-CS1的NPG小鼠皮下移植瘤抗肿瘤效果Example 26: Anti-tumor effect of CS1-UCAR-T cells and CS1-UCAR-CS1-/-T cells on subcutaneous transplanted tumors of human multiple myeloma cells RPMI 8226-CS1 in NPG mice
接种5×10 6过表达CS1的人多发性骨髓瘤细胞RPMI-8226-CS1于5～6周龄，雌性NPG小鼠(来自北京维通达生物技术有限公司)右侧腋部皮下，肿瘤接种日记为D0。当肿瘤体积长至约155mm 3，接种CAR T细胞，小鼠随机分为3组，分别为： 5 × 10 6 human multiple myeloma cells RPMI-8226-CS1 overexpressing CS1 were inoculated subcutaneously in the right axilla of 5-6 week old female NPG mice (from Beijing Weitongda Biotechnology Co., Ltd.), tumor inoculation diary is D0. When the tumor volume grew to about 155mm 3 , the mice were inoculated with CAR T cells, and the mice were randomly divided into 3 groups:
Group1：剂量1×10 6UTD组；Group2：剂量1×10 6hu48G9-UCAR-T组；Group3：剂量1×10 6hu48G9-UCAR-CS1-/-T组； Group1: Dose 1×10 6 UTD group; Group2: Dose 1×10 6 hu48G9-UCAR-T group; Group3: Dose 1×10 6 hu48G9-UCAR-CS1-/-T group;
尾静脉注射相应的CAR T细胞，注射后，每周2次测量体重(包括分组给药及安乐死当天)，并用游标卡尺测量并记录肿瘤长径、短径，计算肿瘤体积，根据肿瘤体积绘制肿瘤生长曲线，并比较各组间肿瘤生长曲线的差异(肿瘤体积：V＝1/2×长径×短径 2)。 The corresponding CAR T cells were injected into the tail vein. After the injection, the body weight was measured twice a week (including group administration and the day of euthanasia), and the long and short diameters of the tumor were measured and recorded with a vernier caliper, the tumor volume was calculated, and the tumor growth was plotted according to the tumor volume. curve, and compare the difference of tumor growth curve among each group (tumor volume: V=1/2×long diameter×short diameter 2 ).
实验结果显示，截至CAR T注射后D25天，小鼠体重正常。与UTD组相比，hu48G9-UCAR-T组的抑瘤率为99.37％，hu48G9-UCAR-CS1-/-T组的抑瘤率为96.49％。说明靶向CS1的通用型CAR-T细胞均能显著抑制肿瘤的生长(图37)。The experimental results showed that up to D25 days after CAR T injection, the mice had normal body weight. Compared with the UTD group, the tumor inhibition rate of the hu48G9-UCAR-T group was 99.37%, and the tumor inhibition rate of the hu48G9-UCAR-CS1-/-T group was 96.49%. It shows that the universal CAR-T cells targeting CS1 can significantly inhibit the growth of tumors (Figure 37).
此外，还检测了靶向CS1的CAR-T细胞可以显著抵抗NK细胞对靶向肿瘤抗原CAR-T或通用型CAR-T细胞的杀伤作用、提高自体或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，从而提高靶向肿瘤抗原CAR-T或通用型CAR-T细胞抗肿瘤活性。In addition, it was also tested that CAR-T cells targeting CS1 can significantly resist the killing effect of NK cells on tumor antigen-targeting CAR-T or universal CAR-T cells, improve autologous or allogeneic immune cells in the presence of host immune cells Persistence and/or graft survival in the presence of tumor antigen-targeting CAR-T or universal CAR-T cells against tumor activity.
