WO2023134718A1 - 靶向gprc5d的嵌合抗原受体及其用途 - Google Patents

靶向gprc5d的嵌合抗原受体及其用途 Download PDF

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Publication number
WO2023134718A1
WO2023134718A1 PCT/CN2023/071869 CN2023071869W WO2023134718A1 WO 2023134718 A1 WO2023134718 A1 WO 2023134718A1 CN 2023071869 W CN2023071869 W CN 2023071869W WO 2023134718 A1 WO2023134718 A1 WO 2023134718A1
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seq
amino acid
acid sequence
sequence shown
binding domain
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PCT/CN2023/071869
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English (en)
French (fr)
Inventor
何晓文
周金财
史中军
陈思晔
杨月
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原启生物科技(上海)有限责任公司
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Publication of WO2023134718A1 publication Critical patent/WO2023134718A1/zh

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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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present application relates to the field of biomedicine, in particular to a novel anti-GPRC5D chimeric antigen receptor, cells containing the chimeric antigen receptor, and their use in treating tumors.
  • Multiple myeloma is the second most common hematologic malignancy and ranks second in cancer-related mortality.
  • MM is a plasma cell malignancy often accompanied by multiple osteolytic lesions, renal impairment, bone marrow infiltration, hypercalcemia, and anemia.
  • the current main treatment for MM is systemic chemotherapy with severe side effects, and it cannot be completely cured.
  • the G protein-coupled receptor family group C 5 member D (GPRC5D) protein is an atypical surface orphan receptor.
  • GPRCD5 like other C5 family receptors, has a short amino-terminus, so it is very similar to the C4 family in conformation. Its expression in normal tissues is limited to hair follicles, but it is also highly expressed specifically in the bone marrow of MM patients, and is highly correlated with plasma cell tumor burden and genetic aberrations.
  • MM With the intervention of new drugs such as protease inhibitors (PIs), immunomodulatory drugs (IMiDs) and monoclonal antibodies (mAbs), the treatment of MM has been expanded, but there is still a very high recurrence rate and drug resistance.
  • PIs protease inhibitors
  • IMDs immunomodulatory drugs
  • mAbs monoclonal antibodies
  • CAR-T chimeric antigen receptor T cell
  • MM patients with low expression of BCMA have the problem of target escape.
  • the specific high expression of GPRC5D on plasma cells makes it an ideal therapeutic target for MM, but the drug therapy products targeting it are not yet mature.
  • the present application provides a chimeric antigen receptor comprising two antigen-binding domains, both of which can target GPRC5D.
  • the present application also provides cells comprising the chimeric antigen receptor.
  • the cells have one or more of the following properties: (1) strong expansion ability, (2) capable of killing target cells expressing GPRC5D, (3) secreting cytokines under the stimulation of target cells, (4) It has strong proliferative ability under the stimulation of target cells, (4) inhibits tumor growth, and (5) has good safety.
  • the application provides a chimeric antigen receptor comprising a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain targeting G protein-coupled receptor family C group 5 member D (GPRC5D) protein, and the second antigen-binding domain targets the GPRC5D protein.
  • GPRC5D G protein-coupled receptor family C group 5 member D
  • said first antigen binding domain and/or said second antigen binding domain of said chimeric antigen receptor comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antigen-binding fragment comprises VHH.
  • said first antigen binding domain of said chimeric antigen receptor comprises at least one CDR of an antibody heavy chain variable region VH comprising SEQ ID NO: 15, SEQ ID NO: 19.
  • the first antigen binding domain of the chimeric antigen receptor comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.
  • said first antigen binding domain of said chimeric antigen receptor comprises HCDR3, and said HCDR3 comprises SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID The amino acid sequence shown in any one of NO:27.
  • said first antigen binding domain of said chimeric antigen receptor comprises HCDR2, and said HCDR2 comprises SEQ ID NO: 34 (IX 1 X 2 X 3 X 4 G X 5 T, wherein, X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, X5 is the amino acid sequence shown in N, S or T).
  • said first antigen binding domain of said chimeric antigen receptor comprises HCDR2, and said HCDR2 comprises SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 and SEQ ID The amino acid sequence shown in any one of NO:26.
  • the first antigen binding domain of the chimeric antigen receptor comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35.
  • said first antigen binding domain of said chimeric antigen receptor comprises HCDR1, and said HCDR1 comprises SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, and SEQ ID The amino acid sequence shown in any one of NO:25.
  • the first antigen binding domain of the chimeric antigen receptor comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, and the HCDR2 comprises SEQ ID NO:34(IX 1 X 2 X 3 X 4 GX 5 T, among them, X 1 is N or T, X 2 is S or W, X 3 is G or S, X 4 is D or G, X 5 is N , S or T), and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.
  • said first antigen binding domain of said chimeric antigen receptor comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2, HCDR3 comprise any group of amino acid sequences selected from the following:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27.
  • the first antigen binding domain of the chimeric antigen receptor comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 11.
  • said first antigen binding domain of said chimeric antigen receptor comprises H-FR2
  • said H-FR2 is located between said HCDR1 and said HCDR2
  • said H-FR2 comprises The amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:28 and SEQ ID NO:23.
  • said first antigen binding domain of said chimeric antigen receptor comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises The amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:29.
  • the first antigen binding domain of the chimeric antigen receptor comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 Comprising the amino acid sequence shown in SEQ ID NO:14.
  • said first antigen binding domain of said chimeric antigen receptor comprises a VH
  • said VH comprises SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID The amino acid sequence shown in any one of NO:30.
  • the first antigen binding domain of the chimeric antigen receptor is a VHH
  • the VHH comprises SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID The amino acid sequence shown in any one of NO:30.
  • said second antigen binding domain of said chimeric antigen receptor comprises at least one CDR of an antibody heavy chain variable region VH comprising SEQ ID NO: 15, SEQ ID NO: 19. SEQ ID NO: 24 and SEQ The amino acid sequence shown in any one of ID NO:30.
  • the second antigen binding domain of the chimeric antigen receptor comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.
  • said second antigen binding domain of said chimeric antigen receptor comprises HCDR3, and said HCDR3 comprises SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID The amino acid sequence shown in any one of NO:27.
  • said second antigen binding domain of said chimeric antigen receptor comprises HCDR2, and said HCDR2 comprises SEQ ID NO: 34 (IX 1 X 2 X 3 X 4 G X 5 T, wherein, X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, X5 is the amino acid sequence shown in N, S or T).
  • said second antigen binding domain of said chimeric antigen receptor comprises HCDR2, and said HCDR2 comprises SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID The amino acid sequence shown in any one of NO:26.
  • the second antigen binding domain of the chimeric antigen receptor comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35.
  • said second antigen binding domain of said chimeric antigen receptor comprises HCDR1, and said HCDR1 comprises SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID The amino acid sequence shown in any one of NO:25.
  • the second antigen binding domain of the chimeric antigen receptor comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35, and the HCDR2 comprises SEQ ID NO:34(IX 1 X 2 X 3 X 4 GX 5 T, among them, X 1 is N or T, X 2 is S or W, X 3 is G or S, X 4 is D or G, X 5 is N , S or T), and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.
  • the second antigen-binding domain of the chimeric antigen receptor comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise any set of amino acid sequences selected from the following group:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:18;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR2 comprises SEQ ID NO: 20 The amino acid sequence shown in ID NO:21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22; and
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27.
  • the second antigen binding domain of the chimeric antigen receptor comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 11.
  • said second antigen binding domain of said chimeric antigen receptor comprises H-FR2
  • said H-FR2 is located between said HCDR1 and said HCDR2
  • said H-FR2 comprises The amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:28 and SEQ ID NO:23.
  • said second antigen binding domain of said chimeric antigen receptor comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises The amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:29.
  • the second antigen binding domain of the chimeric antigen receptor comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 Comprising the amino acid sequence shown in SEQ ID NO:14.
  • said second antigen binding domain of said chimeric antigen receptor comprises a VH
  • said VH comprises SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID The amino acid sequence shown in any one of NO:30.
  • the second antigen binding domain of the chimeric antigen receptor is a VHH
  • the VHH comprises SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID The amino acid sequence shown in any one of NO:30.
  • said first antigen binding domain and said second antigen binding domain of said chimeric antigen receptor comprise HCDR1, HCDR2 and HCDR3 respectively, said first antigen binding domain and said second antigen binding domain
  • the sequence of HCDR1, HCDR2 and HCDR3 of the antigen binding domain is selected from any one of the following amino acid sequences:
  • the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 9
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10;
  • the first antigen-binding domain: the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10
  • the second antigen binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 18;
  • the first antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10
  • the amino acid sequence shown the second antigen binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:20, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:21, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 22;
  • the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10
  • the amino acid sequence shown the second antigen-binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:25, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:26, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 27;
  • the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18
  • the second antigen binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:8, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 10;
  • the first antigen-binding domain: the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:18 The amino acid sequence shown;
  • the first antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:18
  • the second antigen-binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 22;
  • the first antigen-binding domain: the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18 The amino acid sequence shown;
  • the first antigen-binding domain: the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22 The amino acid sequence shown;
  • the first antigen binding domain: the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22 The amino acid sequence shown;
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 18;
  • the first antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22
  • the second antigen binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 22;
  • the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22
  • the second antigen-binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 27;
  • the first antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27
  • the second antigen binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:8, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 10;
  • the first antigen-binding domain the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27;
  • the second antigen-binding domain the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16,
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18;
  • the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27
  • the second antigen binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 22;
  • the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27
  • the second antigen-binding domain: the HCDR1 includes the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 27.
  • said first antigen binding domain and said second antigen binding domain of said chimeric antigen receptor target the same epitope of GPRC5D.
  • said first antigen binding domain and said second antigen binding domain of said chimeric antigen receptor comprise the same or different amino acid sequences.
  • said first antigen binding domain and said second antigen binding domain of said chimeric antigen receptor are directly or indirectly linked.
  • said first antigen binding domain and said second antigen binding domain of said chimeric antigen receptor are linked by a linker.
  • the linker comprises the amino acid sequence of (GGGGS)n, wherein the n is any positive integer from 1-10.
  • the linker comprises the amino acid sequence of (EAAAK)n, wherein the n is any positive integer from 1-10.
  • the chimeric antigen receptor comprises a co-stimulatory signaling domain, wherein the co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA- 1. Ligands of LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40 and MyD88.
  • the co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226,
  • said co-stimulatory signaling domain of said chimeric antigen receptor is an intracellular co-stimulatory signaling domain derived from 4-1BB.
  • the co-stimulatory signaling region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:4.
  • the chimeric antigen receptor comprises an intracellular signaling domain comprising an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10, DAP -12 and a domain containing at least one ITAM.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpesvirus
  • the intracellular signaling domain of the chimeric antigen receptor is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:5.
  • the chimeric antigen receptor comprises a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4- 1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • proteins selected from the group consisting of: CD8, CD28, 4- 1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5,
  • said transmembrane region of said chimeric antigen receptor is a transmembrane region derived from CD8.
  • the transmembrane region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:3.
  • the chimeric antigen receptor further comprises a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • said hinge region of said chimeric antigen receptor is a hinge region derived from CD8.
  • the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:2.
  • the chimeric antigen receptor further comprises low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the low-density lipoprotein receptor-associated protein or fragment thereof is located at the C-terminus of the intracellular signaling region.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-associated protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.
  • the chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • the signal peptide of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:1.
  • the present application also provides one or more isolated nucleic acid molecules encoding said chimeric antigen receptor.
  • the nucleic acid molecule further comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is the EF1 ⁇ promoter.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the vector is a viral vector.
  • the vector is a lentiviral vector.
  • the present application also provides a cell comprising the chimeric antigen receptor, the nucleic acid molecule, and/or the vector.
  • the cells are immune effector cells.
  • the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral Blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • NK cells natural killer cells
  • NKT cells monocytes, dendritic cells
  • monocytes dendritic cells
  • granulocytes lymphocytes
  • leukocytes granulocytes
  • peripheral Blood mononuclear cells embryonic stem cells
  • lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells are T cells.
  • the present application also provides a method for preparing the chimeric antigen receptor, the method comprising culturing the cells under the condition that the chimeric antigen receptor is expressed.
  • the present application also provides a method for preparing the modified immune effector cells, which comprises adding to the immune effector cells Introduce the vector.
  • the present application also provides a pharmaceutical composition, which comprises the chimeric antigen receptor, the nucleic acid molecule, the carrier, and/or the cell, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides the use of the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition in the preparation of a medicament for Prevent, treat and/or alleviate diseases and/or conditions.
  • the diseases and/or disorders include diseases and/or disorders associated with aberrant expression of GPRC5D.
  • the diseases and/or disorders associated with abnormal expression of GPRC5D include tumors.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a hematoma and/or a lymphoma.
  • the tumor comprises myeloma.
  • the myeloma comprises refractory/relapsed multiple myeloma.
  • the present application also provides a method for preventing, treating and/or alleviating diseases and/or conditions, the method comprising administering the cells and/or the drugs to a subject in need combination.
  • the diseases and/or disorders include diseases and/or disorders associated with aberrant expression of GPRC5D.
  • the diseases and/or disorders associated with abnormal expression of GPRC5D include tumors.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a hematoma and/or a lymphoma.
  • the tumor comprises myeloma.
  • the myeloma comprises refractory/relapsed multiple myeloma.
  • Figure 1 shows a schematic diagram of the elements and connection sequences of each part of the chimeric antigen receptor described in the present application.
  • Figure 2A shows the positive rate of T cells after double-binding domain GPRC5D double V H H CAR lentivirus infection.
  • Figure 2B shows the positive rate of T cells after infection with a single V H H CAR lentivirus.
  • Figure 3 shows the in vitro expansion curve of a single V H H CAR-T cell.
  • Figure 4A shows the expression of CD107a in double binding domain GPRC5D double V H H CAR-T cells.
  • Figure 4B shows the expression of CD107a in a single V H H CAR-T cell.
  • Figure 5A shows the specific killing of double binding domain GPRC5D double V H H CAR-T cells.
  • Figure 5B shows the specific killing of a single V H H CAR-T cell.
  • Figure 6A shows the secretion level of double binding domain GPRC5D double V H H CAR-T cytokines.
  • Figure 6B shows the secretion level of individual V H H CAR-T cytokines.
  • Figure 7A shows the targeted proliferation of double binding domain GPRC5D double V H H CAR-T cells.
  • Figure 7B shows the targeted proliferation fold of a single V H H CAR-T cell.
  • Figure 8 shows the flow cytometric detection of CAR-T cell memory phenotype.
  • Figure 9 shows the phenotype of CD8 + CAR-T cells.
  • Figure 10 shows the expression of CD3 + CAR-T cell activation markers.
  • Figure 11 shows the expression of CD3 + CAR-T cell exhaustion markers.
  • Figure 12 shows the confocal imaging of CAR-T cell immune synapse.
  • Figure 13 shows the quantitative analysis of MTOC and synaptic distance.
  • Figure 14 shows the flowchart of the tumor inhibition experiment in NSG mice.
  • Figures 15A-15D show the effect of tumor inhibition in NSG mice, a subcutaneous tumor formation model.
  • Figure 16 shows the effect of tumor suppression in NSG mice, an orthotopic transplantation model.
  • Figure 17 shows the graph of antibody affinity detection.
  • Figure 18 shows a flow chart of antibody binding activity.
  • Figure 19 shows the flow chart of the competitive binding between G5V H H1 and G5V H H2.
  • Figure 20 shows the cross-binding activity of dual binding domain VHH -hFc antibodies to target antigens of different species.
  • antibody as used includes whole antibodies and binding fragments thereof. Typically, a fragment competes with the intact antibody from which it is derived for specific binding to the antigen.
  • antibodies or binding fragments thereof can be chemically conjugated to other proteins, or expressed as fusion proteins with other proteins.
  • the antibodies can be monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the binding protein of the antibody or binding fragment thereof can include GPRC5D.
  • the antibody or binding fragment thereof can be specific for GPRC5D.
  • antigen-binding fragment refers to a portion of an intact antibody and refers to the antigenically determining variable region of an intact antibody.
  • the antigen-binding fragments may include Fab, Fab', F(ab')2, Fv fragments and single-chain Fv fragments, tandem Fv fragments, VHH, bispecific antibodies.
  • the antigen-binding fragment can be a VHH.
  • the antigen-binding fragment can bind GPRC5D.
  • the antigen-binding fragment can be specific for GPRC5D.
  • VHH or VHH " are used interchangeably and generally refer to an antibody comprising the variable antigen binding domain of a heavy chain antibody.
  • VHHs may also be referred to as Nanobodies (Nb) and/or Single Domain Antibodies.
  • Nb Nanobodies
  • the VHH can bind GPRC5D.
  • the VHH can be specific for GPRC5D.
  • the antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • the term "heavy chain constant region” consists of three domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region.
  • the term "light chain constant region” consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors.
  • G protein-coupled receptor family C group member 5 member D (GPRC5D) protein is an atypical surface orphan receptor.
  • GPRCD5 like other C5 family receptors, has a short amino-terminus, so it is very similar to the C4 family in conformation. Its expression in normal tissues is restricted to hair follicles, but it is also prominently expressed in the bone marrow of patients with multiple myeloma and is highly correlated with plasma cell tumor burden and genetic aberrations.
  • GPRC5D in this application may specifically refer to GPRC5D expressed in MM patients.
  • chimeric antigen receptor generally refers to a Recombinant polypeptides of ectodomain, transmembrane region and intracellular domain.
