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WO2022228429A1 - 靶向bcma的嵌合抗原受体及其应用 - Google Patents

靶向bcma的嵌合抗原受体及其应用 Download PDF

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Publication number
WO2022228429A1
WO2022228429A1 PCT/CN2022/089287 CN2022089287W WO2022228429A1 WO 2022228429 A1 WO2022228429 A1 WO 2022228429A1 CN 2022089287 W CN2022089287 W CN 2022089287W WO 2022228429 A1 WO2022228429 A1 WO 2022228429A1
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seq
car
amino acid
acid sequence
sequence shown
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PCT/CN2022/089287
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English (en)
French (fr)
Inventor
陈思晔
徐艳红
郭昊
杨月
李慧姣
许志锋
孙亚男
杨焕凤
何晓文
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原启生物科技(上海)有限责任公司
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Priority to US18/557,494 priority Critical patent/US20240383992A1/en
Priority to CN202280025146.4A priority patent/CN117136199A/zh
Priority to JP2023565910A priority patent/JP2024517719A/ja
Priority to EP22794899.9A priority patent/EP4331678A1/en
Publication of WO2022228429A1 publication Critical patent/WO2022228429A1/zh

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Definitions

  • the present application relates to the field of biomedicine, in particular to a chimeric antigen receptor targeting BCMA and its application.
  • BCMA B cell maturation antigen
  • B cell maturation antigen the B cell maturation antigen, mainly regulates the proliferation and survival of B cells, and the maturation and differentiation of B cells into plasma cells (PCs).
  • PCs plasma cells
  • BCMA molecules are gradually induced to express. Because BCMA molecules are only expressed on the membrane surface of PCs and plasmablasts, and are not expressed on most B cells, hematopoietic stem cells, and other normal tissues, BCMA molecules become one of the most ideal target molecules for the treatment of MM.
  • Chimeric antigen receptor T (CAR-T) cell therapy is a precision targeted therapy emerging in recent years, and it is one of the hottest research directions in the field of tumor immunotherapy.
  • the basic principle of this therapy is to transform T lymphocytes through genetic engineering technology to become CAR-T cells with specific recognition ability, which are expanded and cultured in vitro and then returned to the patient to attack and kill specific tumor cells.
  • CAR-T cell therapy can be applied to BCMA-targeted tumor immunotherapy.
  • the present application provides a chimeric antigen receptor (CAR) targeting BCMA.
  • CAR chimeric antigen receptor
  • the CAR of the present application can be expressed stably and efficiently on the surface of immune effector cells (eg, T cells), and the immune effector cells including the CAR have the following properties One or more of: (1) killing BCMA-positive target cells with high toxicity, (2) massive secretion of cytokines upon stimulation of target cells, (3) higher targeted proliferation ability, and (4) Inhibit tumor growth and eliminate tumors.
  • the application provides a chimeric antigen receptor (CAR) comprising a BCMA binding domain comprising an antibody or antigen-binding fragment thereof that specifically binds BCMA, wherein the antibody comprises a light Strand Complementarity Determining Region 3 (HCDR3), and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25.
  • CAR chimeric antigen receptor
  • HCDR3 light Strand Complementarity Determining Region 3
  • the antibody that specifically binds BCMA comprises a light chain complementarity determining region 2 (HCDR2), and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:41.
  • HCDR2 light chain complementarity determining region 2
  • the HCDR2 of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.
  • the antibody that specifically binds BCMA comprises a light chain complementarity determining region 1 (HCDR1), and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:21.
  • HCDR1 light chain complementarity determining region 1
  • the antibody that specifically binds BCMA comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:21 and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:41 amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25.
  • the antibody that specifically binds BCMA comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 22-24 The amino acid sequence shown in any one, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25.
  • the antibody that specifically binds BCMA comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 are selected from any of the following group of amino acid sequences: (1) HCDR1: SEQ ID NO: 21, HCDR2: SEQ ID NO: 22 and HCDR3: SEQ ID NO: 25; (2) HCDR1: SEQ ID NO: 21, HCDR2: SEQ ID NO: 23 and HCDR3: SEQ ID NO: 25; and (3) HCDR1: SEQ ID NO: 21, HCDR2: SEQ ID NO: 24 and HCDR3: SEQ ID NO: 25.
  • the antibody that specifically binds BCMA comprises a light chain variable region VH comprising a framework region H-FR1, the C-terminus of H-FR1 being directly or N-terminal to the HCDR1 Indirectly linked, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:42.
  • the H-FR1 of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26-29.
  • the VH of the antibody that specifically binds BCMA comprises a framework region H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO : amino acid sequence shown in 43.
  • the H-FR2 that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 30-32.
  • the VH of the antibody that specifically binds BCMA comprises a framework region H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO : amino acid sequence shown in 44.
  • the H-FR3 of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 33-36.
  • the VH of the antibody that specifically binds BCMA comprises the framework region H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID The amino acid sequence shown in NO:37.
  • the antibody that specifically binds BCMA comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:42,
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:43
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:44
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:37 sequence.
  • the H-FR1 of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26-29
  • the H-FR2 comprises SEQ ID NOs: 30-32
  • the amino acid sequence shown in any one the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:33-36
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:37 .
  • the H-FR1, H-FR2, H-FR3, and H-FR4 of the antibody that specifically binds BCMA comprise an amino acid sequence selected from any of the following groups: (1) H-FR1: SEQ ID NO: 26, H-FR2: SEQ ID NO: 30, H-FR3: SEQ ID NO: 33 and H-FR4: SEQ ID NO: 37; (2) H-FR1: SEQ ID NO: 27, H-FR2 : SEQ ID NO: 31, H-FR3: SEQ ID NO: 34 and H-FR4: SEQ ID NO: 37; (3) H-FR1: SEQ ID NO: 28, H-FR2: SEQ ID NO: 32, H-FR3: SEQ ID NO: 35 and H-FR4: SEQ ID NO: 37; and (4) H-FR1: SEQ ID NO: 29, H-FR2: SEQ ID NO: 30, H-FR3: SEQ ID NO:36 and H-FR4: SEQ ID NO:37.
  • the antibody that specifically binds BCMA comprises a light chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO:46.
  • the VH of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5-9.
  • the antibody that specifically binds BCMA comprises a light chain complementarity determining region 3 (LCDR3), and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:40.
  • LCDR3 light chain complementarity determining region 3
  • the LCDR3 of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-16.
  • the antibody that specifically binds BCMA comprises a light chain complementarity determining region 2 (LCDR2), and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:39.
  • LCDR2 light chain complementarity determining region 2
  • the LCDR2 of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13-14.
  • the antibody that specifically binds BCMA comprises a light chain complementarity determining region 1 (LCDR1), and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:38.
  • LCDR1 light chain complementarity determining region 1
  • the LCDR1 of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.
  • the antibody that specifically binds BCMA comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:38 and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:39 amino acid sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:40.
  • the antibody that specifically binds BCMA comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10-12, and the LCDR2 comprises SEQ ID The amino acid sequence shown in any one of NO: 13-14, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 15-16.