在本申请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，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，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。All documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
本申请中涉及的序列如下：The sequences involved in this application are as follows:
Figure PCTCN2022084732-appb-000008
Figure PCTCN2022084732-appb-000008
Figure PCTCN2022084732-appb-000009
Figure PCTCN2022084732-appb-000009
Figure PCTCN2022084732-appb-000010
Figure PCTCN2022084732-appb-000010
Figure PCTCN2022084732-appb-000011
Figure PCTCN2022084732-appb-000011
Figure PCTCN2022084732-appb-000012
Figure PCTCN2022084732-appb-000012

Claims (36)

  1. 一种靶向CS1的抗原结合单元，其特征在于，所述抗原结合单元选自下组：An antigen-binding unit targeting CS1, wherein the antigen-binding unit is selected from the group consisting of:
    (1)抗原结合单元，其包含重链可变区，所述重链可变区包含SEQ ID NO:3、13或23所示的HCDR1，和/或包含SEQ ID NO:4、14、24或32所示的HCDR2，和/或包含SEQ ID NO:5、15、25或41所示的HCDR3；(1) an antigen-binding unit comprising a heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 3, 13 or 23, and/or comprising SEQ ID NO: 4, 14, 24 or HCDR2 shown in 32, and/or comprising HCDR3 shown in SEQ ID NO: 5, 15, 25 or 41;
    (2)抗原结合单元，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:8、18或28所示的LCDR1，和/或包含SEQ ID NO:9、19或29所示的LCDR2，和/或包含SEQ ID NO:10、20或30所示的LCDR3；(2) an antigen binding unit comprising a light chain variable region comprising LCDR1 shown in SEQ ID NO: 8, 18 or 28, and/or comprising SEQ ID NO: 9, 19 or 29 LCDR2 shown, and/or LCDR3 comprising SEQ ID NO: 10, 20 or 30;
    (3)抗原结合单元，包含(1)所述抗原结合单元的重链可变区及(2)所述抗原结合单元的轻链可变区；(3) an antigen-binding unit comprising (1) a heavy chain variable region of the antigen-binding unit and (2) a light chain variable region of the antigen-binding unit;
    (4)抗原结合单元，(1)～(3)中任一项所述的抗原结合单元的变体，且具备与(1)～(3)中任一项所述的抗原结合单元相同或相似的活性。(4) An antigen-binding unit, which is a variant of the antigen-binding unit described in any one of (1) to (3), and has the same antigen-binding unit as that described in any one of (1) to (3) or similar activity.
  2. 如权利要求1所述的抗原结合单元，其特征在于，所述的抗原结合单元选自：The antigen-binding unit of claim 1, wherein the antigen-binding unit is selected from:
    (1)抗原结合单元，其包含SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:8所示的LCDR1、SEQ ID NO:9所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者(1) antigen-binding unit comprising HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, HCDR3 shown in SEQ ID NO:5 and LCDR1 shown in SEQ ID NO:8, SEQ ID NO:8 LCDR2 shown in ID NO: 9, LCDR3 shown in SEQ ID NO: 10; or
    (2)抗原结合单元，其包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3以及SEQ ID NO:18所示的LCDR1、SEQ ID NO:19所示的LCDR2、SEQ ID NO:20所示的LCDR3；或者(2) an antigen-binding unit comprising HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15, and LCDR1 shown in SEQ ID NO: 18, SEQ ID NO: 18 LCDR2 shown in ID NO: 19, LCDR3 shown in SEQ ID NO: 20; or
    (3)抗原结合单元，其包含SEQ ID NO:23所示的HCDR1、SEQ ID NO:24所示的HCDR2、SEQ ID NO:25所示的HCDR3以及SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2、SEQ ID NO:30所示的LCDR3；或者(3) an antigen-binding unit comprising HCDR1 shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, HCDR3 shown in SEQ ID NO:25, and LCDR1 shown in SEQ ID NO:28, SEQ ID NO:28 LCDR2 shown in ID NO: 29, LCDR3 shown in SEQ ID NO: 30; or
    (4)抗原结合单元，其包含SEQ ID NO:3所示的HCDR1、SEQ ID NO:32所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:8所示的LCDR1、SEQ ID NO:9所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者(4) an antigen binding unit comprising HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:32, HCDR3 shown in SEQ ID NO:5 and LCDR1 shown in SEQ ID NO:8, SEQ ID NO:8 LCDR2 shown in ID NO: 9, LCDR3 shown in SEQ ID NO: 10; or