  • a hinge region is included between the extracellular domain and the transmembrane region.
  • the chimeric antigen receptor may also include low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the chimeric antigen receptor can include a signal peptide. Binding of the extracellular domain of the CAR to the target antigen on the surface of the target cell results in clustering of the CAR and delivery of an activation stimulus to the CAR-containing cell.
  • CAR redirects specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis and/or production of molecules capable of mediating death of cells expressing target antigen in a major histocompatibility (MHC)-independent manner .
  • MHC major histocompatibility
  • the extracellular structure can specifically bind GPRC5D.
  • intracellular domain is meant to include any truncated portion sufficient to transduce an activation signal.
  • the intracellular domain may comprise an intracellular signaling domain and/or a co-stimulatory signaling domain.
  • intracellular signaling region refers to an intracellular region that can generate signals that promote immune effector functions of CAR-containing cells (eg, CART cells or CAR-expressing NK cells).
  • the intracellular signaling region may comprise an intracellular signaling region of one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30 , Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and domains containing at least one ITAM.
  • the intracellular signaling region may be a signaling domain derived from CD3 ⁇ .
  • co-stimulatory signaling region refers to a part of the CAR capable of transducing effector signals in the intracellular signaling region.
  • the co-stimulatory signal domain may comprise an intracellular co-stimulatory signal domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40 and MyD88.
  • the co-stimulatory signal domain may be an intracellular co-stimulatory signal domain derived from 4-1BB.
  • the term "transmembrane region” refers to a domain of a peptide, polypeptide or protein capable of spanning the plasma membrane of a cell. These domains can be used to anchor the extracellular domain to the cell membrane.
  • the transmembrane region may comprise a transmembrane domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • the transmembrane region
  • the term "hinge region” refers to a part of an antibody heavy chain polypeptide connecting the CH1 domain and the CH2 domain, for example, from about position 216 to about position 230 of the EU numbering system according to Kabat.
  • the hinge region is usually a dimeric molecule composed of two polypeptides with the same amino acid sequence.
  • the hinge region generally consists of about 25 amino acid residues and is the flexible , allowing the antigen-binding domain to move independently.
  • the hinge region can be subdivided into three structural domains: upper, middle, and lower hinge domains.
  • the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region may be derived from the hinge region of CD8.
  • the term "low-density lipoprotein receptor-related protein” refers to a cell surface protein belonging to endocytic receptors, which is widely distributed in organisms and has a large interstitial space. Difference, the main function is to take cholesterol into the cell for the synthesis of cell proliferation and sterol hormones and bile salts.
  • the low-density lipoprotein receptor-related protein can be from any vertebrate.
  • the Low-density lipoprotein receptor-associated protein or fragments thereof may be located at the C-terminus of the intracellular signaling region.
  • said low-density lipoprotein receptor-associated proteins or fragments thereof may comprise one or more selected from the following group : Low-density lipoprotein receptor-related protein 1-12 and its functional fragment.
  • the low-density lipoprotein receptor-related protein or its fragment can be low-density lipoprotein receptor-related protein 6 or its fragment.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 5 or a fragment thereof.
  • signal peptide refers to the leader sequence at the amino terminus (N-terminus) of the nascent CAR protein, which directs the nascent protein to the endoplasmic reticulum and subsequent surface expression upon translation or after translation.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • nucleic acid molecule includes DNA molecules and RNA molecules.
  • a nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • promoter generally refers to a DNA sequence that regulates the expression of a selected DNA sequence operably linked to the promoter, thereby affecting the expression of the selected DNA sequence in a cell.
  • the nucleic acid molecule may encode an antigen binding protein and/or the chimeric antigen receptor.
  • the nucleic acid molecule can include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be the EF1 ⁇ promoter.
  • the term "vector” generally refers to a molecule to which one or more nucleic acid molecules of the present application can be attached.
  • the vector may be a viral vector.
  • the vector may be a lentiviral vector.
  • the term “cell” refers to a cell into which nucleic acid can be transfected, and the term “cell” includes prokaryotic cells for plasmid propagation, and eukaryotic cells for nucleic acid expression and production of encoded polypeptides.
  • a cell may comprise said chimeric antigen receptor, said nucleic acid molecule and/or said vector.
  • the cells may be immune effector cells.
  • immune effector cells generally refers to immune cells that participate in the immune response and perform effector functions.
  • the exercising effector functions may include clearing foreign antigens or promoting immune effector responses, etc.
  • immune effector cells can include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes cells, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • immune effector cells can be T cells.
  • the term "pharmaceutical composition” generally refers to a chemical or biological composition suitable for administration to a mammalian subject.
  • the pharmaceutical composition may comprise the antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable Carrier.
  • the pharmaceutical composition can be used to prevent, treat and/or alleviate diseases or conditions related to abnormal expression of GPRC5D.
  • the diseases or conditions associated with abnormal expression of GPRC5D may include tumors.
  • the tumors include solid tumors and/or non-solid tumors.
  • the tumor pack may include hematomas and/or lymphomas.
  • the tumor can include myeloma.
  • the application provides a chimeric antigen receptor comprising a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain targeting G protein-coupled receptor family C group 5 member D( GPRC5D) protein, and the second antigen binding domain targets the GPRC5D protein.
  • the first antigen binding domain and/or the second antigen binding domain of the chimeric antigen receptor may comprise an antibody or antigen binding fragment thereof.
  • the antigen-binding fragments include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • such antigen-binding fragments include VHH.
  • the CDR can be divided by IMGT division.
  • the first antigen-binding domain of the chimeric antigen receptor may target the GPRC5D protein.
  • the first antigen binding domain of the chimeric antigen receptor may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH may comprise SEQ ID NO: 15, SEQ ID NO: 19.
  • the first antigen-binding domain of the chimeric antigen receptor may comprise HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.
  • said first antigen binding domain of said chimeric antigen receptor comprises HCDR3, and said HCDR3 comprises any of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 22 and SEQ ID NO: 27 Amino acid sequence shown in one item.
  • the first antigen binding domain of the chimeric antigen receptor may comprise HCDR2, and the HCDR2 may comprise SEQ ID NO: 34 (IX 1 X 2 X 3 X 4 GX 5 T, wherein, X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, X5 is the amino acid sequence shown in N, S or T).
  • the first antigen binding domain of the chimeric antigen receptor can comprise HCDR2, and the HCDR2 can comprise SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:26 The amino acid sequence shown in any one.
  • the first antigen-binding domain of the chimeric antigen receptor may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:35.
  • the first antigen binding domain of the chimeric antigen receptor can comprise HCDR1, and the HCDR1 can comprise SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, and SEQ ID NO:25 The amino acid sequence shown in any one.
  • the first antigen binding domain of the chimeric antigen receptor may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:35, and the HCDR2 may comprise SEQ ID NO:35 ID NO:34(IX 1 X 2 X 3 X 4 GX 5 T, wherein, X 1 is N or T, X 2 is S or W, X 3 is G or S, X 4 is D or G, X 5 is N, S or T), and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the first antigen-binding domain of the chimeric antigen receptor may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise any group of amino acid sequences selected from the following:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:18;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27.
  • the first antigen-binding domain of the chimeric antigen receptor may comprise H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 -FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the first antigen binding domain of the chimeric antigen receptor may comprise H-FR2
  • the H-FR2 is located between the HCDR1 and the HCDR2
  • the H-FR2 may comprise The amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:28 and SEQ ID NO:23.
  • the first antigen binding domain of the chimeric antigen receptor may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise The amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:29.
  • the first antigen-binding domain of the chimeric antigen receptor may comprise H-FR4, and the N of the H-FR4 The end is connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the first antigen binding domain of the chimeric antigen receptor may comprise VH, and the VH may comprise SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID The amino acid sequence shown in any one of NO:30.
  • the first antigen binding domain of the chimeric antigen receptor may be VHH, and the VHH may comprise SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID The amino acid sequence shown in any one of NO:30.
  • the second antigen-binding domain of the chimeric antigen receptor can target the GPRC5D protein.
  • the second antigen binding domain of the chimeric antigen receptor may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH may comprise SEQ ID NO: 15, SEQ ID NO: 19.
  • the second antigen-binding domain of the chimeric antigen receptor may comprise HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.
  • the second antigen binding domain of the chimeric antigen receptor comprises HCDR3, and the HCDR3 comprises any of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 22 and SEQ ID NO: 27 Amino acid sequence shown in one item.
  • the second antigen binding domain of the chimeric antigen receptor may comprise HCDR2, and the HCDR2 may comprise SEQ ID NO: 34 (IX 1 X 2 X 3 X 4 GX 5 T, wherein, X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, X5 is the amino acid sequence shown in N, S or T).
  • the second antigen binding domain of the chimeric antigen receptor can comprise HCDR2, and the HCDR2 can comprise SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:26 The amino acid sequence shown in any one.
  • the second antigen-binding domain of the chimeric antigen receptor may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:35.
  • the second antigen binding domain of the chimeric antigen receptor can comprise HCDR1, and the HCDR1 can comprise SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, and SEQ ID NO:25 The amino acid sequence shown in any one.
  • the second antigen binding domain of the chimeric antigen receptor may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:35, and the HCDR2 may comprise SEQ ID NO:35 ID NO:34(IX 1 X 2 X 3 X 4 GX 5 T, wherein, X 1 is N or T, X 2 is S or W, X 3 is G or S, X 4 is D or G, X 5 is N, S or T), and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the second antigen-binding domain of the chimeric antigen receptor may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise any group of amino acid sequences selected from the following group:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:18;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27.
  • the second antigen-binding domain of the chimeric antigen receptor may comprise H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 -FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the second antigen binding domain of the chimeric antigen receptor may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise The amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:28 and SEQ ID NO:23.
  • the second antigen binding domain of the chimeric antigen receptor may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise The amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:29.
  • the second antigen-binding domain of the chimeric antigen receptor may comprise H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may be Comprising the amino acid sequence shown in SEQ ID NO:14.
  • the second antigen binding domain of the chimeric antigen receptor may comprise VH, and the VH may comprise SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID The amino acid sequence shown in any one of NO:30.
  • the second antigen binding domain of the chimeric antigen receptor may be VHH, and the VHH may comprise SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID The amino acid sequence shown in any one of NO:30.
  • the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor may respectively comprise Contains HCDR1, HCDR2 and HCDR3.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises SEQ ID NO:10
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9 Amino acid sequence shown in ID NO:10.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 comprises SEQ ID NO:10
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the amino acid sequence shown in ID NO:18 comprises the amino acid sequence shown in ID NO:18.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 comprises SEQ ID NO:10
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 comprises SEQ ID NO:10
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:18
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9 Amino acid sequence shown in ID NO:10.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17,
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:18
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:18
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:26 Amino acid sequence shown in ID NO:27.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the amino acid sequence shown in ID NO:18 comprises the amino acid sequence shown in ID NO:18.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:27 amino acid sequence.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9 Amino acid sequence shown in ID NO:10.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:21 Amino acid sequence shown in ID NO:22.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:27
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:26 Amino acid sequence shown in ID NO:27.
  • the first antigen binding domain and the second antigen binding domain of the chimeric antigen receptor may target the same epitope of GPRC5D.
  • the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor may target different epitopes of GPRC5D.
  • the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor may comprise the same or different amino acid sequences.
  • the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor may be directly or indirectly linked.
  • the first antigen binding domain and the second antigen binding domain of the chimeric antigen receptor can be linked by a linker.
  • the linker may comprise the amino acid sequence of (GGGGS)n, wherein the n is any of 1-10 It means a positive integer.
  • the n can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the linker may comprise the amino acid sequence of (EAAAK)n, wherein the n is any positive integer in 1-10.
  • the n can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric antigen receptor may comprise a costimulatory signal domain, wherein the costimulatory signal domain comprises an intracellular costimulatory signal domain derived from one or more proteins selected from the group: CD28 , 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30 , CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40 and MyD88.
  • the costimulatory signal domain comprises an intracellular costimulatory signal domain derived from one or more proteins selected from the group: CD28 , 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1
  • the co-stimulatory signaling domain of the chimeric antigen receptor can be an intracellular co-stimulatory signaling domain derived from 4-1BB.
  • the co-stimulatory signaling region of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO:4.
  • the chimeric antigen receptor may comprise an intracellular signaling domain comprising an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10, DAP- 12 and a domain containing at least one ITAM.
  • the intracellular signaling domain of the chimeric antigen receptor can be a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain of the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO:5.
  • the chimeric antigen receptor may comprise a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group: CD8, CD28, 4-1BB , CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10 , CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • proteins selected from the group: CD8, CD28, 4-1BB , CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICO
  • the transmembrane region of the chimeric antigen receptor can be a transmembrane region derived from CD8.
  • the transmembrane region of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the chimeric antigen receptor may also include a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4 -1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region of the chimeric antigen receptor can be a hinge region derived from CD8.
  • the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:2.
  • the chimeric antigen receptor may also comprise low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or its fragment is located at the C-terminus of the intracellular signaling region.
  • the low density The lipoprotein receptor-associated protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.
  • the chimeric antigen receptor may further comprise a signal peptide.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • the signal peptide of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:1.
  • the low-density lipoprotein receptor-associated protein or its fragments in the chimeric antigen receptor can be connected to the C of the CAR through a self-cleaving peptide (for example, 2A peptides such as T2A, P2A, E2A, etc.). end.
  • a self-cleaving peptide for example, 2A peptides such as T2A, P2A, E2A, etc.
  • the low-density lipoprotein receptor-related protein or its fragments can be connected to the C-terminus of the intracellular signaling region through T2A.
  • the chimeric antigen receptor may sequentially comprise an antigen-binding domain that binds to the GPRC5D protein, the hinge region, the transmembrane domain, the co-stimulatory signal region, and the Intracellular signaling domain.
  • the chimeric antigen receptor may sequentially comprise the antigen-binding domain, the hinge region derived from CD8, the transmembrane region derived from CD8, and the co-stimulatory signal region derived from 4-1BB , and the intracellular signaling domain derived from CD3 ⁇ .
  • the chimeric antigen receptor may sequentially comprise an antigen-binding domain that binds to the GPRC5D protein, the hinge region, the transmembrane domain, the co-stimulatory signal region, the The intracellular signal region and the low-density lipoprotein receptor-related protein or its fragments.
  • the chimeric antigen receptor may sequentially comprise the antigen-binding domain, the hinge region derived from CD8, the transmembrane region derived from CD8, and the co-stimulatory signal region derived from 4-1BB , derived from the intracellular signaling region of CD3 ⁇ and the low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO: 31 or SEQ ID NO: 32.
  • the chimeric antigen receptor may comprise the structure shown in the following table: Among them, A stands for (GGGGS)n; B stands for B:(EAAAK)n. Wherein, n is any positive integer from 1 to 10.
  • the HCDR1 of 3B5 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR2 of 3B5 comprises the amino acid sequence shown in SEQ ID NO:9
  • the HCDR3 of 3B5 comprises the amino acid sequence shown in SEQ ID NO:10
  • the VHH of 3B5 Comprising the amino acid sequence shown in SEQ ID NO:15.
  • the HCDR1 of 3E7 comprises the amino acid sequence shown in SEQ ID NO:16
  • the HCDR2 of 3E7 comprises the amino acid sequence shown in SEQ ID NO:17
  • the HCDR3 of 3E7 comprises the amino acid sequence shown in SEQ ID NO:18
  • the VHH of 3E7 Comprising the amino acid sequence shown in SEQ ID NO:19.
  • the HCDR1 of 4A2 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 of 4A2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 of 4A2 comprises the amino acid sequence shown in SEQ ID NO:22
  • the VHH of 4A2 Comprising the amino acid sequence shown in SEQ ID NO:24.
  • the HCDR1 of 4B3 comprises the amino acid sequence shown in SEQ ID NO:25
  • the HCDR2 of 4B3 comprises the amino acid sequence shown in SEQ ID NO:26
  • the HCDR3 of 4B3 comprises the amino acid sequence shown in SEQ ID NO:27
  • the VHH of 4B3 Comprising the amino acid sequence shown in SEQ ID NO:30.
  • the CD8 ⁇ signal peptide may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the CD8 ⁇ hinge region may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the CD8 ⁇ transmembrane region may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the 4-1BB co-stimulatory signal may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the CD3 ⁇ intracellular signal region may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the Ori may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the present application also provides one or more nucleic acid molecules, which can be nucleotides, deoxynucleotides and/ribonucleotides in isolated forms of any length, and can encode said embedded Synthetic antigen receptors.
  • the nucleic acid molecule can include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be the EF1 ⁇ promoter.
  • the present application also provides a carrier, which may include the nucleic acid molecule.
  • the vector can transform, transduce or transfect host cells, so that the genetic material elements carried by it can be expressed in the host cells.
  • vectors can include promoters, transcripts, enhancers, replicons, selection elements, and reporter genes.
  • a carrier may include components that facilitate entry into cells.
  • the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeats.
  • the vector may be a viral vector.
  • the vector may be a lentiviral vector.
  • the present application also provides cells, which may include the chimeric antigen receptor, the nucleic acid molecule and/or the carrier.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • the cells may be immune effector cells.
  • the cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes , peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells may be T cells.
  • the present application also provides a pharmaceutical composition, which may include the chimeric antigen receptor, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable adjuvant agent.
  • the pharmaceutical composition may also comprise one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition of the present invention may comprise liquid, Freezing and lyophilized compositions.