  • the antibody that specifically binds BCMA comprises LCDR1, LCDR2 and LCDR3, and said LCDR1, LCDR2 and LCDR3 are selected from any of the following group of amino acid sequences: (1) LCDR1: SEQ ID NO: 10, LCDR2: SEQ ID NO: 13 and LCDR3: SEQ ID NO: 15; (2) LCDR1: SEQ ID NO: 11, LCDR2: SEQ ID NO: 13 and LCDR3: SEQ ID NO: 16; (3) LCDR1: SEQ ID NO: 12, LCDR2: SEQ ID NO: 13 and LCDR3: SEQ ID NO: 16; and (4) LCDR1: SEQ ID NO: 12, LCDR2: SEQ ID NO: 14 and LCDR3: SEQ ID NO: 16.
  • the antibody that specifically binds BCMA comprises a light chain variable region VL comprising a framework region L-FR1, the C-terminus of L-FR1 being directly or N-terminal to the LCDR1 Indirectly linked, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:17.
  • the VL of the antibody that specifically binds BCMA comprises a framework region L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO : amino acid sequence shown in 18.
  • the VL of the antibody that specifically binds BCMA comprises a framework region L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO : the amino acid sequence shown in 19.
  • the VL of the antibody that specifically binds BCMA comprises a framework region L-FR4, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID The amino acid sequence shown in NO:20.
  • the antibody that specifically binds BCMA comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17,
  • the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 18
  • the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 19
  • the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 20 sequence.
  • the antibody that specifically binds BCMA comprises a light chain variable region VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO:45.
  • the VL of the antibody that specifically binds BCMA comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.
  • the antibody that specifically binds BCMA comprises VH and VL, and the VH comprises the amino acid sequence set forth in SEQ ID NO:46 and the VL comprises the amino acid sequence set forth in SEQ ID NO:45 .
  • the antibody that specifically binds BCMA comprises VH and VL, and the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5-9, the VL comprises SEQ ID NO: The amino acid sequence shown in any one of 1-4.
  • the antibody that specifically binds BCMA comprises VH and VL, and the VH and VL are selected from any of the following group of amino acid sequences: (1) VH: SEQ ID NO: 5 and VL: SEQ ID NO: 1; (2) VH: SEQ ID NO: 6 and VL: SEQ ID NO: 2; (3) VH: SEQ ID NO: 7 and VL: SEQ ID NO: 2; (4) VH: SEQ ID NO: : 8 and VL: SEQ ID NO: 2; (5) VH: SEQ ID NO: 9 and VL: SEQ ID NO: 3; (6) VH: SEQ ID NO: 8 and VL: SEQ ID NO: 3; ( 7) VH: SEQ ID NO: 9 and VL: SEQ ID NO: 4; and (8) VH: SEQ ID NO: 10 and VL: SEQ ID NO: 4.
  • the BCMA binding domain comprises a scFv
  • the scFv comprises the heavy chain variable region VH and the light chain variable region VL of the antibody that specifically binds BCMA.
  • the C-terminus of the heavy chain variable region VH and the N-terminus of the light chain variable region VL are directly or indirectly linked.
  • the C-terminus of the heavy chain variable region VL and the N-terminus of the light chain variable region VL are directly or indirectly linked.
  • the scFv comprises a linker peptide between VH and VL, and the linker peptide comprises the amino acid sequence set forth in SEQ ID NO:58.
  • the scFv comprises the amino acid sequence set forth in any one of SEQ ID NOs: 49-56.
  • the CAR comprises a transmembrane domain
  • the transmembrane domain comprises a transmembrane domain selected from the group consisting of CD28, CD3e, CD45, CD4, CD5, CD8, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain is derived from CD8, and the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:61.
  • the CAR comprises a costimulatory domain
  • the costimulatory domain comprises a costimulatory domain selected from the group consisting of CD137, CD28, 4-1BB, OX-40, and ICOS .
  • the costimulatory domain is derived from CD137, and the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:62.
  • the CAR comprises an intracellular signaling domain
  • the intracellular signaling domain comprises an intracellular signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:63.
  • the CAR comprises a hinge region, and the hinge region comprises a CD8-derived hinge region.
  • the hinge region is derived from CD8, and the hinge region comprises the amino acid sequence set forth in SEQ ID NO:60.
  • the CAR comprises a signal peptide.
  • the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:59.
  • the CAR comprises L6-Li located C-terminal to the intracellular signaling domain.
  • the L6-Li comprises the amino acid sequence shown in SEQ ID NO:107.
  • the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64-71 and 74. In certain embodiments, the CAR comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 95-102 and 105.
  • the application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the CAR described herein.
  • the isolated nucleic acid molecule further comprises a promoter located 5' to the nucleotide sequence encoding the CAR.
  • the promoter is a constitutive promoter.
  • the promoter is the EF1 ⁇ promoter.
  • the application provides a nucleic acid molecule encoding a CAR, the nucleic acid molecule encoding a CAR comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 95-102 and 105.
  • the application provides vectors comprising the nucleic acid molecules described herein.
  • the vector is selected from the group consisting of plasmids, retroviral vectors and lentiviral vectors.
  • the present application provides immune effector cells comprising the CAR, the nucleic acid molecule, and/or the vector described herein.
  • the immune effector cells are selected from T lymphocytes.
  • the present application provides a method for preparing immune effector cells, which comprises introducing the vector of the present application into the immune effector cells.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the CAR, the nucleic acid molecule, the carrier, and/or the immune effector cell, and a pharmaceutically acceptable carrier.
  • the present application provides the use of the CAR, the nucleic acid molecule, the carrier, the immune effector cell, and/or the pharmaceutical composition in the preparation of a medicament, wherein the medicament is used for the treatment of patients with A disease or disorder associated with BCMA expression.
  • the disease or disorder associated with BCMA expression is cancer or malignancy. In certain embodiments, the disease or disorder associated with BCMA expression is a BCMA-positive tumor. In certain embodiments, the disease or disorder associated with BCMA expression is a solid tumor and/or a non-solid tumor. In certain embodiments, the disease or disorder associated with BCMA expression is myeloma. In certain embodiments, the disease or disorder associated with BCMA expression is multiple myeloma.
  • the application provides a method of treating a disease or disorder associated with BCMA expression comprising administering to a subject in need the CAR, the nucleic acid molecule, the vector, the immune effector cell, and /or the pharmaceutical composition.
  • the disease or disorder associated with BCMA expression is cancer or malignancy. In certain embodiments, the disease or disorder associated with BCMA expression is a BCMA-positive tumor. In certain embodiments, the disease or disorder associated with BCMA expression is a solid tumor and/or a non-solid tumor. In certain embodiments, the disease or disorder associated with BCMA expression is myeloma. In certain embodiments, the disease or disorder associated with BCMA expression is multiple myeloma.
  • the application provides the CAR, the nucleic acid molecule, the vector, the immune effector cell, and/or the pharmaceutical composition for use in the treatment of a disease or disorder associated with BCMA expression.
  • the disease or disorder associated with BCMA expression is cancer or malignancy. In certain embodiments, the disease or disorder associated with BCMA expression is a BCMA-positive tumor. In certain embodiments, the disease or disorder associated with BCMA expression is a solid tumor and/or a non-solid tumor. In certain embodiments, the disease or disorder associated with BCMA expression is myeloma. In certain embodiments, the disease or disorder associated with BCMA expression is multiple myeloma.
