WO2022206941A1 - Cellules modifiées par cs1 et composition de celles-ci - Google Patents

Cellules modifiées par cs1 et composition de celles-ci Download PDF

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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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English (en)
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/zh
Publication of WO2022206941A1 publication Critical patent/WO2022206941A1/fr

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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.

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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une unité de liaison à l'antigène capable de cibler CS1, et son utilisation. La présente invention concerne un immunoconjugué et un récepteur chimérique comprenant l'unité de liaison à l'antigène ciblant CS1, et un acide nucléique codant pour l'unité de liaison à l'antigène ciblant CS1, l'immunoconjugué et le récepteur chimérique. La présente invention concerne en outre l'utilisation de l'unité de liaison à l'antigène ciblant CS1, l'immunoconjugué, le récepteur chimérique et une cellule hôte dans le traitement de maladies.
PCT/CN2022/084732 2021-04-02 2022-04-01 Cellules modifiées par cs1 et composition de celles-ci WO2022206941A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1960750A (zh) * 2004-03-29 2007-05-09 Pdl生物制药公司 抗cs1抗体的治疗用途
CN107429253A (zh) * 2014-12-05 2017-12-01 希望之城公司 Cs1靶向性嵌合抗原受体修饰的t细胞
WO2019241358A2 (fr) * 2018-06-12 2019-12-19 The Regents Of The University Of California Récepteurs antigéniques chimériques bispécifiques à chaîne unique pour le traitement du cancer
US20200080114A1 (en) * 2018-09-07 2020-03-12 Crispr Therapeutics Ag Universal donor cells
WO2020081613A1 (fr) * 2018-10-16 2020-04-23 Intellia Therapeutics, Inc. Compositions et procédés d'immunothérapie

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1960750A (zh) * 2004-03-29 2007-05-09 Pdl生物制药公司 抗cs1抗体的治疗用途
CN107429253A (zh) * 2014-12-05 2017-12-01 希望之城公司 Cs1靶向性嵌合抗原受体修饰的t细胞
WO2019241358A2 (fr) * 2018-06-12 2019-12-19 The Regents Of The University Of California Récepteurs antigéniques chimériques bispécifiques à chaîne unique pour le traitement du cancer
US20200080114A1 (en) * 2018-09-07 2020-03-12 Crispr Therapeutics Ag Universal donor cells
WO2020081613A1 (fr) * 2018-10-16 2020-04-23 Intellia Therapeutics, Inc. Compositions et procédés d'immunothérapie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOSEPH D MALAER, PORUNELLOOR A MATHEW: "CS1 (SLAMF7, CD319) is an effective immunotherapeutic target for multiple myeloma", AMERICAN JOURNAL OF CANCER RESEARCH, E-CENTURY PUBLISHING CORPORATION, US, vol. 7, no. 8, 1 January 2017 (2017-01-01), US , pages 1637 - 1641, XP055485233, ISSN: 2156-6976 *

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