  • the pharmaceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with drug administration, generally safe, non-toxic , and is neither biologically nor otherwise undesirable.
  • the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present application also provides a method for preparing the chimeric antigen receptor.
  • the method may comprise culturing the cell under conditions such that the chimeric antigen receptor is expressed.
  • the present application also provides a method for preparing modified immune effector cells, which may include introducing the carrier into immune cells.
  • the present application also provides the use of the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of medicines, the The above-mentioned medicines can be used for preventing, alleviating and/or treating tumors.
  • the present application also provides a method for preventing, alleviating and/or treating tumors, which may include administering the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cells and/or the pharmaceutical composition.
  • the present application also provides the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition, which are used for prevention, alleviation and/or treatment tumor.
  • the tumor may include a tumor with abnormal expression of GPRC5D.
  • the tumor may include solid tumors and/or non-solid tumors.
  • the tumor may include hematoma and/or lymphoma.
  • the tumor may include myeloma.
  • the myeloma may include refractory/relapsed multiple myeloma.
  • the subject may include humans or non-human animals.
  • the present application provides a kit or a drug delivery device, which includes the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition.
  • the CDRs mutation strategy was determined, and the length of CDR3 was three types: 14, 17 and 21 amino acids.
  • Select the already-marketed Nanobody Caplacizumab (Caplacizumab) as the backbone synthesize the nucleotide sequence of Caplacizumab, and then clone it into the HP153 phage vector, then extract the single-stranded DNA of HP153, and use the method of Kunkel Mutagenesis to mutate the Nanobody CDRs, to obtain double-stranded DNA.
  • the double-stranded DNA was electrotransferred into the competent Escherichia coli SS320 pre-infected with M13KO7 helper phage, and the supernatant of the phage was collected after overnight culture to finally construct a synthetic nanobody library NanoOri_1.0 with a diversity of 1.44 ⁇ 10 10 (Yuanqi Biotechnology Co., Ltd. (Shanghai) Co., Ltd.), as a seed bank for antibody sequence screening.
  • the full-length sequence of GPRC5D purchased from Sino Biological Company (cat: HG24447-UT) was constructed on the company's existing lentiviral vector, and lentiviral transduction was carried out to prepare the overexpression of target cell CHO-GPRC5D and target cell HEK293-GPRC5D cell line.
  • the target cells CHO-GPRC5D and HEK293-GPRC5D were used for alternate panning of the artificially synthesized nanobody library (Yuanqi Biotechnology (Shanghai) Co., Ltd.), and a total of four rounds of panning were performed.
  • the target cells CHO-GPRC5D and HEK293-GPRC5D were selected for alternate screening of cell panning. Mix 500 ⁇ L of phage and 500 ⁇ L of 10% FBS/PBS buffer to a final volume of 1 mL, put it in a 2 mL low-adsorption EP tube, and block it for 1 hour at 4°C by gently rotating or periodically mixing. CHO-GPRC5D cells were harvested by centrifugation at 140 g for 10 min, and washed once with 10 mL of PBS.
  • the number of living cells reached more than 95%, and the CHO-GPRC5D cells were washed three times with 5% FBS/PBS buffer. Then resuspend in 1 mL of 5% FBS/PBS buffer to make the number of cells reach 1 ⁇ 10 7 . Cells were kept on ice during this period.
  • Centrifuge CHO-GPRC5D cells at 140g at 4°C for 2min, resuspend in 1mL phage antibody library that has been blocked, rotate gently or mix periodically at 4°C or room temperature for binding for 2 hours, remove supernatant, and harvest cells Resuspend in 1mL of 5% FBS/PBS buffer, repeat washing twice, after resuspending the cells for the last time, change to a new 2mL low-adsorption EP tube to avoid the phage non-specifically adsorbed to the EP tube from being washed take it off.
  • cell-based mono phage ELISA Cell-based mono phage ELISA
  • 3B5V H H (the amino acid sequence of V H H is shown in SEQ ID NO: 15), 3E7V H H (the amino acid sequence of V H H is shown in SEQ ID NO: 19), 4A2V H H (the amino acid sequence of V H H is shown in SEQ ID NO: 19), 4A2V H H (V H
  • the amino acid sequence of H is shown in SEQ ID NO:24) and 4B3V H H (the amino acid sequence of V H H is shown in SEQ ID NO:30) antibody sequence, by flow cytometry fluorescence sorting technique (FACS), using iQue Screener flow meter (purchased from IntelliCyt Company), using PBS containing 0.1% BSA as a buffer to detect the cell binding activity between the cell surface target antigen (GPRC5D) and the antibody.
  • FACS flow cytometry fluorescence sorting technique
  • iQue Screener flow meter purchased from IntelliCyt Company
  • PBS containing 0.1% BSA as a buffer to detect
  • Use buffer to prepare target cells (MM1S myeloma cells) at a concentration of 1 ⁇ 10 6 cells/mL, and add them to a 96-well pointed bottom plate (corning 3894), 30 ⁇ L per well; use buffer to prepare a detection antibody concentration of 10 ⁇ g/mL, and press Dilute the antibody by 3 times to form 8 concentration gradients; add 30 ⁇ L/well of prepared antibodies of different concentrations to the plated target cells and mix well; incubate at 4°C for 1 hour; add 150 ⁇ L of buffer solution to each well, 300 g Centrifuge for 5 minutes, discard the supernatant and shake the cells loose; repeat the wash once; use the buffer to prepare the fluorescent secondary antibody (ab98593) at a ratio of 1:200, add 30 ⁇ l to the cells and mix well, and incubate at 4°C for 30 minutes; Add 150 ⁇ L of buffer solution to each well, centrifuge at 300 g for 5 minutes, discard the supernatant and shake the cells loose; repeat the washing
  • the binding activity of anti-GPRC5D antibodies to GPRC5D protein on the cell surface is shown in Table 1.
  • ET150-5scFv is a positive control antibody, its sequence can be found in patent application CN107428829A, the amino acid sequence of 3B5V H H is shown in SEQ ID NO:15, the amino acid sequence of 3E7V H H is shown in SEQ ID NO:19, 4A2V H H
  • the amino acid sequence of 4B3V H H is shown in SEQ ID NO:24, and the amino acid sequence of 4B3V H H is shown in SEQ ID NO:30.
  • the results showed that all the anti-GPRC5D V H Hs described in this application can bind to GPRC5D on the cell surface, and the binding ability is comparable to that of the positive control ET150-5scFv, even higher than that of the positive control.
  • the construct preparation steps are as follows: Specifically, two V H H sequences are fused by (GGGGS)n or (EAAAK)n (wherein, n can be a positive integer), and then SphI and NotI endonucleases (purchased from NEB ) Carry out double enzyme digestion on the CAR lentivirus core empty plasmid (self-constructed by Yuanqi Biotechnology (Shanghai) Co., Ltd., containing the new Ori element, hereinafter referred to as the CAR lentiviral empty vector) to generate a 8992bp linearized fragment, which is recovered after tapping
  • the double V H H fragments obtained from the fusion were mixed at a molar ratio of 1:3 for homologous recombination, and then transformed into DH5 ⁇ competent cells. Single colonies were picked for sequencing identification.
  • the connection sequence of the double V H H CAR structure of the double binding domain is shown in Table 2.
  • a double V H H CAR structure combination named OriCAR-017 (ie oriCAR-DG508M), followed by a single domain G5V H H1 CAR (wherein, the amino acid sequence of the targeting part of G5V H H1 is shown in SEQ ID NO: 19 (3E7)) and G5V H H2 CAR (the amino acid sequence of G5V H H2 in the targeting part is shown in SEQ ID NO: 30 (4B3)), the positive control ET150-8 (the amino acid sequence of which is shown in Patent No.
  • Table 2 Exemplary double binding domain GPRC5D double V H H CAR structural order A: (GGGGS)n, wherein, n is an integer greater than or equal to 0, and the amino acid sequence of GGGGS is shown in SEQ ID NO:39 B: (EAAAK)n, wherein, n is an integer greater than or equal to 0, and the amino acid sequence of EAAAK is shown in SEQ ID NO:40
  • the lentiviral vector system used to construct the present invention belongs to the third generation, and the system is composed of four plasmids, namely, the packaging plasmid pRH1 encoding the Gag-Pol protein, the packaging plasmid pVH3 encoding the Rev protein, and the encoding envelope protein
  • the pMH2 plasmid of VSV-G, and the core plasmid that is, each CAR lentiviral plasmid containing the VHH sequence in the above-mentioned Example 2.
  • the expression of the CAR gene in each CAR lentiviral plasmid is regulated by the elongation factor-1 ⁇ (EF-1 ⁇ ) promoter.
  • EF-1 ⁇ elongation factor-1 ⁇
  • the cell culture plate was placed in a 37° C., 5% CO 2 incubator for static culture.
  • NBS solution PBS solution containing 1% neonatal bovine serum
  • centrifuge 500 g at 4°C for 5 min, and discard the supernatant.
  • the titer of each of the above-mentioned CAR viruses containing VHH was in the range of 1-10 ⁇ 10 6 TU/mL.
  • the CAR-T cell preparation method of each combined double-binding domain GPRC5D double V H H CAR antibody sequence and a single V H H GPRC5D antibody sequence is as follows:
  • PBMC peripheral blood mononuclear cells
  • CD3 + T cells with a purity >95% were obtained after PBMCs were sorted by CD3 positive selection magnetic beads.
  • T cell sorting please refer to the product manual (MACS, DS130-050-101).
  • CD3 + T cells were resuspended with T cell complete medium (X-VIVO 15 (Lonza) + 5% FBS + cytokines), and then washed CD3/CD28 Dynabeads (Gibco, 40203D) were added in a 3-fold ratio, Then add complete medium for T cells, adjust the cell density to 1.0-1.2 ⁇ 10 6 cells/mL, and place in a 37°C, 5% CO 2 incubator to activate the culture.
  • T cell complete medium X-VIVO 15 (Lonza) + 5% FBS + cytokines
  • Lentiviral transduction is generally performed 20-24 hours after T cell activation.
  • T cells of each group collect the T cells of each group, centrifuge at 500g for 5 minutes, resuspend the cells with a certain volume of T cell complete medium, take samples and count, add T cell complete medium, and adjust the cells
  • the density is 0.5-0.7 ⁇ 10 6 cells/mL.
  • CAR-T cells were counted every 1-2 days, and T cell complete medium was supplemented according to the actual situation, and the cell density was adjusted to 0.5-1.0 ⁇ 10 6 cells/mL.
  • Dynabeads are removed on the fifth day of culture, and the total culture period is about 12 days.
  • CAR-T cells One of the important ways for CAR-T cells to exert cytotoxicity is through the release of perforin and granzymes.
  • the degranulation effect of CAR-T cells is also an important manifestation of CAR T cell activation.
  • Key indicators of CAR T killing effect In order to detect the ability of CAR-T to activate target cells, we detected the expression level of CD107a in the CD3-positive cells in the co-incubation wells, and the activation and killing ability of CAR-T were judged by the amount of the content.
  • GPRC5D In order to accurately detect the killing ability of CAR-T cells on target cells, we introduced the GPRC5D gene into CHO cells by means of lentivirus, and then sorted GPRC5D positive cells by flow cytometry, that is, CHO-GPRC5D cells.
  • the target cells are CHO-GPRC5D cells, and the negative cells are CHO negative cells.
  • the effector cells cultured to the 9th day were incubated with the target cells for 20 hours at an effector-target ratio of 3:1, and the survival of the target cells (that is, the lethality of CAR-T cells) was judged by the LDH method.
  • Cell killing toxicity can be calculated with the following formula:
  • the target cell killing experiment was carried out in a 96-well plate, the ratio of effector cells to target cells was 1:1, and then the number of effector cells in each 96-well plate was fixed at 2 ⁇ 10 4 , and target cells and effector cells were added in sequence.
  • target cells and effector cells were added in sequence.
  • untreated MM.1S cells were selected as the activated target cells for the detection of the targeted proliferation ability of CAR-T cells.
  • the CAR-T cells are cultured for about 9-12 days.
  • the CAR-positive rate of each group needs to be detected in advance, and then the blank effector T cells are used to adjust the CAR-positive rate of each group to be consistent (the culture medium does not contain any additives. X-VIVO 15 medium).
  • CAR-T cells and MM.1S cells were subjected to the first round of targeted stimulation at an effect-to-target ratio of 1:1. After 5 days of co-incubation, all cells were collected, sampled and counted, and then followed by 1:3 (full CAR- T cell: new MM.1S cell) ratio, carry out the second round of targeted stimulation, and after co-incubating again for 4-5 days, carry out the third round of targeted stimulation (the ratio between the two cells is the same as that of the second round of targeted stimulation) . During the three rounds of targeted stimulation, an appropriate amount of X-VIVO 15 medium was added every 1-2 days according to the growth of the cells.
  • the double-binding domain GPRC5D double V H H CAR-T cells had different degrees of targeted proliferation ability after receiving target cell stimulation, and the proliferation multiples of two rounds of repeated stimulation were between 50-200 times, compared with the control Compared with Mock T cells, there is a significant difference, which indicates that the double binding domain GPRC5D double V H H CAR-T cells can effectively and rapidly expand against specific tumors in vivo.
  • CAR-T cells in each group exhibited Strong cell proliferation ability, after three rounds of target cell stimulation, the proliferation rate can reach 1000-3500 times, compared with the control mock T cells, the proliferation activity is significantly higher, and the expansion efficiency of G5V H H1 CAR T cells under target cell stimulation Highest.
  • OriCAR-017 cells were higher than that of the positive control ET150-8 CAR T cells. This result indicates that the rapid proliferation of OriCAR-017 CAR T cells stimulated by target cells indicates that it can effectively and rapidly expand against specific tumors in vivo.
  • the memory phenotype, activation phenotype, and exhaustion phenotype of the cells were detected on the 12th day after co-culture: four groups were collected respectively Effector CAR T cells (including G5V H H1 CAR-T, G5V H H2 CAR-T, OriCAR-017 cells, and positive control ER150-8) were used to detect memory markers in CD3 + T cells in each group by flow cytometry: including CD45RO, CCR7 , CD62L cell ratio; detection of activated markers in CD3+T cells in each group: including CD25, CD69 cell ratio; detection of exhaustion markers in CD3+T cells in each group: including PD-1, LAG-3, TIM-3 cell ratio .
  • OriCAR-017 and G5V H H1 CAR T cell activation markers CD25 and CD69 were lower than those of G5V H H2 and ET150-8 CAR T cells, that is, T cell activation To a lesser extent, OriCAR-017 T cells had a low autoactivation cell phenotype advantage.
  • OriCAR-017 T cell exhaustion markers LAG-3 and TIM-3 were lower than those of G5V H H2 and ET150-8 CAR-T cells, that is, T cells With less exhaustion, OriCAR-017 T cells have the advantage of a low-exhaustion cell phenotype.
  • Immune synapse is a structure formed by connecting with specific activating receptors. It is a key step for CAR-T cells to recognize antigens, proliferate and activate, and is an important component of the body's cellular and humoral immune responses. part; The formation of immune synapse is related to the effector function of CAR-T cells. At the level of CAR-T, the polarization of the microtubule organizing center (MTOC) of the immune synapse structure was measured to further evaluate the functional advantages of CAR-T cells.
  • MTOC microtubule organizing center
  • CAR-T cells and target cells were co-cultured at a ratio of 1:1 for 5 minutes, then transferred to Nunc chamber slides, incubated for 20 minutes, fixed with 4% PFA at room temperature, washed 3 times with PBS, and immunostained with permeabilization solution (Triton X-100 ), incubate at room temperature; 10% horse serum/immunostaining permeabilization solution (Triton X-100), incubate at room temperature; add 2% horse serum/immunostaining permeabilization solution (Triton X-100) to incubate at room temperature; add Secondary antibody diluted in 2% horse serum/immunostaining permeabilization solution (Triton X-100) was incubated at room temperature; slides were separated, mounted with ProLong Diamond, dried and solidified, laser confocal 100 times oil lens, UV, 488nm, 543nm, 1024x1024, Z-axis scanning 0.4 ⁇ m, superimposed to get 3D images and videos.
  • G5V H H1, G5V H H2 and OriCAR-017 CAR T cells were confocally imaged; among them, the nuclei were stained in blue, ⁇ -tubulin was stained in green, and T cells were stained in red, indicated by white arrows
  • the polarization position of MTOC at the IS the closer the distance between MTOC and IS, the more stable the structure of IS.
  • mice After inoculation of CAR-T (or T) cells, the secretion of cytokines in the peripheral blood of mice was detected on the 7th and 14th days after CAR-T (or T) cell inoculation, and the tumor size and body weight of mice were measured 3 times a week , observed for 3 weeks.
  • the experimental process of tumor inhibition in NSG mice is shown in Figure 14; as shown in Figure 15A, the secreted cytokine IFN- ⁇ was detected on the 7th day after injection, and the IFN- ⁇ produced by the OriCAR-017 CAR-T cell group was significantly higher In G5V H H1 CAR-T, G5V H H2 CAR-T and positive control ET150-8 CAR-T.
  • Figure 15B the body weight of the mice showed a steady growth trend over time, and the body weight curves of all CAR-T cell experimental groups and the Mock T group had the same trend.
  • the mouse coat color after OriCAR-017 injection The light, the behavior and other vital signs of the mice are normal, which proves that OriCAR-017 has good safety in mice.
  • the OriCAR-017 cell group showed a very significant resistance to Tumor effect, tumor inhibition rate can be close to 100%.