  • Figure 1 shows an embodiment of the composition of each element of the CAR in the CAR lentiviral vector and its connection sequence.
  • Figure 2 shows the expansion fold of each group of BCMA CAR-T cells containing the CAR described in this application during the culture process.
  • Figure 3 shows the proportion of CAR-positive cells in each group of BCMA CAR-T cells containing the CAR described in this application.
  • Figure 4 shows the killing toxicity of each group of BCMA CAR-T cells containing the CAR described in this application to target cells, wherein A: MM1s cells, B: K562 cells.
  • Figure 5 shows the secretion of IFN- ⁇ factors after co-incubation of BCMA CAR-T cells containing the CAR described in this application with MM1s cells.
  • Figure 6 shows the targeted expansion ability of each group of BCMA CAR-T cells containing the CAR described in this application for MM1s cells.
  • Figure 7 shows the killing toxicity of BCMA CAR-T cells under the L6-Li CAR structure to MM1s cells.
  • Figure 8 shows the secretion of IFN- ⁇ factors after co-incubation of BCMA CAR-T cells with MM1s cells under the L6-Li CAR structure.
  • Figure 9 shows the ability of BCMA CAR-T cells under the L6-Li CAR structure to inhibit and eliminate tumors in tumor-bearing mice, where A is the experimental group and B is the control group.
  • Figure 10 shows the metabolism of BCMA CAR-T cells under the L6-Li CAR structure in tumor-bearing mice, where A is the experimental group and B is the control group.
  • Figure 11 shows the secretion of IFN- ⁇ cytokines caused by BCMA CAR-T cells under the L6-Li CAR structure in tumor-bearing mice, where A is the experimental group and B is the control group.
  • Figure 12 shows the secretion of IL-2 cytokines caused by BCMA CAR-T cells under the L6-Li CAR structure in tumor-bearing mice.
  • BCMA protein is a member of the tumor necrosis factor receptor family.
  • BCMA may include proteins comprising mutations, eg, may include proteins comprising point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type BCMA.
  • the BCMA can include those from any vertebrate, eg, primates (eg, humans or monkeys), rodents (mice or rats), avian and/or livestock, and the like.
  • BCMA may also include a portion of the complete BCMA protein, so long as the relevant biological activity is retained.
  • the BCMA can be human BCMA whose GenBank accession number is BAB60895.1.
  • human BCMA is typically a 184 amino acid long protein encoded by a 994 nucleotide long primary mRNA transcript (NM_001192.2).
  • binding domain generally refers to (specifically) a given target epitope or a given target site that binds (specifically) to a target molecule (antigen), or associates with said given target epitope or given target site.
  • the term "BCMA binding domain” generally refers to a domain that can specifically bind to a BCMA protein.
  • the BCMA binding domain may comprise a chimeric antigen receptor or fragment thereof, an anti-BCMA antibody or antigen binding fragment thereof capable of specifically binding to a BCMA polypeptide expressed on a B cell.
  • binding domain “binding domain”, “extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain” and “extracellular antigen-specific binding domain” are used interchangeably in this application use, and may comprise, domains or fragments of a CAR that have the ability to specifically bind to a target antigen of interest (eg, BCMA).
  • the BCMA binding domain can be of natural, synthetic, semi-synthetic or recombinant origin.
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide including an antigen binding site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutant and transplanted antibodies.
  • an antibody also includes antibody fragments, such as antigen-binding fragments Fab, F(ab')2, Fv, scFv, Fd, dAb and retained Other antibody fragments with antigen binding function (eg, specific binding to BCMA).
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • scFv generally refers to a single chain antibody, which is an antibody composed of a heavy chain variable region and a light chain variable region directly linked or linked by a linking molecule (eg, a linking peptide).
  • the structure of the scFv is from the N-terminus to the C-terminus, which can be heavy chain variable region-light chain variable region, light chain variable region-heavy chain variable region, heavy chain variable region-connecting peptide-light chain variable region. variable region, or light chain variable region-linking peptide-heavy chain variable region.
  • CAR chimeric antigen receptor
  • CAR can be an antigen receptor formed by fusion of the antigen-binding region of an antibody that recognizes tumor-associated antigen (TAA) and the intracellular signaling domain "immunoreceptor tyrosine activation motif (ITAM)".
  • TAA tumor-associated antigen
  • ITAM intracellular signaling domain
  • the CAR can be combined with an immune effector cell receptor activating intracellular domain based on the antigen (eg, BCMA) specificity of an antibody.
  • Immune effector cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing target antigens.
  • the term "signaling domain” generally refers to a domain located inside a cell capable of transducing a signal.
  • the intracellular signaling domain can transmit a signal into a cell.
  • a signaling domain is any contiguous amino acid sequence used to direct a protein to a target.
  • the intracellular signaling domain may be selected from the group consisting of CD3 ⁇ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain, and OX40 intracellular domain.
  • costimulation generally refers to the source of lymphocyte activation of a second signal, usually by costimulatory molecules on the surface of immune cells (T-cell/B-cell or antigen-presenting/T-cell) surfaces involved in adaptive immunity and its receptors interact.
  • T-cell/B-cell or antigen-presenting/T-cell immune cells
  • full activation of T cells depends on dual signaling and cytokines.
  • the first signal of T cell activation comes from the specific binding of its receptor TCR to the antigen, that is, T cells recognize the antigen;
  • the second signal of T cell activation comes from costimulatory molecules, that is, the costimulatory molecules on APC and the surface of T cells. interactions with the corresponding receptors.
  • costimulatory domain generally refers to any amino acid sequence capable of interacting with a costimulatory molecule to produce a costimulation.
  • the term "hinge region” generally refers to a region in the structure of a CAR located between an antibody or antigen-binding fragment thereof (eg, scFv) that specifically binds BCMA and the transmembrane domain.
  • the hinge region is usually derived from the IgG family, such as IgGl and IgG4, but also some from IgD and CD8.
  • the hinge region has a certain degree of flexibility, which affects the spatial confinement between the CAR molecule and its specific target, which in turn affects the contact between CAR T cells and tumor cells.
  • transmembrane domain generally refers to a sequence in a cell surface protein that spans the cell membrane, which may contain a hydrophobic alpha helix.
  • the transmembrane domain can be linked with the intracellular signal transduction domain and play a role in transmitting signals.
  • the transmembrane domain generally comprises three distinct domains: an N-terminal extracellular domain, an intermediate conserved transmembrane stretch domain, and a C-terminal cytoplasmic domain.
  • the transmembrane domain may also contain an intracellular or cytoplasmic domain.
  • the transmembrane domain may be derived from any type I, type II or type III transmembrane protein.
  • signal peptide generally refers to a leader sequence at the amino terminus (N-terminus) of a nascent CAR protein, which, upon translation or post-translation, guides the nascent protein to the endoplasmic reticulum and subsequent surface expression.
  • the signal peptide is usually cleaved during this process.
  • the signal peptide can be heterologous or homologous to the organism used to produce the polypeptide.
  • L6-Li generally refers to the fusion protein of low-density lipoprotein receptor-related protein and leptin.
  • the L6-Li may comprise the amino acid sequence shown in SEQ ID NO: 107.