    (5)抗原结合单元，其包含SEQ ID NO:23所示的HCDR1、SEQ ID NO:24所示的HCDR2、SEQ ID NO:41所示的HCDR3以及SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2、SEQ ID NO:30所示的LCDR3；(5) an antigen-binding unit comprising HCDR1 shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, HCDR3 shown in SEQ ID NO:41, and LCDR1 shown in SEQ ID NO:28, SEQ ID NO:28 LCDR2 shown in ID NO: 29, LCDR3 shown in SEQ ID NO: 30;
    (6)抗原结合单元，(1)～(5)中任一项所述的抗原结合单元的变体，且具备与(1)～(5)中任一项所述的抗原结合单元相同或相似的活性。(6) An antigen-binding unit that is a variant of the antigen-binding unit according to any one of (1) to (5), and which has the same or the same antigen-binding unit as the antigen-binding unit described in any one of (1) to (5). similar activity.
  3. 如权利要求1所述的抗原结合单元，其特征在于，所述的抗原结合单元选自：The antigen-binding unit of claim 1, wherein the antigen-binding unit is selected from:
    (1)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：1、11、21、31、36或40所示的氨基酸序列；(1) an antigen-binding unit, the heavy chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 1, 11, 21, 31, 36 or 40;
    (2)抗原结合单元，所述的抗原结合单元的轻链可变区具有SEQ ID NO：6、16、26、34、38或43所示的氨基酸序列；(2) an antigen-binding unit, the light chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 34, 38 or 43;
    (3)抗原结合单元，包含(1)所述抗原结合单元的重链可变区及(2)所述抗原结合单元的轻链可变区；(3) an antigen-binding unit comprising (1) a heavy chain variable region of the antigen-binding unit and (2) a light chain variable region of the antigen-binding unit;
    (4)抗原结合单元，(1)～(3)中任一项所述的抗原结合单元的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。(4) An antigen-binding unit, which is a variant of the antigen-binding unit described in any one of (1) to (3), which has the same or similar properties to the antibody described in any one of (1) to (3). active.
  4. 如权利要求3所述的抗原结合单元，其特征在于，所述的抗原结合单元选自：The antigen-binding unit of claim 3, wherein the antigen-binding unit is selected from:
    (1)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：1所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：6所示的氨基酸序列；(1) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 6 the amino acid sequence of ;
    (2)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：11所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：16所示的氨基酸序列；(2) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 11 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 16 the amino acid sequence of ;
    (3)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：26所示的氨基酸序列；(3) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 26 the amino acid sequence of ;
    (4)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：31所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：34所示的氨基酸序列；(4) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 31 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 34 the amino acid sequence of ;
    (5)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：36所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：38所示的氨基酸序列；(5) an antigen-binding unit, the heavy chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 36 and the light chain variable region of the antigen-binding unit has the amino acid sequence shown in SEQ ID NO: 38 the amino acid sequence of ;
    (6)抗原结合单元，所述的抗原结合单元的重链可变区具有SEQ ID NO：40所示的氨基酸序列并且所述抗原结合单元的轻链可变区具有SEQ ID NO：43所示的氨基酸序列；(6) an antigen binding unit, the heavy chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 40 and the light chain variable region of the antigen binding unit has the amino acid sequence shown in SEQ ID NO: 43 the amino acid sequence of ;
    (7)抗原结合单元，(1)～(6)中任一项所述的抗原结合单元的变体，且具备与(1)～(6)中任一项所述的抗原结合单元相同或相似的活性。(7) An antigen-binding unit, which is a variant of the antigen-binding unit described in any one of (1) to (6), which has the same or the same antigen-binding unit as the antigen-binding unit described in any one of (1) to (6). similar activity.