  • mice in the OriCAR-017 cell group achieved 100% survival within the study period, and the survival rate of the mice was much higher than that of the single-binding domain VHH CAR T cell group and the positive control ET150-8 CAR -T cells, indicating that OriCAR-017 may show better anti-tumor activity in clinical practice.
  • B-NSG mice were injected with MM1S-Luciferase target cells into the tail vein, and were randomly divided into groups according to the bioluminescence intensity of the mice, with 5 mice in each group; different doses of OriCAR-017 CAR-T or Mock T cells that were cryopreserved in advance were injected into the tail vein ; After the reinfusion of CAR-T, the bioluminescence changes in the mice were observed every Tuesday and Thursday with the animal in vivo imaging system, and the weight and health status of the mice were observed. Factor detection, BD flow cytometry detection of CD3 + CD8 + T cell ratio.
  • the primary outcome of the study measured the occurrence of treatment-related adverse events assessed by Common Terminology Criteria for Adverse Events (CTCAE) v4.0 within 1-30 days after OriCAR-017 CAR-T cell injection; secondary The results assessed CAR-T-induced anti-myeloma responses, for example, by measuring abnormal immunoglobulin levels in serum, and the number of multiple myeloma cells in the patient's bone marrow, before and after administration of OriCAR-017 CAR-T cells. Efficacy objectives of the study included the proportion of pathological complete responses, 3-year disease-free survival, and 3-year progression-free survival.
  • TEAEs treatment-emergent adverse events
  • CRS cytokine release syndrome
  • 9 cases were grade 1
  • 1 case was grade 2.
  • All CRS resolved rapidly after conventional intervention (tocilizumab and steroids).
  • the median time to onset was 2 days (range, 1-9), and the median duration was 6 days (range, 3-9).
  • the objective response rate (ORR) was 100%
  • MRD was 100% negative
  • the complete remission/strict complete remission (CR/sCR) rate was 60%. 5 patients who had previously received anti-BCMA CAR-T therapy could be evaluated for efficacy, 2 patients achieved sCR, 2 patients achieved VGPR, and 1 patient achieved PR, as shown in Tables 4 and 5.
  • ORR (CR+sCR+VGPR+PR cases)/the number of subjects whose curative effect can be evaluated ⁇ 100%.
  • DCR (CR+sCR+VGPR+PR+MR cases)/the number of subjects whose curative effect can be evaluated ⁇ 100%.
  • the calculation of the percentage is based on the number of subjects enrolled in each dose group in the efficacy evaluable population.
  • the epitope of the GPRC5D V H H domain in OriCAR-017 CAR-T was identified. Construction of an exemplary bivalent GPRC5D CAR with two different anti-GPRC5D VH H domains that specifically bind to different epitopes of GPRC5D.
  • the bivalent/biepitope CAR comprising G5V H H1 and G5V H H2 was named OriCAR-017, which is a double binding domain GPRC5D double V H H CAR listed in exemplary Table 2.
  • the G5V H H1 and G5V H H2 antibody sequences were cloned into the p3.4-hIgG1Fc eukaryotic vector containing the human IgG1Fc fragment (hIgG1Fc) sequence to promote the recombinant expression of GPRC5D V H H-hIgG1Fc to obtain the recombinant protein and purify it. Affinity of G5V H H1-hIgG1Fc and G5V H H2-hIgG1Fc to the affinity N-terminal peptide (Biotin) of GPRC5D.
  • 1-27: MYKDCIESTGDYFLLCDAEGPWGIILE) protein of human GPRC5D was labeled with biotin (EZ-Link Sulfo-NHS-LC-Biotin, Pierce, 21327).
  • biotin EZ-Link Sulfo-NHS-LC-Biotin, Pierce, 21327.
  • PALL molecular interaction analyzer fortebiooctet RED384
  • the biotin-conjugated antigen was used to immobilize the SA sensor at a concentration of 20 nM, and combined for 3 minutes; then combined with the double-diluted antibody (ie, G5V H H1 and G5V H H2 antibody) solution for 5 minutes, and finally dissociated for 5 minutes.
  • the obtained results were analyzed by Octet Data Analysis 9.0 software (fortebio) to calculate the binding strength between antigen and antibody, and obtain KD value, Ka(1/Ms) value and Kd(1/s) value.
  • the binding affinity results of G5V H H1-hIgG1Fc and G5V H H2-hIgG1Fc are shown in Figure 17.
  • the regression equation for the detection of G5V H H1 antibody has a low R2 value (0.7914) for the goodness of fit of the sample data points, and the model is not credible. It can be seen that G5V H H1 does not bind to the N-terminal peptide of GPRC5D, while the G5V H H2 antibody binds to the N-terminal peptide with a KD of 7.69E-08M; further indicating that there are epitopes that G5V H H1 and G5V H H2 bind to the GPRC5D target difference.
  • the differential epitope binding of G5V H H1 and G5V H H2 was further validated by a cell-based competitive binding assay.
  • a stable CHO cell line overexpressing human GPRC5D (CHO-GPRC5D) was used for the assay.
  • G5V H H1 and G5V H H2 were cloned into the eukaryotic vector p3.4-hIgG1Fc containing the human IgG1 Fc fragment (hIgG1Fc) sequence and an expression vector containing a 6 ⁇ His tag to promote GPRC5D V H H-hIgG1Fc
  • G5V H H2-His recombinant proteins were obtained by recombinant expression and purification of GPRC5D V H H-His.
  • PBS buffer to prepare CHO-GPRC5D target cells at a concentration of 1 ⁇ 10 6 cells/mL, and add them to a 96-well conical bottom plate (corning 3894), 30 ⁇ L per well; use PBS buffer to prepare detection antibodies G5V H H1-hIgG1Fc, G5V H H2- The concentration of hIgG1Fc is 30 ⁇ g/mL, and the antibody is diluted 3 times to form 8 concentration gradients (the final concentration is 0); add the prepared antibodies of different concentrations at 30 ⁇ L/well to the plated target cells and mix well; Incubate at 4°C for 1 hour; add 150 ⁇ L of buffer to each well, centrifuge at 300 g for 5 minutes and discard supernatant; repeat washing once; use buffer to prepare goat anti-human IgG (Fc specific, ab98593) fluorescent secondary antibody at a ratio of 1:200 , add 30 ⁇ L per well to the cells and mix well, incubate at 4°C for 30 minutes;
  • G5V H H2-hIgG1Fc was incubated with CHO-GPRC5D cells of 3x10 4 total cells for 1 hour, and the concentration of antibody G5V H H1-His was 80 ⁇ g/mL in PBS buffer solution, and the concentration of antibody G5V H H1-His was 80 ⁇ g/mL, and followed by 3 Doubly dilute the antibody to form 12 concentration gradients (the final concentration is 0); then the serially diluted G5V H H1-His Recombinant antibody was added to the plate wells and incubated at 4°C for another 1h.
  • G5V H H2-hIgG1Fc binds to cells saturatingly, G5V H H1-His still has an antibody gradient dependence, and binds to G5V H H1-His in the absence of G5V H H2-hIgG1Fc The activities were similar, that is, the presence of G5VHH2 -hIgG1Fc did not affect the binding of G5VHH1 -His.
  • G5V H H2-hIgG1Fc when G5V H H2-hIgG1Fc is saturated with cells, G5V H H1-His is added to bind to target cells at the same time, and it is found that G5V H H2-hIgG1Fc has the same effect as G5V H H2-His in the absence of G5V H H1 -His.
  • the binding activity of hIgG1Fc is similar, that is, the presence of G5V H H1-His does not affect the binding of G5V H H1-His.
  • G5V H H1 and G5V H H2 bind to different epitopes of GPRC5D targets, and they do not affect each other.

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Abstract

本申请涉及一种嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域靶向G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白，且所述第二抗原结合域靶向GPRC5D蛋白。本申请还提供了包含所述嵌合抗原受体的细胞及其用途。

Description

靶向GPRC5D的嵌合抗原受体及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种新型抗GPRC5D的嵌合抗原受体，包含所述嵌合抗原受体的细胞，以及它们在治疗肿瘤中的用途。
背景技术
多发性骨髓瘤(Multiple Myeloma，MM)是第二常见的恶性血液病，并且在癌症致死率中居于第2位。MM属于浆细胞恶性肿瘤，常伴有多发性溶骨性损害、肾脏功能损害、骨髓浸润、高钙血症和贫血。MM在目前的主要治疗方法是副作用严重的全身化疗，并且无法彻底治愈。
G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白，是一种非典型的表面孤儿受体。GPRCD5与其他C5家族受体一样拥有过短的氨基末端，因此在构象上与C4家族十分类似。其在正常组织中表达仅限于毛囊，但在MM患者的骨髓中也有特异性高表达，并与浆细胞肿瘤负荷与遗传畸变高度相关。
随着蛋白酶抑制剂(PI)、免疫调节药物(IMiD)和单克隆抗体(mAb)等新型药物的介入，MM的治疗方式得到扩充，但依然有着极高的复发率并产生耐药性。而嵌合抗原受体T细胞(CAR-T)治疗对MM产生了显著疗效和可控的毒性，其中主流的以B细胞成熟抗原(BCMA)为靶点的CAR-T细胞疗法对于BCMA阴性或BCMA低表达的MM病患存在靶向逃逸问题。GPRC5D在浆细胞上的特异性高表达，使其成为了MM的理想治疗靶点，但以其为靶点的药物治疗产品尚未成熟。
发明内容
本申请提供了一种嵌合抗原受体，其包含两个抗原结合域，所述两个抗原结合域均能够靶向GPRC5D。本申请还提供了包含所述嵌合抗原受体的细胞。所述细胞具有下述性质中的一种或多种：(1)扩增能力强，(2)能够杀伤表达GPRC5D的靶细胞，(3)在靶细胞刺激下分泌细胞因子，(4)在靶细胞刺激下有较强的增殖能力，(4)抑制肿瘤生长，以及，(5)安全性好。
一方面，本申请提供了一种嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域靶向G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白，且所述第二抗原结合域靶向GPRC5D蛋白。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域和/或所述第二抗原结合域包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述抗原结合片段包括VHH。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:22和SEQ ID NO:27中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:21和SEQ ID NO:26中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:20和SEQ ID NO:25中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含HCDR1，HCDR2和 HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下述的任意一组氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ IDNO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
(3)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ IDNO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；以及
(4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:12、SEQ ID NO:28和SEQ ID NO:23中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:13或SEQ ID NO:29所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域包括VH，且所述VH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域为VHH，且所述VHH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ  ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:22和SEQ ID NO:27中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:21和SEQ ID NO:26中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:20和SEQ ID NO:25中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下述的任意一组氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
(3)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ  ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；以及
(4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:12、SEQ ID NO:28和SEQ ID NO:23中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:13或SEQ ID NO:29所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域包括VH，且所述VH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第二抗原结合域为VHH，且所述VHH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域分别包含HCDR1，HCDR2和HCDR3，所述第一抗原结合域和所述第二抗原结合域的HCDR1，HCDR2和HCDR3的序列选自下述任一组氨基酸序列：