  • the nucleic acid molecule encoding the L6-Li may comprise the nucleotide sequence shown in SEQ ID NO: 106.
  • promoter generally refers to a deoxyribonucleic acid (DNA) sequence that enables transcription of a particular gene.
  • the promoter can be recognized by RNA polymerase and begin to transcribe synthetic RNA.
  • RNA ribonucleic acid
  • promoters can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription).
  • the promoter includes a core promoter region and a regulatory region, which is located in the regulatory sequence that controls gene expression, upstream of the gene transcription initiation site (5' direction of the DNA antisense strand), and has no compiling function itself. According to their mode of action and function, they are divided into three categories: constitutive promoters (continuous activity in most or all tissues), specific promoters (tissue specific or developmental stage specific) and inducible promoters (reduced by External chemical or physical signal regulation).
  • the term "specific binding” generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which may determine the target in the presence of a heterogeneous population of molecules, including biomolecules The presence.
  • an antibody that specifically binds a target is an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets.
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include, but does not require, exclusive binding.
  • nucleic acid molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs thereof. In certain embodiments, the nucleic acid molecules described herein can be isolated from their natural environment.
  • the nucleic acid molecules described herein can be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) ) recombinantly produced by cloning, (iii) purified, eg, by enzymatic cleavage and fractionation by gel electrophoresis, or (iv) synthesized, eg, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • nucleic acids encoding the antibodies or antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including, but not limited to, manipulation using restriction fragments or using synthetic oligonucleotides.
  • methods known in the art including, but not limited to, manipulation using restriction fragments or using synthetic oligonucleotides.
  • overlap extension PCR see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.
  • polypeptide or “protein” are used interchangeably and generally refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acid residues is an analog or mimetic of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers, homologues.
  • the term may also include modified amino acid polymers, eg, by addition of sugar residues to form glycoproteins or modified by phosphorylation.
  • Polypeptides and proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of the native protein, or deletions, additions, or deletions of one or more amino acids of the native sequence and/or substituted molecules.
  • polypeptide and “protein” specifically include deleted, added and/or substituted sequences of one or more amino acids of the antigen binding proteins described herein.
  • homologue generally refers to an amino acid sequence or nucleotide sequence that has some homology to a wild-type amino acid sequence and a wild-type nucleotide sequence.
  • homoology may be equivalent to sequence "identity”.
  • homologous sequences can include amino acid sequences that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will contain the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
  • a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • a vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes of P1 origin (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses.
  • Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Viruses (eg SV40).
  • a vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain an origin of replication site.
  • the carrier may also contain components to facilitate its entry into the cell, such as viral particles, liposomes or protein coats, but not only these substances.
  • immune effector cells generally refers to cells involved in an immune response, eg, promoting an immune effector response.
  • immune effector cells include T cells, eg, alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient, a human patient.
  • a pharmaceutical composition described herein which may comprise an immune effector cell, CAR, nucleic acid molecule or vector described herein, and optionally a pharmaceutically acceptable carrier.
  • a pharmaceutical composition contains an active ingredient form that is effectively biologically active and does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is to be administered.
  • pharmaceutically acceptable carrier generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • disease or disorder associated with BCMA expression includes, but is not limited to, diseases associated with BCMA expression, or disorders associated with BCMA-expressing cells, including tumor cells of various cancers.
  • Individuals with a disease or disorder associated with BCMA expression have abnormal BCMA expression (eg, BCMA overexpression), or cells that have abnormal BCMA expression (eg, BCMA overexpression) compared to a normal body or normal cells.
  • a tumor generally refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • a tumor can be a solid tumor or a non-solid tumor (eg, a hematological tumor).
  • BCMA-positive tumor generally refers to a tumor that overexpresses BCMA protein compared to normal cells.
  • BCMA-positive tumors can include non-solid tumors.
  • BCMA-positive tumors can include myeloma.
  • BCMA-positive tumors can include multiple myeloma.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides a chimeric antigen receptor (CAR) comprising a BCMA-binding domain comprising an antibody or antigen-binding fragment thereof that specifically binds BCMA.
  • CAR chimeric antigen receptor
  • the antibody or antigen-binding fragment thereof that specifically binds BCMA in the CAR may comprise a heavy chain complementarity determining region 3 (HCDR3), and the HCDR3 may comprise an amino acid sequence as shown in SEQ ID NO: 25 : DIRYVMDY (SEQ ID NO: 25).
  • HCDR3 heavy chain complementarity determining region 3
  • DIRYVMDY amino acid sequence as shown in SEQ ID NO: 25 : DIRYVMDY (SEQ ID NO: 25).
  • the sequence can be divided according to Kabat's rule.
  • the antibody or antigen-binding fragment thereof may comprise a heavy chain complementarity determining region 2 (HCDR2)
  • SEQ ID NO: 41 WINTETREPX 10 YAYDFRX 17 (SEQ ID NO: 41) NO:41
  • X 10 A or T
  • the sequence can be divided according to Kabat's rule.
  • the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 22-24.
  • the antibody or antigen-binding fragment thereof may comprise a heavy chain complementarity determining region 1 (HCDR1), and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:21: DYTIN (SEQ ID NO:21 ).
  • the sequence can be divided according to Kabat's rule.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise SEQ ID NO: 21, SEQ ID NO: 41 and SEQ ID NO: 25, respectively, in order amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 21, and the HCDR2 may comprise SEQ ID NO: 22-24 The amino acid sequence shown in any one of the above, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 25, respectively, in order amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 25, respectively, in sequence amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise SEQ ID NO: 21, SEQ ID NO: 24 and SEQ ID NO: 25, respectively, in order amino acid sequence shown.
  • SEQ ID NO: 40 LQSRX 5 FPRX 9
  • the sequence can be divided according to Kabat's rule.
  • the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 15-16.
  • the antibody or antigen-binding fragment thereof may comprise a light chain complementarity determining region 2 (LCDR2)
  • the sequence can be divided according to Kabat's rule.
  • the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 13-14.
  • the antibody or antigen-binding fragment thereof may comprise a light chain complementarity determining region 1 (LCDR1)
  • the sequence can be divided according to Kabat's rule.
  • the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 10-12.
  • the antibody or antigen-binding fragment thereof may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 may comprise SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, respectively, in sequence amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 10-12, and the LCDR2 may comprise SEQ ID NO: 10-12 The amino acid sequence shown in any one of ID NOs: 13-14, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 15-16.