  5. 抗原结合单元，与权利要求1-4中任一项所述的抗原结合单元识别相同的抗原决定部位；或结合CS1蛋白的Ig-like V-type结构域；或结合CS1蛋白的Ig-like C2-type结构域。An antigen-binding unit, which recognizes the same epitope as the antigen-binding unit according to any one of claims 1-4; or binds the Ig-like V-type domain of the CS1 protein; or binds the Ig-like C2 of the CS1 protein -type field.
  6. 如权利要求1-5任一所述抗原结合单元，其特征在于，所述抗原结合单元是杂交瘤抗体、人源化抗体、嵌合抗体或全人抗体；或者所述抗原结合单元是单克隆抗体；或者所述抗原结合单元是全抗、scFv、Fv片段、Fab片段、Fab’片段、(Fab’) 2片段、Fd片段、dAb片段、单域抗体、多功能抗体或scFv-Fc抗体。 The antigen-binding unit according to any one of claims 1-5, wherein the antigen-binding unit is a hybridoma antibody, a humanized antibody, a chimeric antibody or a fully human antibody; or the antigen-binding unit is a monoclonal antibody an antibody; or the antigen binding unit is a whole antibody, scFv, Fv fragment, Fab fragment, Fab' fragment, (Fab') 2 fragment, Fd fragment, dAb fragment, single domain antibody, multifunctional antibody or scFv-Fc antibody.
  7. 免疫辍合物，其特征在于，所述免疫辍合物包括：权利要求1-6任一所述的抗原结 合单元；以及与之连接的功能性分子，所述功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。An immunoconjugate, characterized in that, the immunoconjugate comprises: the antigen binding unit according to any one of claims 1-6; and a functional molecule connected thereto, the functional molecule is selected from: targeting Molecules of tumor surface markers, molecules that inhibit tumors, molecules of surface markers that target immune cells, or detectable markers.
  8. 嵌合受体，其特征在于，所述嵌合受体的胞外域包含权利要求1-6任一所述的抗原结合单元，所述嵌合受体包括：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。A chimeric receptor, characterized in that the extracellular domain of the chimeric receptor comprises the antigen-binding unit according to any one of claims 1-6, and the chimeric receptor comprises: a chimeric antigen receptor (CAR), Chimeric T cell receptor, T cell antigen coupler (TAC), or a combination thereof.
  9. 如权利要求8所述的嵌合受体，其特征在于，所述嵌合受体包含顺序连接的：权利要求1-6任一所述的抗原结合单元、跨膜区和胞内信号区。The chimeric receptor of claim 8, wherein the chimeric receptor comprises sequentially linked: the antigen binding unit of any one of claims 1-6, a transmembrane region and an intracellular signal region.
  10. 如权利要求9所述的嵌合受体，所述的胞内信号区选自：CD3ζ、FcεRIγ、CD27、CD28、CD137、CD134、MyD88、CD40的胞内信号区序列或其组合；和/或所述的跨膜区包含CD8或CD28的跨膜区。The chimeric receptor of claim 9, wherein the intracellular signal region is selected from the group consisting of: CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, MyD88, CD40 intracellular signal region sequences or combinations thereof; and/or The transmembrane region comprises the transmembrane region of CD8 or CD28.