(1)第一抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述 HCDR3包含SEQ ID NO:10所示的氨基酸序列；
(2)第一抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
(3)第一抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；
(4)第一抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；
(5)第一抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
(6)第一抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
(7)第一抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；
(8)第一抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；
(9)第一抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
(10)第一抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
(11)第一抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；
(12)第一抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；
(13)第一抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
(14)第一抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述 HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
(15)第一抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；以及
(16)第一抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域靶向GPRC5D的相同表位。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域包含相同或不同的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域直接或间接相连。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域通过连接子相连。
在某些实施方式中，所述连接子包含(GGGGS)n的氨基酸序列，其中所述n为1-10中的任意正整数。
在某些实施方式中，所述连接子包含(EAAAK)n的氨基酸序列，其中所述n为1-10中的任意正整数。
在某些实施方式中，所述嵌合抗原受体包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA- 1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述嵌合抗原受体的所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，所述嵌合抗原受体的所述共刺激信号区域包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述嵌合抗原受体的所述胞内信号传导域为源自CD3ζ的信号传导结构域。
在某些实施方式中，所述嵌合抗原受体的所述胞内信号传导域包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述嵌合抗原受体的所述跨膜区为源自CD8的跨膜区。
在某些实施方式中，所述嵌合抗原受体的所述跨膜区包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述嵌合抗原受体的所述铰链区为源自CD8的铰链区。
在某些实施方式中，所述嵌合抗原受体的所述铰链区包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含低密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段位于所述胞内信号区域的C端。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含信号肽。
在某些实施方式中，所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述嵌合抗原受体的所述信号肽包含SEQ ID NO:1所示的氨基酸序列。
另一方面，本申请还提供了一种或多种分离的核酸分子，其编码所述嵌合抗原受体。
在某些实施方式中，所述核酸分子还包含启动子。
在某些实施方式中，所述启动子为组成型启动子。
在某些实施方式中，所述启动子为EF1α启动子。
另一方面，本申请还提供了载体，其包含所述的核酸分子。
在某些实施方式中，所述载体为病毒载体。
在某些实施方式中，所述载体为慢病毒载体。
另一方面，本申请还提供了细胞，其包含所述嵌合抗原受体，所述核酸分子，和/或所述载体。
在某些实施方式中，所述细胞为免疫效应细胞。
在某些实施方式中，所述细胞包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
在某些实施方式中，所述细胞为T细胞。
另一方面，本申请还提供了制备所述嵌合抗原受体的方法，所述方法包括在使得所述嵌合抗原受体表达的条件下，培养所述细胞。
另一方面，本申请还提供了制备经修饰的免疫效应细胞的方法，其包括向免疫效应细胞 中引入所述的载体。
另一方面，本申请还提供了药物组合物，其包含所述嵌合抗原受体，所述核酸分子，所述载体，和/或所述细胞，以及可选地药学上可接受的载体。
另一方面，本申请还提供了所述嵌合抗原受体，所述核酸分子，所述载体，所述细胞，和/或所述药物组合物在制备药物中的用途，所述药物用于预防、治疗和/或缓解疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括与GPRC5D异常表达相关的疾病和/或病症。
在某些实施方式中，所述与GPRC5D异常表达相关的疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括血液瘤和/或淋巴瘤。
在某些实施方式中，所述肿瘤包括骨髓瘤。
在某些实施方式中，所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
另一方面，本申请还提供了一种预防、治疗和/或缓解疾病和/或病症的方法，所述方法包括向有需要的受试者施用所述的细胞，和/或所述的药物组合物。
在某些实施方式中，所述疾病和/或病症包括与GPRC5D异常表达相关的疾病和/或病症。
在某些实施方式中，所述与GPRC5D异常表达相关的疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括血液瘤和/或淋巴瘤。
在某些实施方式中，所述肿瘤包括骨髓瘤。
在某些实施方式中，所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请所述嵌合抗原受体各部分的元件及连接顺序示意图。
图2A显示的是双结合域GPRC5D double VHH CAR慢病毒感染后T细胞的阳性率。
图2B显示的是单个VHH CAR慢病毒感染后T细胞的阳性率。
图3显示的是单个VHH CAR-T细胞的体外扩增曲线。
图4A显示的是双结合域GPRC5D double VHH CAR-T细胞CD107a表达。
图4B显示的是单个VHH CAR-T细胞CD107a表达。
图5A显示的是双结合域GPRC5D double VHH CAR-T细胞特异性杀伤。
图5B显示的是单个VHH CAR-T细胞特异性杀伤。
图6A显示的是双结合域GPRC5D double VHH CAR-T细胞因子分泌水平。
图6B显示的是单个VHH CAR-T细胞因子分泌水平。
图7A显示的是双结合域GPRC5D double VHH CAR-T细胞的靶向增殖倍数。
图7B显示的是单个VHH CAR-T细胞的靶向增殖倍数。
图8显示的是CAR-T细胞记忆性表型流式检测图。
图9显示的是CD8+CAR-T细胞表型。
图10显示的是CD3+CAR-T细胞激活性marker表达。
图11显示的是CD3+CAR-T细胞耗竭性marker表达。
图12显示的是CAR-T细胞免疫突触共聚焦成像图。
图13显示的是MTOC与突触距离的定量分析。
图14显示的是NSG小鼠体内抑瘤实验流程图。
图15A-15D显示的是皮下成瘤模型NSG小鼠体内抑瘤效果图。
图16显示的是原位移植模型NSG小鼠体内抑瘤效果图。
图17显示的是抗体亲和力检测图。
图18显示的是抗体结合活性流式图。
图19显示的是G5VHH1与G5VHH2竞争性结合流式图。
图20显示的是双结合域VHH-hFc抗体与不同种属靶抗原的交叉结合活性。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
使用的术语“抗体”包括完整抗体和其结合片段。通常，片段与其来源的完整抗体竞争性地与抗原特异性结合。任选，抗体或其结合片段可以与其它蛋白质化学结合，或者与其它蛋白质以融合蛋白质的形式表达。例如，所述抗体可以是单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。例如，所述抗体或其结合片段的结合蛋白质可以包括GPRC5D。例如，所述抗体或其结合片段对GPRC5D可以有特异性。
术语“抗原结合片段”是指完整抗体的一部分并且是指完整抗体的抗原决定可变区。例如，所述抗原结合片段的可以包括Fab、Fab'、F(ab')2、Fv片段和单链Fv片段、串联Fv片段、VHH、双特异性抗体。例如，所述抗原结合片段可以是VHH。例如，所述抗原结合片段可以结合GPRC5D。例如，所述抗原结合片段对GPRC5D可以有特异性。
在本申请中，术语“VHH”或“VHH”可以互换使用，通常是指包含重链抗体的可变抗原结合结构域的抗体。VHH也可称为纳米抗体(Nanobody)(Nb)和/或单域抗体。例如，所述VHH可以结合GPRC5D。例如，所述VHH对GPRC5D可以有特异性。
在本申请中，所述抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链。每条重链由重链可变区(VH)和重链恒定区组成。术语“重链恒定区”由三个结构域CH1，CH2和CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区组成。术语“轻链恒定区”由一个结构域CL组成。VH和VL区可以进一步细分为高变区，称为互补决定区(CDR)，散布有更保守的区域，称为构架区(FR)。每个VH和VL由以下列顺序从氨基末端到羧基末端排列的三个CDR和四个FR组成:FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合。
在本申请中，术语“G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白”是一种非典型的表面孤儿受体。GPRCD5与其他C5家族受体一样拥有过短的氨基末端，因此在构象上与C4家族十分类似。其在正常组织中表达仅限于毛囊，但在多发性骨髓瘤患者的骨髓中也有显著表达，并与浆细胞肿瘤负荷与遗传畸变高度相关。例如，在本申请中的GPRC5D可以特指在MM患者体内表达的GPRC5D。
在本申请中，术语“嵌合抗原受体”(CAR)通常是指至少包含特异性地结合抗原或靶标的胞 外结构域、跨膜区以及胞内结构域的重组多肽。例如，所述胞外结构域与所述跨膜区之间包括铰链区。例如，所述嵌合抗原受体还可以包括低密度脂蛋白受体相关蛋白或其片段。例如，所述嵌合抗原受体可以包括信号肽。CAR的胞外结构域与靶细胞表面上的靶抗原结合导致CAR聚簇并将激活刺激传送给含CAR细胞。CAR重定向免疫效应细胞的特异性，并且触发了增殖、细胞因子产生、能够以不依赖主要组织相容性(MHC)的方式介导表达靶抗原的细胞死亡的分子的吞噬作用和/或产生。例如，所述胞外结构可以特异性结合GPRC5D。
在本申请中，术语“胞内结构域”指意思是包括足以转导活化信号的任何截短部分的细胞内结构域。所述胞内结构域可以包括细胞内信号区域和/或共刺激信号区域。术语“细胞内信号区域”指可以产生促进含CAR细胞(例如CART细胞或表达CAR的NK细胞)的免疫效应子功能的信号的胞内区域。例如，所述胞内信号区域可以包括选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。例如，所述胞内信号区域可以是源自CD3ζ的信号传导结构域。术语“共刺激信号区域”是指所述胞内信号区域中能够转导效应信号的CAR的一部分。例如，所述共刺激信号区域可以包括源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。例如，所述共刺激信号区域可以是源自4-1BB的胞内共刺激信号区域。
在本申请中，术语“跨膜区”指能够跨越细胞质膜的肽、多肽或蛋白质的结构域。可采用这些结构域将胞外结构域锚定在细胞膜上。例如，所述跨膜区可以包含选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。例如，所述跨膜区可以源自CD8的跨膜区。
在本申请中，术语“铰链区”表示抗体重链多肽中连接CH1结构域和CH2结构域的一部分，例如，从根据Kabat的EU编号系统的约第216位至约第230位。铰链区通常是由两条具有相同氨基酸序列的多肽组成的二聚体分子。铰链区一般包括约25个氨基酸残基，是柔性 的，允许抗原结合区独立的移动。铰链区可以细分为3个结构域：上、中、下铰链结构域。例如，所述铰链区可以包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。例如，所述铰链区可以源自CD8的铰链区。
在本申请中，术语“低密度脂蛋白受体相关蛋白(Low-density lipoproteinreceptor-related protein)指一种属于内吞性受体的细胞表面蛋白，在生物体内广泛分布并有很大的组织间差异性，主要功能是摄取胆固醇进入细胞内用以细胞增殖和固醇类激素及胆汁酸盐的合成。例如，所述低密度脂蛋白受体相关蛋白可以来自于任何脊椎动物。例如，所述低密度脂蛋白受体相关蛋白或其片段可以位于所述胞内信号区域的C端。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白6或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白5或其片段。
在本申请中，术语“信号肽”是指处于新生CAR蛋白的氨基末端(N-末端)的前导序列，其在翻译时或在翻译后将新生蛋白引导到内质网并后续表面表达。例如，所述信号肽源自CD8蛋白的信号肽。
在本申请中，术语“核酸分子”包括DNA分子和RNA分子。一个核酸分子可以是单链或双链的，但优选为双链DNA。术语“启动子”通常是指一个DNA序列，可以调节与启动子操作性连接的所选DNA序列的表达，从而影响细胞中所选DNA序列的表达。例如，所述核酸分子可以编码包括所属抗原结合蛋白和/或所述嵌合抗原受体。例如，所述核酸分子可以包括启动子。例如，所述启动子可以是组成型启动子。例如，所述启动子可以是EF1α启动子。
在本申请中，术语“载体”通常是指可以附着本申请的一种或多种核酸分子的分子。例如，所述载体可以是病毒载体。例如，所述载体可以是慢病毒载体。
在本申请中，术语“细胞”指可以向其转染核酸的细胞，术语“细胞”包括用于质粒繁殖的原核细胞，和用于核酸表达和编码多肽产生的真核细胞。例如，细胞可以包括所述的嵌合抗原受体，所述的核酸分子和/或所述的载体。例如，所述细胞可以是免疫效应细胞。术语“免疫效应细胞”通常是指参与免疫应答，行使效应功能的免疫细胞。例如，所述行使效应功能可以包括清除异物抗原或促进免疫效应子应答等。例如，免疫效应细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细 胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。例如，免疫效应细胞可以是T细胞。
在本申请中，术语“药物组合物”通常指适合施用于哺乳动物个体的化学或生物组合物。例如，所述药物组合物可以包括所述抗原结合蛋白，所述嵌合抗原受体，所述多肽，所述核酸分子，所述载体和/或所述细胞，以及可选的药学上可接受的载体。所述药物组合物可以用于预防、治疗和/或缓解与GPRC5D异常表达相关的疾病或病症。例如，所述与GPRC5D异常表达相关的疾病或病症可以包括肿瘤。例如，所述肿瘤包括实体瘤和/或非实体瘤。例如，所述肿瘤包可以括血液瘤和/或淋巴瘤。例如，所述肿瘤可以包括骨髓瘤。
发明详述
一方面，本申请提供一种嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域靶向G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白，且所述第二抗原结合域靶向GPRC5D蛋白。
在某些实施方式中，所述嵌合抗原受体的所述第一抗原结合域和/或所述第二抗原结合域可以包含抗体或其抗原结合片段。例如，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。例如，所述抗原结合片段包括VHH。
在本申请中，所述CDR可以通过IMGT划分方式进行划分。
第一抗原结合域
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可靶向GPRC5D蛋白。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含抗体重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含HCDR3，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。例如，所述嵌合抗原受体的所述第一抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:22和SEQ ID NO:27中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含HCDR2，且所述HCDR2可包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。例如，所述嵌合抗原受体的所述第一抗原结合域可包含HCDR2，且所述HCDR2可包含SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:21和SEQ ID NO:26中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含HCDR1，且所述HCDR1可包含SEQ ID NO:35所示的氨基酸序列。例如，所述嵌合抗原受体的所述第一抗原结合域可包含HCDR1，且所述HCDR1可包含SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:20和SEQ ID NO:25中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含HCDR1，HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含HCDR1，HCDR2和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下述的任意一组氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
(3)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；以及
(4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1可包含SEQ ID NO:11所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可包含SEQ ID NO:12、SEQ ID NO:28和SEQ ID NO:23中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可包含SEQ ID NO:13或SEQ ID NO:29所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包含H-FR4，所述H-FR4的N 末端与所述HCDR3的C末端相连，且所述H-FR4可包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可包括VH，且所述VH可包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域可为VHH，且所述VHH可包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
第二抗原结合域
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可靶向GPRC5D蛋白。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含抗体重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含HCDR3，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。例如，所述嵌合抗原受体的所述第二抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:22和SEQ ID NO:27中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含HCDR2，且所述HCDR2可包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。例如，所述嵌合抗原受体的所述第二抗原结合域可包含HCDR2，且所述HCDR2可包含SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:21和SEQ ID NO:26中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含HCDR1，且所述HCDR1可包含SEQ ID NO:35所示的氨基酸序列。例如，所述嵌合抗原受体的所述第二抗原结合域可包含HCDR1，且所述HCDR1可包含SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:20和SEQ ID NO:25中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含HCDR1，HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含HCDR1，HCDR2和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下述的任意一组氨基酸序列：