  • the antibody or antigen-binding fragment thereof may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 may comprise SEQ ID NO: 10, SEQ ID NO: 13 and SEQ ID NO: 15, respectively, in sequence amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 may comprise SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 16, respectively, in sequence amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 may comprise SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 16, respectively, in sequence amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 may comprise SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, respectively, in sequence amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may respectively comprise SEQ ID NO: 21 in sequence , SEQ ID NO: 41, SEQ ID NO: 25, SEQ ID NO: 38, SEQ ID NO: 39 and the amino acid sequences shown in SEQ ID NO: 40.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 21, and the HCDR2 may comprise The amino acid sequence shown in any one of SEQ ID NOs: 22-24, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 25, and the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 10-12
  • the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 13-14, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 15-16.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may respectively comprise SEQ ID NO: 21 in sequence , SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 10, SEQ ID NO: 13 and the amino acid sequences shown in SEQ ID NO: 15.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may respectively comprise SEQ ID NO: 21 in sequence , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 11, SEQ ID NO: 13 and the amino acid sequences shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may respectively comprise SEQ ID NO: 21 in sequence , SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 11, SEQ ID NO: 13 and the amino acid sequences shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may respectively comprise SEQ ID NO: 21 in sequence , SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 12, SEQ ID NO: 13 and the amino acid sequences shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may respectively comprise SEQ ID NO: 21 in sequence , SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 12, SEQ ID NO: 13 and the amino acid sequences shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may respectively comprise SEQ ID NO: 21 in sequence , SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 12, SEQ ID NO: 14 and the amino acid sequences shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may respectively comprise SEQ ID NO: 21 in sequence , SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 12, SEQ ID NO: 14 and the amino acid sequences shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof may comprise a framework region H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ
  • the sequence can be divided according to Kabat's rule.
  • the H-FR1 of the antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in any one of SEQ ID NOs: 26-29.
  • the sequence can be divided according to Kabat's rule.
  • the H-FR2 of the antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in any one of SEQ ID NOs: 30-32.
  • the antibody or antigen-binding fragment thereof may comprise a framework region H-FR3 located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO: 44
  • the sequence can be divided according to Kabat's rule.
  • the H-FR3 of the antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in any one of SEQ ID NOs: 33-36.
  • the antibody or antigen-binding fragment thereof may comprise a framework region H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise SEQ ID NO: Amino acid sequence shown in 37: WGQGTLVTVSS (SEQ ID NO: 37).
  • the sequence can be divided according to Kabat's rule.
  • the antibody or antigen-binding fragment thereof may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 may be The amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 37 are respectively included in sequence.
  • the antibody or antigen-binding fragment thereof may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise any one of SEQ ID NOs: 26-29
  • the amino acid sequence shown the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 30-32
  • the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 33-36
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the antibody or antigen-binding fragment thereof may comprise H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 comprise Amino acid sequences selected from any of the following groups: (1) H-FR1: SEQ ID NO: 26, H-FR2: SEQ ID NO: 30, H-FR3: SEQ ID NO: 33 and H-FR4: SEQ ID NO: 33 37; (2) H-FR1: SEQ ID NO: 27, H-FR2: SEQ ID NO: 31, H-FR3: SEQ ID NO: 34 and H-FR4: SEQ ID NO: 37; (3) H- FR1: SEQ ID NO: 28, H-FR2: SEQ ID NO: 32, H-FR3: SEQ ID NO: 35 and H-FR4: SEQ ID NO: 37; and (4) H-FR1: SEQ ID NO: 29, H-FR2: SEQ ID NO:30, H-FR3: SEQ ID NO:36 and H-FR4
  • the VH of the antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in any one of SEQ ID NOs: 5-9.
  • the antibody or antigen-binding fragment thereof may comprise a framework region L-FR1, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 may comprise SEQ Amino acid sequence shown in ID NO: 17: EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 17).
  • the sequence can be divided according to Kabat's rule.
  • the antibody or antigen-binding fragment thereof may comprise a framework region L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise SEQ ID NO: 18 Amino acid sequence shown: WYQQKPGQAPRLLIY (SEQ ID NO: 18).
  • the sequence can be divided according to Kabat's rule.
  • the antibody or antigen-binding fragment thereof may comprise a framework region L-FR3 located between the LCDR2 and the LCDR3, and the L-FR3 may comprise SEQ ID NO: 19 Amino acid sequence shown: GIPARFSGSGSGTDFTLTISSLEPEDAAIYYC (SEQ ID NO: 19).
  • the sequence can be divided according to Kabat's rule.
  • the antibody or antigen-binding fragment thereof may comprise a framework region L-FR4, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 may comprise SEQ ID NO: Amino acid sequence shown in 20: FGQGTKLEIK (SEQ ID NO: 20).
  • the sequence can be divided according to Kabat's rule.
  • the antibody or antigen-binding fragment thereof may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1, L-FR2, L-FR3 and L-FR4 may be The amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 are respectively included in sequence.
  • the sequence can be divided according to Kabat's rule.
  • the VL of the antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-4.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:46, and the VL may comprise the amino acid shown in SEQ ID NO:45 sequence.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 5-9, and the VL may comprise SEQ ID NO : the amino acid sequence shown in any one of 1-4.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:5, and the VL may comprise the amino acid shown in SEQ ID NO:1 sequence.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:6, and the VL may comprise the amino acid shown in SEQ ID NO:2 sequence.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:7, and the VL may comprise the amino acid shown in SEQ ID NO:2 sequence.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:8, and the VL may comprise the amino acid shown in SEQ ID NO:2 sequence.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:9, and the VL may comprise the amino acid shown in SEQ ID NO:3 sequence.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:8, and the VL may comprise the amino acid shown in SEQ ID NO:3 sequence.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:9, and the VL may comprise the amino acid shown in SEQ ID NO:4 sequence.
  • the antibody or antigen-binding fragment thereof may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:8, and the VL may comprise the amino acid shown in SEQ ID NO:4 sequence.
  • the antibody or antigen-binding fragment thereof may be an scFv, and the scFv may comprise the VH and the VL.
  • the C-terminus of the VH and the N-terminus of the VL may be directly linked, eg, in-frame.
  • the C-terminus of the VH and the N-terminus of the VL may be linked indirectly, eg, via a linker (eg, a linker peptide).
  • the C-terminus of the VL and the N-terminus of the VH may be directly linked, eg, in-frame.
  • the C-terminus of the VL and the N-terminus of the VH may be linked indirectly, eg, via a linker (eg, a linker peptide).
  • the BCMA domain of the CAR may comprise VH, VL and a connecting peptide
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:5
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:1
  • the amino acid sequence, the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO:58.
  • the BCMA domain of the CAR may comprise VH, VL and a connecting peptide
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 6
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 2
  • the amino acid sequence of the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the BCMA domain of the CAR may comprise VH, VL and a connecting peptide
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:7
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:2
  • the amino acid sequence of the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the BCMA domain of the CAR may comprise VH, VL and a connecting peptide
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 8
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 2
  • the amino acid sequence of the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the BCMA domain of the CAR may comprise VH, VL and a connecting peptide
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:9
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:3
  • the amino acid sequence of the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the BCMA domain of the CAR may comprise VH, VL and a connecting peptide
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 8
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 3
  • the amino acid sequence of the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the BCMA domain of the CAR may comprise VH, VL and a connecting peptide
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:9
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:4
  • the amino acid sequence of the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the BCMA domain of the CAR may comprise VH, VL and a connecting peptide
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 8
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 4
  • the amino acid sequence of the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the BCMA domain of the CAR may comprise VH, VL and a linker peptide
  • the C-terminus of the VL may be linked to the N-terminus of the VH through a linker peptide
  • the linker peptide may comprise SEQ ID NO : amino acid sequence shown in 58.
  • the BCMA domain of the CAR may comprise a scFv, and the scFv may comprise the amino acid sequence shown in any one of 39-49.