  11. 如权利要求10所述的嵌合受体，其特征在于，所述的嵌合受体包括：The chimeric receptor of claim 10, wherein the chimeric receptor comprises:
    权利要求1-6任一所述的抗原结合单元、CD8/CD28的跨膜区和CD3ζ；或The antigen binding unit of any one of claims 1-6, the transmembrane region of CD8/CD28, and CD3ζ; or
    权利要求1-6任一所述的抗原结合单元、CD8/CD28的跨膜区、CD137的胞内信号区和CD3ζ；或The antigen binding unit of any one of claims 1-6, the transmembrane region of CD8/CD28, the intracellular signal region of CD137, and CD3ζ; or
    权利要求1-6任一所述的抗原结合单元、CD8/CD28的跨膜区、CD28的胞内信号区和CD3ζ；或The antigen binding unit of any one of claims 1-6, the transmembrane region of CD8/CD28, the intracellular signal region of CD28, and CD3ζ; or
    权利要求1-6任一所述的抗原结合单元、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3ζ。The antigen binding unit of any one of claims 1-6, the transmembrane region of CD8/CD28, the intracellular signal region of CD28, CD137 and CD3ζ.
  12. 如权利要求11所述的嵌合受体，其特征在于，所述的嵌合受体的氨基酸序列如SEQ ID NO：45、46或47所示。The chimeric receptor of claim 11, wherein the amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 45, 46 or 47.
  13. 编码权利要求1-6任一所述的抗原结合单元、权利要求7所述的免疫缀合物、权利要求8-12任一所述的嵌合受体的核酸。A nucleic acid encoding the antigen-binding unit of any one of claims 1-6, the immunoconjugate of claim 7, and the chimeric receptor of any one of claims 8-12.
  14. 一种表达载体，其包含权利要求13所述的核酸。An expression vector comprising the nucleic acid of claim 13.
  15. 一种病毒，其包含权利要求14所述的表达载体。A virus comprising the expression vector of claim 14.
  16. 包括权利要求1-6任一所述的抗原结合单元、权利要求7所述的免疫缀合物、和/或权利要求8-12任一所述的嵌合受体的组合物，其特征在于，所述组合物对表达CS1的细胞具有细胞毒性。A composition comprising the antigen-binding unit of any of claims 1-6, the immunoconjugate of claim 7, and/or the chimeric receptor of any of claims 8-12, characterized in that , the composition is cytotoxic to cells expressing CS1.
  17. 如权利要求16所述的组合物，其特征在于，所述表达CS1的细胞是肿瘤细胞和/或病原体细胞。The composition of claim 16, wherein the CS1-expressing cells are tumor cells and/or pathogen cells.
  18. 一种宿主细胞，其包含权利要求14所述的表达载体或基因组中整合有权利要求13所述的核酸。A host cell comprising the expression vector of claim 14 or the nucleic acid of claim 13 integrated into the genome.
  19. 如权利要求18所述的宿主细胞，其特征在于，其表达权利要求8-12任一所述的嵌 合受体。The host cell according to claim 18, characterized in that it expresses the chimeric receptor according to any one of claims 8-12.
  20. 如权利要求18或19所述的宿主细胞，其特征在于，所述宿主细胞包括T细胞、细胞毒性T淋巴细胞、NK细胞、NKT细胞、DNT细胞、调节性T细胞、NK92细胞、干细胞衍生的免疫效应细胞或其组合。The host cell of claim 18 or 19, wherein the host cell comprises T cells, cytotoxic T lymphocytes, NK cells, NKT cells, DNT cells, regulatory T cells, NK92 cells, stem cell-derived cells Immune effector cells or a combination thereof.
  21. 如权利要求20所述的宿主细胞，其特征在于，所述T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；The host cell of claim 20, wherein the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells;
    优选地，所述T细胞为自体或同种异体T细胞；Preferably, the T cells are autologous or allogeneic T cells;
    优选地，所述T细胞为原代T细胞；Preferably, the T cells are primary T cells;
    优选地，所述T细胞来源于人的自体T细胞。Preferably, the T cells are derived from human autologous T cells.
  22. 如权利要求20或21所述的宿主细胞，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)、αβT细胞、γδT细胞或其组合。The host cell of claim 20 or 21, wherein the T cells comprise memory stem-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells T cells (Tregs), effector memory T cells (Tem), αβ T cells, γδ T cells, or a combination thereof.