(5)所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
(6)所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
(7)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；以及
(8)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1可包含SEQ ID NO:11所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可包含SEQ ID NO:12、SEQ ID NO:28和SEQ ID NO:23中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可包含SEQ ID NO:13或SEQ ID NO:29所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4可包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可包括VH，且所述VH可包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第二抗原结合域可为VHH，且所述VHH可包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
嵌合抗原受体
在本申请中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域可分别包 含HCDR1，HCDR2和HCDR3。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示 的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示 的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列。
例如，在第一抗原结合域中：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；在第二抗原结合域中：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域可靶向GPRC5D的相同表位。在本申请中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域可靶向GPRC5D的不同表位。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域可包含相同或不同的氨基酸序列。
在本申请中，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域可直接或间接相连。例如，所述嵌合抗原受体的所述第一抗原结合域和所述第二抗原结合域可通过连接子相连。
在本申请中，所述连接子可包含(GGGGS)n的氨基酸序列，其中所述n为1-10中的任 意正整数。例如，所述n可以为1，2，3，4，5，6，7，8，9或10。
在本申请中，所述连接子可包含(EAAAK)n的氨基酸序列，其中所述n为1-10中的任意正整数。例如，所述n可以为1，2，3，4，5，6，7，8，9或10。
在本申请中，所述嵌合抗原受体可包含共刺激信号区域，其中所述共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。例如，所述嵌合抗原受体的所述共刺激信号区域可为源自4-1BB的胞内共刺激信号区域。例如，所述嵌合抗原受体的所述共刺激信号区域可包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述嵌合抗原受体可包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。例如，所述嵌合抗原受体的所述胞内信号传导域可为源自CD3ζ的信号传导结构域。例如，所述嵌合抗原受体的所述胞内信号传导域可包含SEQ ID NO:5所示的氨基酸序列。
在本申请中，所述嵌合抗原受体可包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。例如，所述嵌合抗原受体的所述跨膜区可为源自CD8的跨膜区。例如，所述嵌合抗原受体的所述跨膜区可包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述嵌合抗原受体还可包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。例如，所述嵌合抗原受体的所述铰链区可为源自CD8的铰链区。例如，所述嵌合抗原受体的所述铰链区包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述嵌合抗原受体还可包含低密度脂蛋白受体相关蛋白或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段位于所述胞内信号区域的C端。例如，所述低密度 脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述嵌合抗原受体还可包含信号肽。例如，所述信号肽源自CD8蛋白的信号肽。例如，所述嵌合抗原受体的所述信号肽包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述嵌合抗原受体中的所述低密度脂蛋白受体相关蛋白或其片段可通过自剪切肽(例如，T2A、P2A、E2A等2A肽)连接至CAR的C端。例如，所述低密度脂蛋白受体相关蛋白或其片段可以通过T2A与胞内信号区域的C端连接。
在本申请中，自N端至C端，所述嵌合抗原受体可依次包含结合GPRC5D蛋白的抗原结合域、所述铰链区、所述跨膜域、所述共刺激信号区域和所述胞内信号区域。例如，自N端至C端，所述嵌合抗原受体可依次包含所述抗原结合域，源自CD8的铰链区，源自CD8的跨膜区，源自4-1BB的共刺激信号区域，和源自CD3ζ的胞内信号区域。
在本申请中，自N端至C端，所述嵌合抗原受体可依次包含结合GPRC5D蛋白的抗原结合域、所述铰链区、所述跨膜域、所述共刺激信号区域，所述胞内信号区域和所述低密度脂蛋白受体相关蛋白或其片段。例如，自N端至C端，所述嵌合抗原受体可依次包含所述抗原结合域，源自CD8的铰链区，源自CD8的跨膜区，源自4-1BB的共刺激信号区域，源自CD3ζ的胞内信号区域和所述低密度脂蛋白受体相关蛋白或其片段。
例如，所述嵌合抗原受体可以包含SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列。
在本申请中，所述嵌合抗原受体可以包含下表所示的结构：

其中，A代表(GGGGS)n；B代表B:(EAAAK)n。其中，n为1至10中的任意正整数。
例如，3B5的HCDR1包含SEQ ID NO:8所示的氨基酸序列，3B5的HCDR2包含SEQ ID NO:9所示的氨基酸序列，3B5的HCDR3包含SEQ ID NO:10所示的氨基酸序列，3B5的VHH包含SEQ ID NO:15所示的氨基酸序列。
例如，3E7的HCDR1包含SEQ ID NO:16所示的氨基酸序列，3E7的HCDR2包含SEQ ID NO:17所示的氨基酸序列，3E7的HCDR3包含SEQ ID NO:18所示的氨基酸序列，3E7的VHH包含SEQ ID NO:19所示的氨基酸序列。
例如，4A2的HCDR1包含SEQ ID NO:20所示的氨基酸序列，4A2的HCDR2包含SEQ ID NO:21所示的氨基酸序列，4A2的HCDR3包含SEQ ID NO:22所示的氨基酸序列，4A2的VHH包含SEQ ID NO:24所示的氨基酸序列。
例如，4B3的HCDR1包含SEQ ID NO:25所示的氨基酸序列，4B3的HCDR2包含SEQ ID NO:26所示的氨基酸序列，4B3的HCDR3包含SEQ ID NO:27所示的氨基酸序列，4B3的VHH包含SEQ ID NO:30所示的氨基酸序列。
例如，所述CD8α信号肽可包含SEQ ID NO:1所示的氨基酸序列。
例如，所述CD8α铰链区可包含SEQ ID NO:2所示的氨基酸序列。
例如，所述CD8α跨膜区可包含SEQ ID NO:3所示的氨基酸序列。
例如，所述4-1BB共刺激信号可包含SEQ ID NO:4所示的氨基酸序列。
例如，所述CD3ζ胞内信号区可包含SEQ ID NO:5所示的氨基酸序列。
例如，所述Ori可包含SEQ ID NO:7所示的氨基酸序列。
核酸分子
另一方面，本申请还提供了一种或多种核酸分子，所述核酸分子可以是任意长度的分离形式的核苷酸、脱氧核苷酸和/核糖核苷酸，可以编码所述的嵌合抗原受体。
例如，所述核酸分子可以包括启动子。例如，所述启动子可以是组成型启动子。例如，所述启动子可以是EF1α启动子。
载体
另一方面，本申请还提供了一种载体，所述载体可以包括所述的核酸分子。所述载体可以转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达。例如，载体可以包括启动子、转录子、增强子、复制子、选择元件和报告基因。例如，载体可以包括协助进入细胞的成分。为了使所述核酸分子在载体中复制，所述核酸分子的5’端和3’端还可以包含长末端重复序列。
例如，所述载体可以是病毒载体。例如，所述载体可以是慢病毒载体。
细胞
另一方面，本申请还提供了细胞，所述细胞可以包括所述的嵌合抗原受体、所述的核酸分子和/或所述的载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。
在某些实施方案中，所述的细胞可以是免疫效应细胞。在某些实施方案中，所述的细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。例如，所述的细胞可以是T细胞。
药物组合物
另一方面，本申请还提供了一种药物组合物，可以包括所述嵌合抗原受体、所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的佐剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物可以包括液体、 冷冻和冻干组合物。
在某些实施方案中，所述药学上可接受的佐剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
制备方法
另一方面，本申请还提供了制备所述嵌合抗原受体的方法。所述方法可以包括在使得所述嵌合抗原受体表达的条件下，培养所述细胞。
本申请还提供了制备经修饰的免疫效应细胞的方法，所述方法可以包括向免疫细胞中引入所述载体。
用途
另一方面，本申请还提供了所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物可以用于预防、缓解和/或治疗肿瘤。
另一方面，本申请还提供了预防、缓解和/或治疗肿瘤的方法，所述方法可以包括向受试者施用所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物。
另一方面，本申请还提供了所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物，其用于预防、缓解和/或治疗肿瘤。
在本申请中，所述肿瘤可以包括GPRC5D异常表达的肿瘤。
在本申请中，所述肿瘤可以包括实体瘤和/或非实体瘤。
在本申请中，所述肿瘤可以包括血液瘤和/或淋巴瘤。
在本申请中，所述肿瘤可以包括骨髓瘤。在本申请中，所述骨髓瘤可以包括难治性/复发性多发性骨髓瘤。
在本申请中，所述受试者可以包括人类或非人类动物。
另一方面，本申请提供了试剂盒或给药装置，其包括所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而 不用于限制本申请发明的范围。
实施例
实施例1利用人工合成纳米抗体库NanoOri_1.0筛选抗GPRC5D抗体
比对PDB数据库(Protein Data Bank)中296条无重复序列的纳米抗体及其结构，确定CDRs突变策略，其中CDR3的长度为三种：14，17和21个氨基酸。选取已经上市的纳米抗体卡拉西单抗(Caplacizumab)为骨架，全基因合成Caplacizumab的核苷酸序列，然后将其克隆至HP153噬菌体载体，之后提取HP153的单链DNA，利用Kunkel Mutagenesis的方法突变纳米抗体的CDRs，获得双链DNA。将双链DNA电转至M13KO7辅助噬菌体预感染的感受态大肠杆菌SS320，过夜培养后收集噬菌体上清，最终构建出多样性为1.44×1010的人工合成纳米抗体库NanoOri_1.0(原启生物科技(上海)有限责任公司)，作为种子库进行抗体序列筛选。
将从Sino Biological公司购买的GPRC5D全长序列(cat:HG24447-UT)构建到公司已有的慢病毒载体上，进行慢病毒转导，制备靶细胞CHO-GPRC5D和靶细胞HEK293-GPRC5D的过表达细胞株。利用靶细胞CHO-GPRC5D和HEK293-GPRC5D进行人工合成纳米抗体库(原启生物科技(上海)有限责任公司)的交替淘选，共进行四轮淘选。
从人工合成纳米抗体库NanoOri_1.0里取一支加入PEG/NaCl，重新沉淀使用。选取靶细胞CHO-GPRC5D和HEK293-GPRC5D用作细胞淘选的交替筛选。取500μL的噬菌体和500μL的10％FBS/PBS缓冲液混合到终体积1mL，装于2mL的低吸附的EP管中，4℃下轻轻旋转或者周期性混匀进行封闭1小时。140g离心10min收获CHO-GPRC5D细胞，用10mL的PBS洗涤一次。进行计数，活细胞数达到95％以上，用5％FBS/PBS缓冲液洗涤CHO-GPRC5D细胞三次。然后重悬于1mL的5％FBS/PBS缓冲液，使细胞数目达到1×107个。期间细胞置于冰上。4℃下140g离心CHO-GPRC5D细胞2min，重悬于已经封闭好的1mL的噬菌体抗体库中，4℃或者室温下轻轻旋转或者周期性混匀进行结合2小时，移去上清，收获细胞重悬于1mL的5％FBS/PBS缓冲液中，重复洗涤二次，最后一次重悬细胞以后，换到新的2mL的低吸附的EP管中，避免非特异性吸附到EP管的噬菌体被洗脱下来。
用1mL的100nM的Gly-HCl(pH2.2)重悬细胞，室温孵育10min后4℃下140g离心2min，把上清转移到新的EP管中，加入25μL的2M Tris中和液。把中和液约1mL再用约500μL的7.5％FBS/PBS缓冲液中和。
离心3份相同的吸附细胞CHO，处理方法同CHO-GPRC5D细胞。把洗脱下来后中和好 的噬菌体重悬于CHO细胞中。4℃下轻轻旋转，或者周期性混匀进行结合30min。4℃下140g离心2min，取上清用于下一次吸附过程，一共3次。最后一次的噬菌体上清用于感染TG1细胞。同样的方法进行第二轮细胞淘选，靶细胞CHO-GPRC5D的数目为5×106个，洗涤次数为3次。采用同样的操作方法进行第3轮和第4轮细胞淘选，靶细胞换成HEK293-GPRC5D，细胞数目为1×106个，洗涤次数增加为10次。
4轮淘洗之后，观察到输出的噬菌体针对靶细胞有显著的特异性富集。利用基于细胞的单噬菌体ELISA(Cell-based mono phage ELISA)对淘选得到的噬菌体抗体克隆进行鉴定：在96孔ELISA板上以3×104细胞/孔包被CHO-GPRC5D靶细胞，4℃过夜。然后用5％脱脂奶粉封闭非特异性结合位点，充分洗涤后，取单克隆的噬菌体上清(预混脱脂奶粉)加入到96孔板，孵育2小时，充分洗涤后，加入Anti-M13-HRP，反应45min，充分洗涤后加入TMB显色，室温下作用5-10min后，用2M硫酸终止反应，于450nm处测定各孔OD值。
在淘选之后，通过ELISA鉴定获得了34株能特异性结合人GPRC5D的噬菌体抗体克隆，测序后获得单链VHH基因序列。
示例性地选择3B5VHH(VHH的氨基酸序列如SEQ ID NO:15所示)、3E7VHH(VHH的氨基酸序列如SEQ ID NO:19所示)、4A2VHH(VHH的氨基酸序列如SEQ ID NO:24所示)和4B3VHH(VHH的氨基酸序列如SEQ ID NO:30所示)抗体序列，通过流式细胞荧光分选技术(FACS)，使用iQue Screener流式仪(购自IntelliCyt公司)，用含有0.1％BSA的PBS作为缓冲液进行细胞表面靶抗原(GPRC5D)与抗体的细胞结合活性检测。
使用缓冲液配置浓度为1x106细胞/mL的靶细胞(即MM1S骨髓瘤细胞)，加入96孔尖底板(corning 3894)，每孔30μL；使用缓冲液配置检测抗体浓度为10μg/mL，并按3倍比稀释抗体，形成8个浓度梯度；将配置好的不同浓度的抗体按30μL/孔加入已铺好的靶细胞中混匀；4℃冰箱孵育1小时；每孔加150μL缓冲液，300g离心5分钟，弃上清后将细胞震松散；重复洗涤一次；使用缓冲液按1:200配比配置荧光二抗(ab98593)，每孔30μl加入细胞中混匀，4℃冰箱孵育30分钟；每孔加150μL缓冲液，300g离心5分钟，弃上清后将细胞震松散；重复洗涤一次；每孔加35μL缓冲液混匀后用流式仪器检测。抗GPRC5D抗体与细胞表面GPRC5D蛋白结合活性如表1所示。其中，ET150-5scFv为阳性对照抗体，其序列可见专利申请CN107428829A，3B5VHH的氨基酸序列如SEQ ID NO:15所示，3E7VHH的氨基酸序列如SEQ ID NO:19所示，4A2VHH的氨基酸序列如SEQ ID NO:24所示，4B3VHH的氨基酸序列如SEQ ID NO:30所示。结果显示，本申请所述的抗GPRC5D VHH均能结合细胞表面的GPRC5D，且结合能力与阳性对照ET150-5scFv相当，甚至高于阳性对照。
表1.抗GPRC5D抗体与细胞表面GPRC5D蛋白的结合
实施例2 CAR慢病毒表达载体构建
为了获得具有更强亲和力及更好抗肿瘤效果的抗GPRC5D CAR-T细胞产品，我们将其中4个在体外功能验证中具有较好靶细胞杀伤活性、较高靶向增殖能力及较低tonic信号水平的3B5VHH、3E7VHH、4A2VHH和4B3VHH抗体序列，通过不同的肽接头组合到CAR结构上以构建双结合域GPRC5D double VHH CAR。示例性双结合域double VHH CAR构建体如表1所示。构建体制备步骤如下：具体而言，使两个VHH序列通过(GGGGS)n或者(EAAAK)n融合(其中，n可以为正整数)，然后用SphI和NotI内切酶(购自NEB)对CAR慢病毒核心空载质粒(原启生物科技(上海)有限责任公司自构，包含Ori新元件，以下简称CAR慢病毒空载体)进行双酶切产生8992bp线性化片段，割胶回收后与融合所得的double VHH片段，按1:3的摩尔比例混合进行同源重组后，转化DH5α感受态细胞。挑取单菌落，进行测序鉴定。双结合域double VHH CAR结构的连接顺序详见表2。
通过对32种不同组合的双结合域double VHH CAR结构进行CAR-T体外生物学功能验证，含CAR-T细胞体外增殖活性、靶向杀伤能力、细胞因子分泌水平等，示例性选择其中一种double VHH CAR结构组合，命名为OriCAR-017(即oriCAR-DG508M)，随后与单一结构域的G5VHH1 CAR(其中，靶向部分的G5VHH1的氨基酸序列如SEQ ID NO:19所示(3E7))和G5VHH2 CAR(其中，靶向部分的G5VHH2的氨基酸序列如SEQ ID NO:30所示(4B3))、阳性对照ET150-8(其氨基酸序列见专利号：US10633426B2)进行CAR-T体外生物学功能实验，各CAR慢病毒载体各部分元件及连接顺序详见图1。其中，CD8α信号肽的氨基酸序列如SEQ ID NO:1所示，CD8α铰链区的氨基酸序列如SEQ ID NO:2所示，CD8α跨膜区的氨基酸序列如SEQ ID NO:3所示，4-1BB共刺激信号的氨基酸序列如SEQ ID NO:4所示，CD3ζ胞内信号传导区的氨基酸序列如SEQ ID NO:5所示，Ori新元件的氨基酸序列如SEQ ID NO: 7所示。接头A的示例性选择序列为(GGGGS)5，接头B的示例性选择序列为(EAAAK)3。
表2示例性双结合域GPRC5D double VHH CAR结构顺序

A:(GGGGS)n，其中，n为大于或等于0的整数，GGGGS的氨基酸序列如SEQ ID NO:39所
示
B:(EAAAK)n，其中，n为大于或等于0的整数，EAAAK的氨基酸序列如SEQ ID NO:40所
示
实施例3慢病毒的包装及病毒滴度测定
作为示例，用于构建本发明的慢病毒载体系统属于第三代，该系统共由四个质粒组成，即编码Gag-Pol蛋白的包装质粒pRH1、编码Rev蛋白的包装质粒pVH3和编码包膜蛋白VSV-G的pMH2质粒，以及核心质粒，即上述实施例2中的含VHH序列的各个CAR慢病毒质粒。各个CAR慢病毒质粒中CAR基因由延长因子-1α(EF-1α)启动子调控其表达。
3.1慢病毒包装：
(1)取生长状态良好、约2.5×106个293T细胞于5mL 293T细胞完全培养基(含10％FBS 的高糖DMEM)中，混匀后接种于6cm培养皿中(确保20-24h后细胞汇合度在80-90％)，37℃、5％CO2的培养箱内培养过夜。
(2)约20-24h后，取300uL Opti-MEM培养基，加入总共6.9ug的三种质粒(按质量比pRH1:pMH2:pVH3:CAR慢病毒核心质粒＝1:1:2:2的比例加入)，混匀后加入20.7uL的FuGENEHD转染试剂(Promega，E2311)，小心混匀后，室温静置15-20min，然后将混合液小心滴加至6cm培养皿中，小心摇动培养皿，37℃、5％CO2的培养箱内培养过夜。
(3)转染约20-24h后，小心弃去6cm培养皿中的上层液体，然后缓慢加入5mL 293T细胞完全培养基。
(4)转染约48h后，收取6cm培养皿中的上层液体(含死细胞和碎片)，3000g离心5min后，将去除细胞碎片的上层病毒液转移至新的离心管中，分装后，保存于-80℃。
3.2慢病毒滴度检测：
(1)快速解冻以上制备的病毒液。
(2)取生长状态良好的293T细胞(一般培养20代以内的细胞)，弃去上层废液，PBS洗涤细胞后，用0.25％的胰酶(GIBCO)，于37℃消化约3min，待细胞消化完全后加入一定体积的293T细胞完全培养基终止反应，取样计数，然后将细胞密度调整为2.0×105/mL，并加入终浓度为10ug/mL的polybrene，再按每孔2.5mL的接种量，将细胞接种于六孔板中。
(3)然后往六孔板中分别加入上述病毒液，一般每种病毒液做三个上样梯度，2uL、5uL和10uL，并且需增加1-2个不加入病毒液的空白孔作为流式检测时的阴性对照。
(4)细胞培养板置于37℃，5％CO2培养箱中静置培养。
(5)细胞静置培养48h后，弃去上层废液，PBS洗涤细胞后，用0.25％的胰酶(GIBICO)，于37℃消化约3分钟，待细胞消化完全后加入一定体积的293T细胞完全培养基终止反应，取样计数，然后取约5×105个细胞至1.5ml无菌离心管，做好标记。
(6)用NBS溶液(含1％新生牛血清的PBS溶液)补齐至1.2-1.5mL，4℃，500g，离心5min，弃上清。
(7)每管加入1mL NBS，轻轻吹打混匀，4℃，500g，离心5min，充分弃去上清。