  • the scFv may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the CAR includes an extracellular BCMA domain and may also include an intracellular domain.
  • the CAR can comprise an intracellular costimulatory signaling domain, which can provide a stimulatory signal.
  • the costimulatory signaling domains may include, but are not limited to, the group consisting of costimulatory signaling domains in CD137, CD28, 4-1BB, OX-40, and ICOS, and combinations thereof.
  • the costimulatory domain can be a costimulatory domain from CD137.
  • the costimulatory domain may comprise the amino acid sequence set forth in SEQ ID NO:62
  • the CAR can comprise an intracellular signaling domain, which can comprise a domain with at least one ITAM motif.
  • the intracellular signaling domain can transmit activation signals to the interior of the cell.
  • Exemplary signaling domains can be derived from signaling domains selected from the group consisting of, but not limited to, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30 activation region, Epstein - Barr virus (EBV) LMP2A, Simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10 and DAP-12, and variants of the above.
  • the intracellular signaling domain can be a signaling domain from CD3 ⁇ .
  • the intracellular signaling domain may comprise the amino acid sequence set forth in SEQ ID NO:63.
  • the CAR can comprise a transmembrane domain, which is a sequence in a cell surface protein that spans the cell membrane, which can comprise a hydrophobic alpha helix.
  • the transmembrane domain can be derived from CD28 with good stability.
  • the transmembrane domain can be derived from any type I transmembrane protein.
  • the transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix.
  • the transmembrane domain may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain may be the transmembrane domain from CD8.
  • the transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO:61.
  • the CAR can comprise a hinge region, which can be located between the extracellular targeting moiety and the transmembrane domain.
  • the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgGl, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8alpha, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region can be derived from CD8.
  • the hinge region may comprise the amino acid sequence set forth in SEQ ID NO:60.
  • the CAR may further comprise a signal peptide, eg, human CD8 signal peptide, at the N-terminus of the BCMA binding domain.
  • a signal peptide eg, human CD8 signal peptide
  • the signal peptide may comprise the amino acid sequence set forth in SEQ ID NO:59.
  • the CAR may sequentially comprise a BCMA targeting moiety (eg, the antigen binding protein, and for example, the scFv), the hinge region, the transmembrane domain, The costimulatory signaling region and the signaling domain.
  • a BCMA targeting moiety eg, the antigen binding protein, and for example, the scFv
  • the CAR may comprise the scFv, the CD8 hinge region, the CD8 transmembrane domain, the CD137 costimulatory signaling region, and the CD3 ⁇ signaling domain, in order from the N-terminus to the C-terminus.
  • the CAR can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 64-71.
  • the C-terminus of the CAR can be linked to leptin and/or functional fragments thereof, and/or low-density lipoprotein receptor-related protein or fragments thereof.
  • the leptin can include secreted leptin.
  • the CAR, the leptin and/or functional fragments thereof, and/or the low-density lipoprotein receptor-related protein or fragments thereof can be processed by self-cleaving peptides (eg, T2A, P2A, 2A peptides such as E2A) are linked.
  • self-cleaving peptides eg, T2A, P2A, 2A peptides such as E2A
  • L6-Li can be attached at the C-terminus of the CAR.
  • the L6-Li can comprise the amino acid sequence shown in SEQ ID NO:107.
  • the nucleic acid molecule encoding the L6-Li can comprise the nucleotide sequence shown in SEQ ID NO:106.
  • the CAR may sequentially comprise a BCMA targeting moiety (eg, the antigen binding protein, and for example, the scFv), the hinge region, the transmembrane domain, The costimulatory signaling region, the signaling domain, and the L6-Li.
  • a BCMA targeting moiety eg, the antigen binding protein, and for example, the scFv
  • the CAR may comprise, in order, the scFv, the CD8 hinge region, the CD8 transmembrane domain, the CD137 costimulatory signaling region, the CD3 ⁇ signaling domain, and all described L6-Li.
  • the CAR can comprise the amino acid sequence set forth in SEQ ID NO:74.
  • Nucleic acid molecule Nucleic acid molecule, vector, cell, preparation method and pharmaceutical composition
  • the application also provides isolated one or more nucleic acid molecules that can encode the chimeric antigen receptors (CARs) described herein.
  • the isolated nucleic acid molecule(s) described herein may be nucleotides, deoxyribonucleotides or ribonucleotides in isolated form of any length, or analogs isolated from their natural environment or synthetically synthesized , but can encode the chimeric antigen receptor (CAR) described in this application.
  • the nucleic acid molecule can comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 95-102 and 105.
  • the present application also provides vectors, which may comprise the nucleic acid molecules described herein.
  • the vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell.
  • vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes of P1 origin (PAC); bacteriophages such as lambda phage or M13 phage and Animal viruses, etc.
  • Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Viruses (eg SV40).
  • the vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • the carrier may also include components to assist its entry into cells, such as viral particles, liposomes or protein coats, but not only these materials.
  • the present application also provides immune effector cells, which can comprise the CAR described in the present application, the nucleic acid molecule described in the present application or the vector described in the present application.
  • the cells may include progeny of a single cell. Progeny may not necessarily be identical (in morphology or in genome) to the original parent cell due to natural, accidental or intentional mutation.
  • the cells may also include cells transfected in vitro with the vectors of the present invention.
  • the cells can be mammalian cells.
  • the immune effector cells include T lymphocytes, eg, ⁇ / ⁇ T lymphocytes and ⁇ / ⁇ T lymphocytes; in certain embodiments, the immune effector cells may include natural killer ( NK) cells, natural killer T (NKT) cells, mast cells and bone marrow-derived phagocytes.
  • NK natural killer
  • NKT natural killer T
  • the application provides a modified immune effector cell comprising a chimeric antigen receptor and/or its coding elements, or a T cell receptor and/or its coding elements, and the modified immune cell Also comprising: leptin and/or a functional fragment thereof; and/or, a leptin receptor and/or a functional fragment thereof, wherein the modified immune cells are The expression level of the leptin receptor and/or its functional fragment is increased.
  • the immune effector cells may comprise leptin and/or functional fragments thereof, and/or, leptin receptors and/or functional fragments thereof, and low-density lipoprotein receptor-related protein or fragments thereof .
  • modification of immune effector cells reference can be made to WO2021057932A1, and the descriptions in WO2021057932A1 can also be applied to the present application.
  • the present application also provides a method for preparing the immune effector cells described in the present application, the method may include introducing the isolated nucleic acid molecule described in the present application or the vector described in the present application into the immune effector cells.
  • the present application also provides compositions, which may comprise the immune effector cells described herein.
  • the composition may also include, optionally, a pharmaceutically acceptable carrier.
  • the acceptable ingredients of the composition are not toxic to the recipient at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.
  • the pharmaceutically acceptable adjuvant can include any and all solvents, dispersion media, isotonic and absorption delaying agents compatible with the immune effector cells, generally safe, nontoxic, and is neither biologically nor otherwise undesirable.
  • the composition may comprise parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present application also provides the chimeric antigen receptor described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the immune effector cells described in the present application and/or the application described in the present application.
  • the disease or disorder associated with the expression of BCMA is cancer or malignancy, eg, the disease or disorder associated with the expression of BCMA may include BCMA-positive tumors.