  23. 如权利要求18-22任一所述的宿主细胞，其特征在于，所述细胞包含：The host cell of any one of claims 18-22, wherein the cell comprises:
    编码TCR蛋白的基因的敲除和/或内源性TCR分子低表达或不表达，和/或Knockout of genes encoding TCR proteins and/or low or no expression of endogenous TCR molecules, and/or
    编码MHC蛋白的基因的敲除和/或内源性MHC低表达或不表达。Knockout of genes encoding MHC proteins and/or low or no endogenous MHC expression.
  24. 如权利要求23所述的宿主细胞，其特征在于，采用CRISPR/Cas9技术敲除内源性MHC分子B2M和内源性TCR。The host cell of claim 23, wherein the endogenous MHC molecule B2M and endogenous TCR are knocked out by using CRISPR/Cas9 technology.
  25. 如权利要求24所述的宿主细胞，其特征在于，敲除B2M所使用的gRNA包括SEQ ID NO:84、85、86和/或87所示序列，敲除TCR使用的gRNA包括SEQ ID NO:76、77、78、79、80、81、82和/或83所示序列。The host cell of claim 24, wherein the gRNA used for knocking out B2M comprises the sequences shown in SEQ ID NO: 84, 85, 86 and/or 87, and the gRNA used for knocking out TCR comprises SEQ ID NO: Sequences shown at 76, 77, 78, 79, 80, 81, 82 and/or 83.
  26. 如权利要求18-25任一所述的宿主细胞，其特征在于，所述细胞包含编码CS1蛋白的基因的敲除和/或内源性CS1分子低表达或不表达。The host cell of any one of claims 18-25, wherein the cell comprises a knockout of a gene encoding CS1 protein and/or low or no expression of endogenous CS1 molecule.
  27. 如权利要求26所述的宿主细胞，其特征在于，采用CRISPR/Cas9技术敲除所述细胞CS1基因，所使用的gRNA选自SEQ ID NO:88、89、90、91、92、93、94和/或95所示序列。The host cell of claim 26, wherein the cell CS1 gene is knocked out using CRISPR/Cas9 technology, and the gRNA used is selected from SEQ ID NOs: 88, 89, 90, 91, 92, 93, 94 and/or the sequence shown in 95.
  28. 如权利要求18-27任一所述的宿主细胞，其特征在于，所述宿主细胞结合表达CS1的细胞，不显著结合不表达CS1的细胞。The host cell of any one of claims 18-27, wherein the host cell binds to cells expressing CS1 and does not significantly bind cells that do not express CS1.
  29. 如权利要求18-28任一所述的宿主细胞，其特征在于，其还携带外源的细胞因子的编码序列；或The host cell according to any one of claims 18-28, characterized in that it also carries a coding sequence for an exogenous cytokine; or
    其还表达另一种嵌合受体；或It also expresses another chimeric receptor; or
    其还表达趋化因子受体；或It also expresses a chemokine receptor; or
    其还表达安全开关。It also expresses a safety switch.
  30. 联合用药，其特征在于，权利要求1-6任一所述的抗原结合单元、权利要求7所述的免疫缀合物、权利要求8-12任一所述的嵌合受体、权利要求16或17所述的组合物、权利要求18-29任一所述的宿主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；Combination medication, characterized in that the antigen-binding unit described in any one of claims 1-6, the immunoconjugate described in claim 7, the chimeric receptor described in any one of claims 8-12, and claim 16 Or the composition of 17, the host cell of any one of claims 18-29 is administered in combination with an agent that enhances its function, preferably, in combination with a chemotherapeutic agent;
    和/或与改善其相关的一种或多种副作用的药剂联合施用；and/or in combination with an agent that ameliorates one or more side effects associated therewith;
    和/或与表达靶向CS1之外的嵌合受体的宿主细胞联合施用。and/or in combination with host cells expressing chimeric receptors targeting other than CS1.