(8)每管加入50μl NBS和0.8μg Biotin-Goat anti-human F(ab)2抗体(109-066-006)，用移液枪轻轻吹打混匀(阴性对照不加一抗)，4℃孵育60min。
(9)孵育完毕直接加入1ml NBS重悬细胞，4℃，500g，离心5min，弃去上清。
(10)每管加入50μl NBS和0.5μg APC streptavidin(554067)荧光二抗(阴性对照不加此蛋白)，4℃孵育30min。
(11)重复步骤(9)。
(12)每管加入200μl NBS重悬细胞。
(13)BD FACS CantoII流式仪上检测荧光表达百分比，采用FlowJo-V10软件数据处理。
仅当各测试管阳性率-阴性对照阳性率为5％-20％时，结果可用；病毒滴度计算公式如下：
经检测，上述各个含VHH的CAR病毒滴度范围在1-10×106TU/mL。
实施例4 T细胞的感染和GPRC5D CAR-T细胞体外扩增
各组合的双结合域GPRC5D double VHH CAR抗体序列及单个VHH GPRC5D抗体序列的CAR-T细胞制备方法如下：
(1)通过Ficoll密度梯度离心法，从单采机采集的血样中分离得到人外周血单个核细胞(PBMC)(单采血由上海原能细胞集团方舟计划志愿者提供)。
(2)PBMC经CD3阳选磁珠分选后得到纯度>95％的CD3+T细胞，T细胞分选具体方法见产品说明书(MACS，DS130-050-101)。
(3)用T细胞完全培养基(X-VIVO15(Lonza)+5％FBS+细胞因子)重悬CD3+T细胞，再按3倍比例加入已经洗涤的CD3/CD28 Dynabeads(Gibco，40203D)，然后补加T细胞完全培养基，调整细胞密度为1.0-1.2×106细胞/mL，置于37℃，5％CO2培养箱中激活培养。
(4)一般在T细胞激活20-24h后，进行慢病毒转导。
(5)收集激活20-24h的T细胞，500g离心5min后，用一定体积的T细胞完全培养基重悬细胞后取样计数，按照病毒感染复数(MOI)为3的比例，分别加入实施例3中已测定滴度的病毒液，然后加入polybrene至终浓度为5μg/mL，再补加T细胞完全培养基，调整细胞密度为0.6-1.0×106/mL，再将细胞置于37℃，5％CO2培养箱中培养，另取一份T细胞不做慢病毒感染，作为阴性对照组。
(6)病毒转导约20-24h后，收集各组的T细胞，500g离心5min后，用一定体积的T细胞完全培养基重悬细胞后取样计数，补加T细胞完全培养基，调整细胞密度为0.5-0.7×106细胞/mL。
(7)此后，每1-2天对CAR-T细胞进行计数处理，并根据实际情况补加T细胞完全培养基，将细胞密度调整为0.5-1.0×106细胞/mL。
(8)一般情况下，在培养第5天时去除Dynabeads，总培养周期为12天左右。
实验结果如图2A-2B所示，经激活培养扩增，双结合域GPRC5D double VHH CAR慢病 毒感染后的T细胞CAR阳性率介于55％-75％之间；单个VHH GPRC5D CAR阳性率介于80％-90％之间；CAR基因在T细胞上稳定表达，且各组之间无显著性差异；如图3所示，在体外CD3/CD28刺激后，CAR-T细胞稳定增殖，特别是在共培养的第9天开始，G5VHH1 CART和OriCAR-017细胞表现出比G5VHH2 CAR-T和阳性对照ET150-8 CAR-T细胞更为优越增殖潜力，累积扩增倍数分别为1000倍和200倍；可以用于细胞学功能实验。
实施例5 GPRC5D CAR-T细胞的脱颗粒分子CD107a的表达水平测定
CAR-T细胞发挥细胞毒性的重要途经之一是通过释放穿孔素和颗粒酶，CAR-T细胞的脱颗粒效应也是CART细胞活化的重要表现，因此，细胞脱颗粒功能分子CD107a的表达水平是评价CAR T杀伤效应的关键指标。为了检测CAR-T针对靶细胞的激活能力，我们检测共孵育孔中CD3阳性细胞中CD107a的表达含量，通过含量的多少来判断CAR-T的激活及杀伤能力。
如图4A-4B所示，不同的双结合域GPRC5D double VHH CAR-T细胞与靶细胞以1:1的效靶比共培养4h后，各组CAR-T细胞CD107a表达的水平不均一，均高于Mock T对照细胞的CD107a表达；其中，OriCAR-017的CD107a表达阳性率在28％左右，与两个单域抗体CART细胞组和阳性对照ET150-8组相比有相对较高的CD107a表达阳性率，实验说明了OriCAR-017针对特异性靶细胞有明显的脱颗粒效应，预示着有良好的活化和效应杀伤潜力。
实施例6 GPRC5D CAR-T细胞的体外效应杀伤活性评估
为了准确地检测CAR-T细胞对于靶细胞的杀伤能力，我们将GPRC5D基因通过慢病毒的方式导入CHO细胞中，再通过了流式分选仪分选出GPRC5D阳性细胞，即CHO-GPRC5D细胞。靶细胞即为CHO-GPRC5D细胞，阴性细胞即为CHO阴性细胞。将培养到第9天的效应细胞，分别以效靶比为3:1的比例，与靶细胞孵育20小时，通过LDH方法判断靶细胞的存活情况(即CAR-T细胞的杀伤力)。
细胞的杀伤毒性可以用以下公式进行计算：
如图5A-5B所示，和空白效应细胞T细胞相比，结果显示，双结合域GPRC5D double VHH CAR-T细胞对靶细胞有明显的杀伤效果；单个VHH CAR-T细胞对靶细胞有明显的杀伤效果，与Mock T细胞相比，单域抗体G5VHH1 CAR-T、单域抗体G5VHH2 CAR-T、双表位靶向的OriCAR-017和阳性对照ET150-8均展示出显著的对靶细胞的杀伤效果。特别是在效 靶比1：9时，OriCAR-017对MM.1S靶细胞的杀伤率明显高于单域抗体CAR-T和阳性对照ET150-8，达到70％。
实施例7 GPRC5D CAR-T细胞体外杀伤后的细胞因子分泌
参照实施例6，在96孔板中进行靶细胞杀伤实验，效应细胞和靶细胞的比例为1:1，然后固定每个96孔的效应细胞数2×104个，依次加入靶细胞和效应细胞，每组每个效靶比下设置2个重复孔，另外每组均需设置2个单独效应细胞孔，检测效应细胞的本底因子分泌量。
在靶细胞杀伤后的24小时左右，将96孔板离心，收集各个孔的上层清液；将效应细胞离心去上清，用X-VIVO稀释至3×105细胞/mL，每孔取100μL使用。将无靶细胞(即不与肿瘤细胞共培养的CAR-T细胞)及与MM.1S细胞均按效靶比为1：1的比例混合于圆底96孔板中，共孵育24小时后，吸取上清，用R&D Cytokine Kit检测上清液中IL-2和IFN-γ的含量。
如图6A-6B所示，在MM.1S靶细胞刺激下，双结合域GPRC5D double VHH CAR-T细胞的IL-2和IFN-γ分泌水平均高于Mock T细胞组；各组CAR-T组的IFN-γ分泌均明显高于Mock T细胞组，且相对于两个单域抗体CAR-T细胞组和阳性对照ET150-8组，双表位靶向的OriCAR-017具有更高的IFN-γ分泌水平，说明OriCAR-017的较强的抗原特异性活化效果。此外，各组CAR-T组的IL-2分泌均明显高于Mock T细胞，但是OriCAR-017的IL-2分泌水平与单域抗体CAR-T细胞相比较低，也说明OriCAR-017有一定的活化增殖特性。
实施例8 GPRC5D CAR-T细胞的靶向增殖能力
本实施例CAR-T细胞的靶向增殖能力检测选用未经处理的MM.1S细胞作为激活靶细胞。
一般在CAR-T细胞培养9-12天左右进行，需提前检测各组的CAR阳性率，再用空白效应T细胞将各组的CAR阳性率调成一致(培养基为不含任何添加物的X-VIVO15培养基)。
将CAR-T细胞和MM.1S细胞，以效靶比1:1的比例进行第一轮靶向刺激，在共孵5天后，收集所有细胞，取样计数，再按照1:3(全CAR-T细胞:新MM.1S细胞)比例，进行第二轮靶向刺激，再次共孵育4-5天后，进行第三轮靶向刺激(两种细胞间的比例与第二轮靶向刺激相同)。三轮靶向刺激期间，按照细胞生长情况每1-2天补加适量X-VIVO15培养基。
如图7A所示，双结合域GPRC5D double VHH CAR-T细胞在接受靶细胞刺激后存在不同程度的靶向增殖能力，两轮反复刺激的增殖倍数在50-200倍之间，与对照Mock T细胞相比具有显著性差异，预示了双结合域GPRC5D double VHH CAR-T细胞在生物体内针对特异性肿瘤可有效迅速扩增。如图7B所示，各组CAR-T细胞在接受MM.1S靶细胞刺激后表现出 强大的细胞增殖能力，三轮靶细胞刺激后的增殖倍数可达1000-3500倍，与对照mock T细胞相比增殖活性明显增高，其中G5VHH1 CAR T细胞在靶细胞刺激下的扩增效率最高。此外，OriCAR-017细胞靶向增殖扩增效率高于阳性对照ET150-8 CAR T细胞。此结果表明OriCAR-017 CAR T细胞在靶细胞刺激迅速增殖预示其在生物体内针对特异性肿瘤可有效迅速扩增。
实施例9 GPRC5D CAR-T细胞的细胞表型分析
慢病毒感染激活后的CD3+T细胞在获得GPRC5D CAR表达检测阳性结果后，在共同培养后第12天进行细胞的记忆性表型、激活性表型、耗竭性表型检测：分别收集四组效应CAR T细胞(包括G5VHH1 CAR-T、G5VHH2 CAR-T、OriCAR-017细胞、阳性对照ER150-8)进行流式检测各组CD3+T细胞中记忆性Markers：包括CD45RO、CCR7、CD62L细胞比例；检测各组CD3+T细胞中激活性markers：包括CD25、CD69细胞比例；检测各组CD3+T细胞中耗竭性markers：包括PD-1、LAG-3、TIM-3细胞比例。
如表3和图8，图9所示，CAR-T细胞培养第12天时，与G5VHH2(TSCM：7.79％；TCM：51.1％)和ET150-8 CAR-T细胞(TSCM：3.91％；TCM：43.1％)相比，OriCAR-017(TSCM：22.7％；TCM：25.7％)和G5VHH1 CAR-T细胞(TSCM：22.8％；TCM：34.4％)表现出较高干细胞记忆(TSCM)表型，较低的中央记忆T细胞(TCM)表型，T细胞分化程度低。如图10所示，CAR T细胞培养第12天时，OriCAR-017和G5VHH1 CAR T细胞激活性marker CD25和CD69的表达水平低于G5VHH2和ET150-8 CAR T细胞，即T细胞激活程度较低，OriCAR-017 T细胞具有低自激活细胞表型优势。如图11所示，CAR-T细胞培养第12天时，OriCAR-017 T细胞耗竭性marker LAG-3和TIM-3的表达水平低于G5VHH2和ET150-8 CAR-T细胞，即T细胞耗竭程度较低，OriCAR-017 T细胞具有低耗竭细胞表型优势。
表3记忆性T细胞亚群比例
  TSCM％ TCM％ TEM％ TEF％
G5VHH1 22.8 34.4 27.2 15.6
G5VHH2 7.79 51.1 34.3 6.76
OriCAR-017 22.7 25.7 23.1 28.5
ET150-8 3.91 43.1 47 6.06
实施例10 GPRC5D CAR-T细胞的免疫突触机制研究
免疫突触(Immune synapse,IS)是通过与特定的激活受体连接后形成的结构，是CAR-T细胞识别抗原，增殖和活化的关键步骤，是机体细胞免疫反应和体液免疫反应的重要组成部分； 免疫突触的形成与CAR-T细胞的效应功能相关，在CAR-T水平上，测量免疫突触结构微管组织中心(MTOC)的极化，以进一步评估CAR-T细胞的功能优势。CAR-T细胞与靶细胞按1：1比例共培养5分钟后转移至Nunc chamber slides中，孵育20分钟，4％PFA室温固定，PBS清洗3次后，免疫染色通透液(Triton X-100)，室温孵育；10％马血清/免疫染色通透液(Triton X-100)，室温孵育；加入2％马血清/免疫染色通透液(Triton X-100)稀释的一抗室温孵育；加入2％马血清/免疫染色通透液(Triton X-100)稀释的二抗室温孵育；分离载玻片，ProLong Diamond封片，干燥凝固后激光共聚焦100倍油镜，紫外、488nm、543nm，1024x1024，Z轴扫描0.4μm，叠加得到3D图像和影片。
如图12所示，G5VHH1、G5VHH2与OriCAR-017 CAR T细胞共聚焦成像；其中，细胞核染色为蓝色，γ-微管蛋白染色为绿色，T细胞染色为红色，白色箭头表示IS处微管组织中心MTOC的极化位置，MTOC与IS的距离越近，则IS结构越稳定。如图13所示，与G5VHH1和G5VHH2 CAR-T细胞相比，OriCAR-017细胞对IS的MTOC极化水平明显增加，表现为MTOC与IS的距离减小，从而获得了更优越、更稳定的IS结构，因而具有更优越的功能优势，表明OriCAR-017细胞介导的细胞毒性优于单靶向的GPRC5D VHH1和GPRC5DVHH2CAR-T细胞。
实施例11.GPRC5D CAR-T细胞小鼠体内实验
我们进一步在NSG小鼠体内验证OriCAR-017 CAR-T细胞的消瘤、抑瘤作用。具体而言实验分为5组，Mock T细胞、G5VHH1 CAR-T、G5VHH2 CAR-T、ET150-8 CAR-T和OriCAR-017 CAR-T细胞组，每组5只小鼠。先在NSG小鼠皮下，按1×107细胞/只的剂量接种MM1S细胞，约2周后(肿瘤大小为50-150mm3)，尾静脉注射已培养10-12天的CAR-T(或T)细胞，剂量均为1.6×106CAR-T细胞/只的剂量(以T细胞调整每只小鼠的总接种细胞数一致)。接种CAR-T(或T)细胞后，CAR-T(或T)细胞接种后第7天和第14天检测小鼠外周血中细胞因子分泌，每周测量小鼠瘤体大小和体重3次，观察3周。
NSG小鼠体内抑瘤实验流程如图14所示；如图15A所示，在注射后第7天检测分泌的细胞因子IFN-γ，OriCAR-017 CAR-T细胞组产生的IFN-γ显著高于G5VHH1 CAR-T、G5VHH2 CAR-T和阳性对照ET150-8 CAR-T。关于安全性，如图15B所示，小鼠体重随着时间呈现稳定增长趋势，所有CAR-T细胞实验组与Mock T组的小鼠体重曲线趋势一致，此外，OriCAR-017注射后小鼠毛色光亮，小鼠行为等生命体征正常，证实OriCAR-017在小鼠体内具有良好的安全性。关于体内抑瘤效果，如图15C所示，单结合域的G5VHH1 CAR-T和G5VHH2 CAR-T细胞，以及阳性对照ET150-8 CAR-T细胞相比，OriCAR-017细胞组显示了非常显著的抗 肿瘤效果，抑瘤率可接近100％。如图15D所示，OriCAR-017细胞组在研究时间内小鼠达到100％生存，其小鼠生存率远高于单结合域的VHH CAR T细胞组和Positive control阳性对照ET150-8 CAR-T细胞，预示着OriCAR-017在临床上可能表现出较好的抗肿瘤活性。
为了进一步模拟血液瘤在人体内的分布，我们基于活体成像技术，进一步在B-NSG小鼠体内验证OriCAR-017细胞的消瘤、抑瘤作用。B-NSG小鼠，尾静脉注射MM1S-Luciferase靶细胞，按小鼠生物发光强度，随机分组，每组5只；尾静脉注射提前冻存的不同剂量的OriCAR-017 CAR-T或Mock T细胞；CAR-T回输后，每周二、周四用动物活体成像系统观察小鼠体内生物发光变化情况，观察小鼠的体重及健康状态，期间进行3次小鼠眼球或者尾静脉采血用于细胞因子检测，BD流式检测CD3+CD8+T细胞比例。
如图16所示，与Mock T对比，不同剂量的OriCAR-017冻存细胞均显示了明显的抗肿瘤活性，极大降低小鼠的肿瘤负荷。其中，低剂量组在回输后的第7天就表现出有效的肿瘤清楚效果，而OriCAR-017中剂量和高剂量组在回输后短时间内(第4天)就表现出明显的肿瘤抑制活性；特别是高剂量组，在回输后可快速清除肿瘤，并持续抑制肿瘤生长，在第21天，高剂量组的肿瘤完全清除率可达60％。
实施例12.OriCAR-017 CAR-T在难治性/复发性多发性骨髓瘤患者中的临床研究
进行单臂、开放标签、I期多中心临床研究，以确定OriCAR-017 CAR-T细胞在治疗诊断为难治性或复发性多发性骨髓瘤(“r/r MM”)的人类患者中的安全性和有效性。临床试验登记信息为www.clinicaltrials.gov，标识符为NCT05016778。
该研究中，用来源于患者的自体T细胞的OriCAR-017 CAR-T细胞治疗难治性/复发性多发性骨髓瘤患者。将1×106/kg、3×106/kg、6×106/kg CAR+T细胞剂量通过静脉内注射施用于每名患者。在研究的第1-30天期间，监测患者的不良事件，并获得患者样品以用于评估。在CAR T施用之后随访所有患者至少36个月。研究的主要结果测量了在OriCAR-017 CAR-T细胞注射后1-30天内通过通用不良事件术语标准(Common Terminology Criteria for Adverse Events，CTCAE)v4.0评估的治疗相关不良事件的发生；次要结果评估了CAR-T诱导的抗骨髓瘤响应，例如通过在OriCAR-017 CAR-T细胞的施用之前和之后测定血清中的异常免疫球蛋白水平，和患者骨髓中多发性骨髓瘤细胞的数量。研究的功效目标包括病理性完全响应的比例、3年无疾病存活率、3年无进展存活率。2022年ASCO年会(2022 ASCO Annual Meeting,https://ascopubs.org/doi/abs/10.1200/JCO.2022.40.16_suppl.8004)会中详细介绍该研究。截至2022年4月30日，12例患者纳入研究并接受单采，10例患者已完成OriCAR-017 CAR-T输注，中位年龄为64岁(范围，41-71)，中位既往治疗线数为5.5线(范围，3-17)。5例(50.0％) 患者既往接受过抗BCMA CAR-T治疗。未出现剂量限制性毒性。最常见的治疗期间出现的不良事件(TEAE)为中性粒细胞减少症、血小板减少症、白细胞减少症、淋巴细胞减少症和贫血。未报告神经系统毒性。所有患者均出现细胞因子释放综合征(CRS)，有9例等级为1级，1例等级为2级。常规干预(托珠单抗和类固醇)后，所有CRS均迅速缓解。中位发病时间为2天(范围，1-9)，中位持续时间为6天(范围，3-9)。客观缓解率(ORR)为100％，MRD是100％阴性，完全缓解/严格意义的完全缓解(CR/sCR)率为60％。既往接受过抗BCMA CAR-T治疗的5例患者可进行疗效评估，2例达到sCR，2例达到VGPR，1例达到PR，具体如表4和5所示。
表4患者入组信息
  1x10^6/kg 3x10^6/kg 6x10^6/kg 合计
  (N＝3) (N＝4) (N＝3) (N＝10)
年龄(岁)        
例数 3 4 3 10
均数(标准差) 69.0(3.46) 53.5(10.34) 64.0(3.61) 61.3(9.51)
中位数 71 54 63 64
Q1-Q3 65.0-71.0 45.5-61.5 61.0-68.0 58.0-68.0
最小值-最大值 65-71 41-65 61-68 41-71
年龄分组，n(％)        
<65岁 0 3(75.0) 2(66.7) 5(50.0)
≥65岁 3(100) 1(25.0) 1(33.3) 5(50.0)
性别，n(％)        
男性 1(33.3) 2(50.0) 2(66.7) 5(50.0)
女性 2(66.7) 2(50.0) 1(33.3) 5(50.0)
民族，n(％)        
汉族 3(100) 4(100) 3(100) 10(100)
其他 0 0 0 0
表5 OriCAR-017 CAR-T治疗效果
  1x10^6/kg 3x10^6/kg 6x10^6/kg 合计
  (N＝3) (N＝4) (N＝3) (N＝10)
疗效评价,n(％)        
sCR 3(100) 3(75.0) 0 6(60.0)
CR 0 0 0 0
VGPR 0 0 3(100) 3(30.0)
PR 0 1(25.0) 0 1(10.0)
MR 0 0 0 0
SD 0 0 0 0
PD 0 0 0 0
ORR,n(％) 3(100) 4(100) 3(100) 10(100)
95％CI[1] 29.2,100.0 39.8,100.0 29.2,100.0 69.2,100.0
DCR,n(％) 3(100) 4(100) 3(100) 10(100)
95％CI[1] 29.2,100.0 39.8,100.0 29.2,100.0 69.2,100.0
ORR＝(CR+sCR+VGPR+PR例数)/疗效可评估人群受试者例数×100％。
DCR＝(CR+sCR+VGPR+PR+MR例数)/疗效可评估人群受试者例数×100％。
百分比的计算基于疗效可评估人群中每个剂量组入组的受试者例数。
[1]基于Clopper Pearson方法计算双侧对应CI。
实施例13.GPRC5D CAR-T细胞中两个VHH域的差异表位结合实验
对OriCAR-017 CAR-T中的GPRC5D VHH域的表位进行鉴定。构建具有特异性结合至GPRC5D的不同表位的两个不同抗GPRC5D VHH域的示例性二价GPRC5D CAR。将包含G5VHH1和G5VHH2的二价/双表位CAR命名为OriCAR-017，是示例性表2中列出的一种双结合域的GPRC5D double VHH CAR。
13.1 FORTEBIO抗体测定
将G5VHH1和G5VHH2抗体序列克隆至含有人IgG1Fc片段(hIgG1Fc)序列的p3.4-hIgG1Fc的真核载体中，促进GPRC5D VHH-hIgG1Fc的重组表达获得重组蛋白并纯化，测定 G5VHH1-hIgG1Fc和G5VHH2-hIgG1Fc对GPRC5D的亲和力N端肽(Biotin)的亲和力。简而言之，用生物素(EZ-Link Sulfo-NHS-LC-Biotin,Pierce,21327)标记人GPRC5D的N端肽(>sp|Q9NZD1|1-27：MYKDCIESTGDYFLLCDAEGPWGIILE)蛋白。通过生物膜干涉(BLI)技术，使用分子相互作用分析仪fortebiooctetRED384(PALL)来进行抗原抗体之间的结合动力学分析(抗原、抗体稀释均使用0.1％BSA及0.02％吐温20的PBS缓冲液)。使用浓度为20nM经过生物素偶联的抗原与SA传感器固定，结合3min；然后再与对倍稀释的抗体(即G5VHH1和G5VHH2抗体)溶液结合5min，最后解离5min。得到的结果通过Octet Data Analysis9.0软件(fortebio)进行数据分析，来计算出抗原与抗体的结合强度，得到KD值、Ka(1/Ms)值和Kd(1/s)值。G5VHH1-hIgG1Fc和G5VHH2-hIgG1Fc的结合亲和力结果如图17所示，G5VHH1抗体检测的回归方程对于样本数据点的拟合优度R2值(0.7914)低，模型不可信，可以看出G5VHH1不与GPRC5D的N端肽结合，而G5VHH2抗体与N端肽结合，KD为7.69E-08M；进一步表明G5VHH1、G5VHH2与GPRC5D靶点结合的表位存在差异。
13.2竞争性结合测定
通过基于细胞的竞争性结合测定来进一步验证G5VHH1和G5VHH2的差异表位结合。使用过表达人GPRC5D的稳定CHO细胞系(CHO-GPRC5D)用于测定。简而言之，将G5VHH1和G5VHH2克隆至含有人IgG1Fc片段(hIgG1Fc)序列的p3.4-hIgG1Fc的真核载体及含有6×His标签的表达载体，促进GPRC5D VHH-hIgG1Fc和GPRC5D VHH-His的重组表达纯化获得G5VHH1-hIgG1Fc、G5VHH2-hIgG1Fc、G5VHH1-His、G5VHH2-His重组蛋白。