  • the cancer or malignancy can comprise a solid tumor or a non-solid tumor.
  • the cancer or malignancy can be a non-solid tumor.
  • the cancer or malignancy may comprise myeloma, eg, the myeloma may be multiple myeloma.
  • the present application also provides a method of preventing, alleviating or treating tumors, the method may include administering the CAR, nucleic acid molecule, vector, immune effector cell and/or drug described in the present application to a subject in need combination.
  • the administration can be carried out by different means, eg intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the subject may include humans and non-human animals.
  • the subject can include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys can include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the obtained positive antibody sequences were subjected to affinity detection and sequencing identification.
  • the screened candidate antibodies were After 1-2 rounds of affinity evolution, and again affinity detection and flow binding activity identification, a total of 8 phage antibody clones that can specifically bind to human BCMA were obtained.
  • the light chain variable region (VL) nucleic acid sequence and The heavy chain variable region (VH) nucleic acid sequence, the VL and VH nucleic acid sequences are linked to form a single-chain antibody (scFv) form, and named as hBCMA-01, hBCMA-02, hBCMA-15, hBCMA-17, hBCMA-19, hBCMA-20, hBCMA-22, hBCMA-23; the scFv sequence of the positive control is from the patent US 2017/ huBCMA-10 in 0226216A1.
  • the nucleic acid sequences of all the above 9 (including huBCMA-10) scFvs were delivered to Nanjing GenScript Biotechnology Co., Ltd.
  • CAR lentiviral empty vector self-constructed by Yuanqi Biotechnology (Shanghai) Co., Ltd., hereinafter referred to as CAR lentiviral empty vector
  • CAR lentiviral vector including L6 -Li element
  • WO2021057932A1 the amino acid sequence of L6-Li is shown in SEQ ID NO: 107) (self-constructed by Yuanqi Biotechnology (Shanghai) Co., Ltd., hereinafter referred to as CAR-L6-Li lentiviral vector ) were double digested, the former produced a 7749bp linearized fragment and the latter a 9605bp linearized fragment.
  • Example 1 After gel tapping and recovery, use the generated 9605bp linearized fragment and the nucleic acid sequence fragments of the scFv of hBCMA-22 and huBCMA-10 in Example 1 (containing homology arms at both ends), and mix them in a molar ratio of 1:3 (volumes are not equal to each other). more than 10 ⁇ l), after homologous recombination, transformed E. coli DH5 ⁇ competent cells. Single clone colonies were picked, shaken, plasmids were extracted and sent for sequencing identification.
  • CAR lentiviral empty vector and h(u)BCMA-XX core plasmid (hereinafter referred to as h(u)BCMA-XX plasmid) are shown in Figure 1 for the elements and connection sequences of CAR and CAR-L6-Li.
  • the amino acid sequence and nucleic acid sequence of each partial element are shown in Table 2.
  • the CAR part of the CAR lentiviral empty vector sequentially comprises a signal peptide-CD8 hinge region-CD8 transmembrane region-CD137 costimulatory domain-CD3 ⁇ intracellular signaling domain.
  • the CAR vector is based on the CAR lentiviral empty vector, and a scFv targeting BCMA is inserted between the signal peptide and the CD8 hinge region.
  • the CAR-L6-Li vector is based on the CAR vector, and is connected to the 3' end of CD3 ⁇ . Li sequence. The 5' end of each vector was linked to the EF1 ⁇ promoter. The sequence of the CAR portion in each plasmid vector is shown in Table 3.
  • Nucleotide sequence (SEQ ID NO) Amino acid sequence (SEQ ID NO) hBCMA-01 CAR 95 64 hBCMA-02 CAR 96 65 hBCMA-15 CAR 97 66 hBCMA-17 CAR 98 67 hBCMA-19 CAR 99 68 hBCMA-20 CAR 100 69 hBCMA-22 CAR 101 70 hBCMA-23 CAR 102 71 huBCMA-10 CAR 103 72 huBCMA-10-L6-Li CAR 104 73 huBCMA-22-L6-Li CAR 105 74
  • the lentiviral vector system used to construct the present invention belongs to the third generation, and the system consists of three plasmids, namely, the packaging plasmid psPAX2 (Addgene, plasmid number #12260) encoding Gag-Pol protein and Rev protein, the encoding plasmid The PMD2.G plasmid (Addgene, plasmid number #12259) of membrane protein VSV-G, and the core plasmid, namely each CAR lentiviral plasmid containing the scFv sequence in the above Example 1-2 (i.e.
  • the expression of the CAR gene in each CAR lentiviral plasmid is regulated by the elongation factor-1 ⁇ (EF-1 ⁇ ) promoter.
  • the packaging process of lentivirus is as follows:
  • the detection of lentivirus titers is performed as follows:
  • 293T cells in good growth state (generally cultured cells within 20 passages), discard the upper waste liquid, wash the cells with PBS, digest them with 0.25% trypsin (GIBICO) at 37°C for about 3 minutes, wait for After the cells were completely digested, a certain volume of 293T cell complete medium was added to terminate the reaction, sampled and counted, then the cell density was adjusted to 2.0 ⁇ 10 5 /mL, and polybrene with a final concentration of 10ug/mL was added, and then 2.5mL per well was added. Cells were seeded in six-well plates.
  • GEBICO trypsin
  • the cell culture plate was placed in a 37°C, 5% CO2 incubator for static culture.
  • Lentivirus titer number of plated cells ⁇ (positive rate of test tube - positive rate of control tube)/volume of inoculated virus liquid (mL).
  • the titers of the above-mentioned CAR viruses containing scFv ranged from 5-15 ⁇ 10 6 IU/mL
  • PBMC peripheral blood mononuclear cells
  • CD3 + T cells with a purity > 90% were obtained after PBMC were sorted by CD3 positive magnetic beads.
  • T sorting please refer to the product manual (MACS, DS130-050-101).
  • T cell complete medium X-VIVO 15 (Lonza) medium with 5% FBS and 300 IU/mL interleukin 2 (Xingil)
  • the amount of cells was added to the washed CD3/CD28Dynabeads (Gibco, 40203D), then supplemented with T cell complete medium, adjusted the cell density to 1.0-1.2 ⁇ 10 6 /mL, and placed the cells at 37 °C, 5% CO 2 culture Activation of the culture in the box (recorded as D0).
  • Lentiviral transduction is generally performed 20-24 hours after T cell activation.
  • T cells activated for 20-24 hours, centrifuge at 500g for 5 minutes, resuspend the cells with a certain volume of T cell complete medium, and then sample and count them.
  • the virus liquid whose titer has been determined in Example 3 is then added with polybrene to a final concentration of 5 ⁇ g/ml, and then T cell complete medium is added to adjust the cell density to 0.6-1.0 ⁇ 10 6 /mL, and then the cells are placed in Culture in a 37°C, 5% CO 2 incubator, and take another portion of T cells without lentivirus infection as a negative control group.
  • T cells About 20-24 hours after virus transduction, collect T cells from each group, centrifuge at 500g for 5 minutes, resuspend the cells with a certain volume of T cell complete medium, sample and count, add T cell complete medium, and adjust the cells
  • the density is 0.5-0.7 x 106 /mL.
  • Dynabeads are removed when culturing D7, and the total culturing period is about 12 days.