  31. 一种制备权利要求1-6任一所述的抗原结合单元、权利要求7所述免疫缀合物、权利要求8-12任一所述的嵌合受体、和/或权利要求16或17所述的组合物的方法，所述方法包含在适于表达所述抗原结合单元、免疫缀合物、嵌合受体的条件下培养权利要求18-29任一所述的宿主细胞，以及分离出由所述宿主细胞表达的所述抗原结合单元、免疫缀合物、嵌合受体、和/或组合物。A kind of preparation of the antigen binding unit described in any one of claims 1-6, the immunoconjugate described in claim 7, the chimeric receptor described in any one of claims 8-12, and/or claim 16 or 17 The method for the composition, the method comprising culturing the host cell of any one of claims 18-29 under conditions suitable for expressing the antigen binding unit, immunoconjugate, chimeric receptor, and isolating The antigen binding units, immunoconjugates, chimeric receptors, and/or compositions expressed by the host cells are produced.
  32. 一种药物组合物，其特征在于，其包括：A pharmaceutical composition, characterized in that it comprises:
    权利要求1-6中任一所述的抗原结合单元或编码该抗原结合单元的核酸；或The antigen-binding unit of any one of claims 1-6 or a nucleic acid encoding the antigen-binding unit; or
    权利要求7所述的免疫辍合物或编码该辍合物的核酸；或The immunoconjugate of claim 7 or the nucleic acid encoding the conjugate; or
    权利要求8-12任一所述的嵌合受体或编码该嵌合受体的核酸；或The chimeric receptor of any one of claims 8-12 or a nucleic acid encoding the chimeric receptor; or
    权利要求18-29任一所述的宿主细胞；The host cell of any one of claims 18-29;
    以及任选的，药学上可接受的载体或赋型剂。And optionally, a pharmaceutically acceptable carrier or excipient.
  33. 一种治疗/诊断疾病的方法，其包括向有需要的受试者给予有效量的如权利要求1-6任一所述的抗原结合单元、或如权利要求7所述的免疫缀合物、或如权利要求18-29任一所述的宿主细胞、或如权利要求32所述的组合物；A method for treating/diagnosing a disease, comprising administering to a subject in need an effective amount of the antigen-binding unit according to any one of claims 1-6, or the immunoconjugate according to claim 7, Or the host cell of any one of claims 18-29, or the composition of claim 32;
    优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤；优选所述肿瘤是多发性骨髓瘤；Preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors; preferably the tumor is multiple myeloma;
    优选地，所述受试者是人；Preferably, the subject is a human;
    优选地，其中所述宿主细胞对于所述受试者是自体的或同种异体的T细胞。Preferably, wherein the host cells are autologous or allogeneic T cells to the subject.
  34. 如权利要求1-6任一所述的抗原结合单元、或权利要求7所述的免疫缀合物、或权利要求18-29任一所述的宿主细胞、或权利要求32所述的组合物在治疗和/或诊断疾病中的应用，其特征在于，所述疾病表达CS1；优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤，优选所述肿瘤是多发性骨髓瘤。The antigen-binding unit of any one of claims 1-6, or the immunoconjugate of claim 7, or the host cell of any one of claims 18-29, or the composition of claim 32 Use in the treatment and/or diagnosis of a disease, characterized in that the disease expresses CS1; preferably, the disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors, preferably the tumor is multiple myeloid tumor.
  35. 如权利要求1-6任一所述的抗原结合单元、或权利要求7所述的免疫缀合物、或权利要求18-29任一所述的宿主细胞、或权利要求32所述的组合物用于制备杀伤NK细胞的药物的用途。The antigen-binding unit of any one of claims 1-6, or the immunoconjugate of claim 7, or the host cell of any one of claims 18-29, or the composition of claim 32 Use for preparing a drug for killing NK cells.
  36. 如权利要求书35所述的用途，其特征在于，提高自体或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。The use according to claim 35, wherein the persistence and/or transplantation survival rate of autologous or allogeneic immune cells in the presence of host immune cells is improved.
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