使用PBS缓冲液配置浓度为1x106细胞/mL的CHO-GPRC5D靶细胞，加入96孔尖底板(corning 3894)，每孔30μL；使用PBS缓冲液配置检测抗体G5VHH1-hIgG1Fc、G5VHH2-hIgG1Fc浓度为30μg/mL，并按3倍比稀释抗体，形成8个浓度梯度(最后浓度为0)；将配置好的不同浓度的抗体按30μL/孔加入已铺好的靶细胞中混匀；4℃冰箱孵育1小时；每孔加150μL缓冲液，300g离心5分钟弃上清；重复洗涤一次；使用缓冲液按1:200配比配置山羊抗人IgG(Fc特异性，ab98593)荧光二抗，每孔30μL加入细胞中混匀，4℃冰箱孵育30分钟；每孔加170μL缓冲液，300g离心5分钟弃上清；重复洗涤一次；每孔加35μL缓冲液混匀后使用iQue Screener流式仪(购自IntelliCyt公司)检测。根据流式亲和力结合实验的分析结果获知G5VHH1-hIgG1Fc、G5VHH2-hIgG1Fc的结合活性EC80值，如图18所示。
根据EC80所对应的抗体浓度0.65ng/mL G5VHH2-hIgG1Fc与3x104细胞总数的CHO-GPRC5D细胞孵育1h，使用PBS缓冲液配置检测抗体G5VHH1-His浓度为80μg/mL，并按3倍比稀释抗体，形成12个浓度梯度(最后浓度为0)；然后将连续稀释的G5VHH1-His 重组抗体加入板孔，并在4℃下再孵育1h。在孵育之后，每孔加140μL缓冲液，300g离心5分钟弃上清；重复洗涤一次；使用缓冲液按1:200配比配置山羊抗人IgG(Fc特异性，ab98593)荧光二抗，每孔30μL加入细胞中混匀，4℃冰箱孵育30分钟；每孔加170μL缓冲液，300g离心5分钟弃上清；重复洗涤一次；每孔加35μL缓冲液混匀后使用iQue Screener流式仪(购自IntelliCyt公司)检测。作为测定对照，根据上述的相同程序，在不存在G5VHH2-hIgG1Fc的情况下用CHO-GPRC5D细胞直接孵育连续稀释的G5VHH1-His用于检测G5VHH1-His与CHO-GPRC5D细胞的结合。
如图19所示，在G5VHH2-hIgG1Fc与细胞饱和结合下，G5VHH1-His仍旧具有抗体梯度依赖性，且与在不存在G5VHH2-hIgG1Fc的情况下的G5VHH1-His结合活性相近，即G5VHH2-hIgG1Fc的存在不影响G5VHH1-His的结合。此外，在G5VHH2-hIgG1Fc与细胞饱和结合下，同时加入G5VHH1-His与靶细胞进行结合，发现G5VHH2-hIgG1Fc与在不存在G5VHH1-His的情况下的G5VHH2-hIgG1Fc结合活性相近，即G5VHH1-His的存在不影响G5VHH1-His的结合。综上所述，G5VHH1与G5VHH2与GPRC5D靶点结合的表位不同，两者互不影响。
实施例14.CAR抗体序列与不同种属靶抗原的交叉结合研究
使用表达人GPRC5D，食蟹猴GPRC5D(SEQ ID NO:36)，小鼠GPRC5D(SEQ ID NO:37)，人GPRC5D对照亚型GPRC5A(SEQ ID NO:38)的稳定转染CHO细胞，通过流式鉴定评估不同抗GPRC5D抗体对人，食蟹猴和鼠GPRC5D的结合。简而言之，使用PBS缓冲液配置浓度为1x106细胞/mL的CHO-HuGPRC5D、CHO-CyGPRC5D、CHO-MuGPRC5D、CHO-HuGPRC5A靶细胞，加入96孔V底板，每孔30μL；使用PBS缓冲液配置检测抗体Double-VHH hFc浓度为30μg/mL，并按3倍比稀释抗体，形成8个浓度梯度(最后浓度为0)；将配置好的不同浓度的抗体按30μL/孔加入已铺好的靶细胞中混匀；4℃冰箱孵育1小时；每孔加150μL缓冲液，300g离心5分钟弃上清；重复洗涤一次；使用缓冲液按1:200配比配置Dyna650-山羊抗人IgG(Fc特异性，ab98593)荧光二抗，每孔30μL加入细胞中混匀，4℃冰箱孵育30分钟；每孔加170μL缓冲液，300g离心5分钟弃上清；重复洗涤一次；每孔加35μL缓冲液混匀后使用iQue Screener流式仪(购自IntelliCyt公司)检测。
流式结合活性检测结果如图20所示，结合特性汇总如表6所示，可知双结合域VHH-hFc抗体显示双结合域VHH-hFc抗体与人GPRC5D存在强结合活性，与小鼠GPRC5D、食蟹猴GPRC5D存在相对较弱的结合活性；未检测到双结合结构域VHH-hFc抗体与空白的CHO细胞的结合，未检测到双结合结构域VHH-hFc抗体与表达人GPRC5D同一家族不同亚型的 GPRC5A的CHO细胞的结合，说明了应用于本产品的双结合域VHH-hFc抗体的高度特异性，可以减少各种脱靶副作用的发生风险。
表6双结合域VHH-hFc抗体与不同种属靶抗原的交叉结合特性汇总
人GPRC5A 人GPRC5D 小鼠GPRC5D 食蟹猴GPRC5D
- ++ + +
注：-不存在结合活性；+存在相对较弱结合活性；++存在较强结合活性。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (91)

  1. 嵌合抗原受体，其包含第一抗原结合域和第二抗原结合域，所述第一抗原结合域靶向G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白，且所述第二抗原结合域靶向GPRC5D蛋白。
  2. 根据权利要求1所述的嵌合抗原受体，其中所述第一抗原结合域和/或所述第二抗原结合域包含抗体或其抗原结合片段。
  3. 根据权利要求2所述的嵌合抗原受体，其中所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
  4. 根据权利要求2-3中任一项所述的嵌合抗原受体，其中所述抗原结合片段包括VHH。
  5. 根据权利要求1-4中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:22和SEQ ID NO:27中任一项所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:21和SEQ ID NO:26中任一项所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:35所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:20和SEQ ID NO:25中任一项所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含 SEQ ID NO:33所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含HCDR1，HCDR2和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下述的任意一组氨基酸序列：
    (1)所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
    (2)所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
    (3)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；以及
    (4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
  14. 根据权利要求10-13中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:11所示的氨基酸序列。
  15. 根据权利要求10-14中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:12、SEQ ID NO:28和SEQ ID NO:23中任一项所示的氨基酸序列。
  16. 根据权利要求8-15中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:13或SEQ ID NO:29所示的氨基酸序列。
  17. 根据权利要求6-16中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:14所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的嵌合抗原受体，其中所述第一抗原结合域包括VH，且所述VH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的嵌合抗原受体，其中所述第一抗原结合域为VHH，且所述VHH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR3，且所述HCDR3包含SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:22和SEQ ID NO:27中任一项所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR2，且所述HCDR2包含SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:21和SEQ ID NO:26中任一项所示的氨基酸序列。
  25. 根据权利要求1-24中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:35所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR1，且所述HCDR1包含SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:20和SEQ ID NO:25中任一项所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:34(IX1X2X3X4GX5T，其中，X1为N或T，X2为S或W，X3为G或S，X4为D或G，X5为N、S或T)所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:33所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含HCDR1，HCDR2和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下述的任一组氨基酸序列：
    (1)所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID  NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
    (2)所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
    (3)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；以及
    (4)所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
  29. 根据权利要求25-28中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:11所示的氨基酸序列。
  30. 根据权利要求25-29中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:12、SEQ ID NO:28和SEQ ID NO:23中任一项所示的氨基酸序列。
  31. 根据权利要求23-30中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:13或SEQ ID NO:29所示的氨基酸序列。
  32. 根据权利要求21-31中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:14所示的氨基酸序列。
  33. 根据权利要求1-32中任一项所述的嵌合抗原受体，其中所述第二抗原结合域包括VH，且所述VH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的嵌合抗原受体，其中所述第二抗原结合域为VHH，且所述VHH包含SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:30中任一项所示的氨基酸序列。
  35. 根据权利要求1-34中任一项所述的嵌合抗原受体，其中所述第一抗原结合域和所述第二抗原结合域分别包含HCDR1，HCDR2和HCDR3，所述第一抗原结合域和所述第二抗原结合域的HCDR1，HCDR2和HCDR3的序列选自下述任一组氨基酸序列：
    (1)第一抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
    (2)第一抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
    (3)第一抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；
    (4)第一抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；
    (5)第一抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
    (6)第一抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
    (7)第一抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述 HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；
    (8)第一抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；
    (9)第一抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
    (10)第一抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
    (11)第一抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；
    (12)第一抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；
    (13)第一抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID  NO:27所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:8所示的氨基酸序列，所述HCDR2包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:10所示的氨基酸序列；
    (14)第一抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:18所示的氨基酸序列；
    (15)第一抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；以及
    (16)第一抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列；第二抗原结合域：所述HCDR1包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:27所示的氨基酸序列。
  36. 根据权利要求1-35中任一项所述的嵌合抗原受体，其中所述第一抗原结合域和所述第二抗原结合域靶向GPRC5D的相同表位。
  37. 根据权利要求1-36中任一项所述的嵌合抗原受体，其中所述第一抗原结合域和所述第二抗原结合域包含相同或不同的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的嵌合抗原受体，其中所述第一抗原结合域和所述第二抗原结合域直接或间接相连。
  39. 根据权利要求1-38中任一项所述的嵌合抗原受体，其中所述第一抗原结合域和所述第二抗原结合域通过连接子相连。
  40. 根据权利要求39所述的嵌合抗原受体，其中所述连接子包含(GGGGS)n的氨基酸序列，其中所述n为1-10中的任意正整数。
  41. 根据权利要求39所述的嵌合抗原受体，其中所述连接子包含(EAAAK)n的氨基酸序列，其中所述n为1-10中的任意正整数。
  42. 根据权利要求1-40中任一项所述的嵌合抗原受体，其包含共刺激信号区域，其中所述共 刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  43. 根据权利要求41所述的嵌合抗原受体，其中所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
  44. 根据权利要求42-43中任一项所述的嵌合抗原受体，其中所述共刺激信号区域包含SEQ ID NO:4所示的氨基酸序列。
  45. 根据权利要求1-44中任一项所述的嵌合抗原受体，其包括胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
  46. 根据权利要求45所述的嵌合抗原受体，其中所述胞内信号传导域为源自CD3ζ的信号传导结构域。
  47. 根据权利要求45-46中任一项所述的嵌合抗原受体，其中所述胞内信号传导域包含SEQ ID NO:5所示的氨基酸序列。
  48. 根据权利要求1-47中任一项所述的嵌合抗原受体，其包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  49. 根据权利要求48所述的嵌合抗原受体，其中所述跨膜区为源自CD8的跨膜区。
  50. 根据权利要求48-49中任一项所述的嵌合抗原受体，其中所述跨膜区包含SEQ ID NO:3所示的氨基酸序列。
  51. 根据权利要求1-50中任一项所述的嵌合抗原受体，其还包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  52. 根据权利要求51所述的嵌合抗原受体，其中所述铰链区为源自CD8的铰链区。
  53. 根据权利要求51-52中任一项所述的嵌合抗原受体，其中所述铰链区包含SEQ ID NO:2所示的氨基酸序列。
  54. 根据权利要求1-53中任一项所述的嵌合抗原受体，其还包含低密度脂蛋白受体相关蛋白或其片段。
  55. 根据权利要求54所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段位于所述胞内信号区域的C端。
  56. 根据权利要求54-55中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  57. 根据权利要求54-56中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
  58. 根据权利要求54-57中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:7所示的氨基酸序列。
  59. 根据权利要求1-58中任一项所述的嵌合抗原受体，其还包含信号肽。
  60. 根据权利要求59所述的嵌合抗原受体，其中所述信号肽源自CD8蛋白的信号肽。
  61. 根据权利要求59-60中任一项所述的嵌合抗原受体，其中所述信号肽包含SEQ ID NO:1所示的氨基酸序列。
  62. 一种或多种分离的核酸分子，其编码权利要求1-61中任一项所述的嵌合抗原受体。
  63. 根据权利要求62所述的核酸分子，其还包含启动子。
  64. 根据权利要求63所述的核酸分子，其中所述启动子为组成型启动子。
  65. 根据权利要求63-64中任一项所述的核酸分子，其中所述启动子为EF1α启动子。
  66. 载体，其包含权利要求62-65中任一项所述的核酸分子。
  67. 根据权利要求66所述的载体，其为病毒载体。
  68. 根据权利要求66-67中任一项所述的载体，其为慢病毒载体。
  69. 细胞，其包含权利要求1-61中任一项所述的嵌合抗原受体，权利要求62-65中任一项所述的核酸分子，和/或权利要求66-68中任一项所述的载体。
  70. 根据权利要求69所述的细胞，其为免疫效应细胞。
  71. 根据权利要求69-70中任一项所述的细胞，其包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
  72. 根据权利要求69-71中任一项所述的细胞，其为T细胞。
  73. 制备要求权利要求1-61中任一项所述的嵌合抗原受体的方法，所述方法包括在使得权利要求1-61中任一项所述的嵌合抗原受体表达的条件下，培养根据权利要求69-72中任一项所述的细胞。
  74. 制备经修饰的免疫效应细胞的方法，其包括向免疫效应细胞中引入权利要求66-68中任一项所述的载体。
  75. 药物组合物，其包含权利要求1-61中任一项所述的嵌合抗原受体，权利要求62-65中任一项所述的核酸分子，权利要求66-68中任一项所述的载体，和/或权利要求69-72中任一项所述的细胞，以及可选地药学上可接受的载体。
  76. 权利要求1-61中任一项所述的嵌合抗原受体，权利要求62-65中任一项所述的核酸分子，权利要求66-68中任一项所述的载体，权利要求69-72中任一项所述的细胞，和/或权利要求75所述的药物组合物，在制备药物中的用途，所述药物用于预防、治疗和/或缓解疾病和/或病症。
  77. 根据权利要求76所述的用途，其中所述疾病和/或病症包括与GPRC5D异常表达相关的疾病和/或病症。
  78. 根据权利要求77所述的用途，其中所述与GPRC5D异常表达相关的疾病和/或病症包括肿瘤。
  79. 根据权利要求78所述的用途，其中所述肿瘤包括实体瘤。
  80. 根据权利要求78所述的用途，其中所述肿瘤包括非实体瘤。
  81. 根据权利要求79-80中任一项所述的用途，其中所述肿瘤包括血液瘤和/或淋巴瘤。
  82. 根据权利要求78-81中任一项所述的用途，其中所述肿瘤包括骨髓瘤。
  83. 根据权利要求82所述的用途，其中所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
  84. 一种预防、治疗和/或缓解疾病和/或病症的方法，所述方法包括向有需要的受试者施用权利要求69-72中任一项所述的细胞，和/或权利要求75所述的药物组合物。
  85. 根据权利要求84所述的方法，其中所述疾病和/或病症包括与GPRC5D异常表达相关的疾病和/或病症。
  86. 根据权利要求85所述的方法，其中所述与GPRC5D异常表达相关的疾病或病症包括肿瘤。
  87. 根据权利要求86所述的方法，其中所述肿瘤包括实体瘤。
  88. 根据权利要求86所述的方法，其中所述肿瘤包括非实体瘤。
  89. 根据权利要求87-88中任一项所述的方法，其中所述肿瘤包括血液瘤和/或淋巴瘤。
  90. 根据权利要求87-89中任一项所述的方法，其中所述肿瘤包括骨髓瘤。
  91. 根据权利要求90所述的方法，其中所述骨髓瘤包括难治性/复发性多发性骨髓瘤。
PCT/CN2023/071869 2022-01-14 2023-01-12 靶向gprc5d的嵌合抗原受体及其用途 WO2023134718A1 (zh)

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