  • the total expansion fold of BCMA CAR-T cells cultured for 12 days was between 100-1600-fold, and the CAR-positive rate after infection was between 65% and 90%. , can be used for cytological function experiments.
  • MM1s cells are myeloma cells that express BCMA
  • K562 cells are tumor cells that do not express BCMA.
  • Luciferase enzyme was indirectly detected by the substrate (principle See Promega, E2510 for details) to determine the targeted killing ability of CAR-T cells.
  • MM1s-Luci and K562-Luci methods for overexpressing Luciferase are as follows:
  • Luciferase-GFP (abbreviated as Luci-GFP) lentivirus was packaged according to the method of Example 3, and the titer of the Luciferase-GFP lentivirus was detected through the GFP fluorescence channel with reference to the method of Example 4.
  • the in vitro killing activity of CAR-T cells should be detected.
  • the positive rate of CAR in each group should be determined in advance, and 0.5-1.0 ⁇ 10 6 cells should be taken.
  • the detection method please refer to (6)- (13).
  • Cells infected with CAR lentivirus are effector cells
  • T cells not infected with lentivirus are blank effector cells
  • MM1s-Luci-GFP cells and K562-Luci-GFP cells are target cells.
  • the killing activity test is carried out 5-6 hours after killing. That is, first mix the cell suspension in the 96-well plate, then evenly remove 1/2 volume of the cell suspension into the 96-well full white plate, then add an equal volume of luciferase substrate, and protect from light at room temperature for 10-15min Then, read the corresponding fluorescence value on the microplate reader. For specific detection methods, see Luciferase detection system (Promega, E2510).
  • cytotoxicity of CAR-T cells to target cells can be calculated by the following formula:
  • BCMA CAR-T cells in each group had stronger killing activity against BCMA-expressing MM1s cells, and showed an obvious effector-target ratio dependence.
  • K562 cells that do not express BCMA there is no killing effect, which proves that the tumor cell killing effect of CAR-T cells is specific.
  • the present application The hBCMA-17, hBCMA-19, hBCMA-20, hBCMA-22 and hBCMA-23 were stronger than the positive control huBCMA-10, and the cytotoxicity to target cells was greater than 20% under the effect-to-target ratio of 1:1.
  • the effect-to-target ratio is 3:1, the killing toxicity to target cells is greater than 70%.
  • Example 6 the target cell killing experiment was carried out in a 96-well plate, the ratio of effector cells and target cells was 1:3, and then the number of effector cells in each 96-well was fixed at 2 ⁇ 10 4 , and target cells and effector cells were added in sequence. Three replicate wells were set for each effector-target ratio in each group, and three separate effector cell wells were required for each group to detect the background factor secretion of effector cells.
  • the 96-well plate was centrifuged to collect the supernatant of each well (if not detected immediately, the supernatant should be stored in a -80°C refrigerator).
  • IFN- ⁇ cytokine secretion was detected by Elisa method (R&D, DY285) as follows:
  • each reagent including IFN- ⁇ standard, capture antibody, detection antibody, Streptavidin-HRP, etc.
  • Other reagents including buffer, blocking solution, diluent, substrate solution,
  • stop solution please refer to R&D, DY285 for details.
  • untreated MM1s cells were selected as target cells for the target proliferation detection of CAR-T cells in this example.
  • the CAR-T cell culture is carried out for about 9-12 days. It is necessary to detect the CAR positive rate of each group in advance, and then use blank effector T cells to adjust the CAR positive rate of each group to the same (the medium is a medium without any additives). X-VIVO 15 medium).
  • CAR-T cells and MM1s cells were subjected to the first round of targeted stimulation at the ratio of effector to target ratio of 1:1. After co-incubating for 4-5 days, all cells were collected, sampled and counted, and then 0.5:1 (total CAR- T cells: new MM1s cells), a second round of targeted stimulation was performed, and after another 4-5 days of co-incubation, a third round of targeted stimulation was performed (the ratio between the two cells was the same as the second round of targeted stimulation) . During the three rounds of targeted stimulation, an appropriate amount of X-VIVO 15 medium was supplemented every 1-2 days according to the cell growth.
  • the nucleic acid sequences of the scFvs of hBCMA-22 and hu-BCMA-10 were constructed into CAR lentiviral vectors containing L6-Li to obtain hBCMA-22-L6-Li and hu-BCMA-10- Lentiviral vector of L6-Li.
  • the in vitro killing activity of CAR-T cells and the amount of IFN- ⁇ secretion after killing were detected after CAR-T cells were expanded in vitro and during the period from D8 to D12. For details, see Implementation Example 3-7.
  • the specific killing abilities of hBCMA-22-L6-Li and hu-BCMA-10-L6-Li CAR-T cells to MM1s cells were basically the same under the three effector-target ratios.
  • the effector-target ratio is 3:1, the killing toxicity of CAR-T cells to target cells is about 90%; when the effector-target ratio is 1:1, the killing toxicity of CAR-T cells is about 40%.
  • the IFN- ⁇ secretion capacity of these two groups of CAR-T cells after co-culture with MM1s cells also basically tends to be consistent.
  • the secretion of IFN- ⁇ could be stimulated to be about 5000 pg/mL; under the effect-target ratio of 1:6, CAR-T cells and target cells could stimulate the secretion of about 5000 pg/mL. After co-culture of target cells for 48 hours, the amount of IFN- ⁇ secretion that can be stimulated is about 17500 pg/mL.
  • Example 10 BCMA CAR-T cells under the structure of hBCMA-22-L6-Li CAR can inhibit tumor and eliminate tumors in tumor-bearing mice and their metabolism in mice
  • mice 6-week-old NSG mice were taken, and each was inoculated with 1 ⁇ 10 7 MM1s cells subcutaneously. After 14 days, the subcutaneous tumor volume of the mice was about 150-250 mm 3 .
  • the mice were divided into 2 groups (8 mice in each group), respectively. Inoculated with freshly prepared hBCMA-22-L6-Li group CAR-T cells (2 ⁇ 10 6 CAR + /mouse) and negative control T cells (the number of cells inoculated per mouse in T cell group was the same as that of hBCMA-22-L6-Li The total number of cells corresponding to 2 ⁇ 10 6 CAR + in the group was the same), and was defined as day 0 after CAR-T injection.
  • mice After inoculation of CAR-T cells, the tumor size was measured with vernier calipers twice a week, the body weight of the mice was weighed, and the coat color, excrement, diet and water intake, body movement and death of the mice were observed. On the 7th, 14th and 21st days after CAR-T injection, tail vein blood was collected from 5 mice in each group to detect cytokine secretion (Th1/2/17CBA to detect IFN- ⁇ and IL-2 cytokines). ) and the proportion of human T cells (hCD3 and hCD8), mice were sacrificed when the tumor volume was greater than 3000 mm 3 , and all the remaining mice were sacrificed on day 90 to end the experiment.
  • cytokine secretion Th1/2/17CBA to detect IFN- ⁇ and IL-2 cytokines
  • a and B of Figure 10 show that CAR-T cells in the hBCMA-22-L6-Li group (taking hCD3 + cells as an example, the change trend of the proportion of hCD8 + cells is basically the same as that of hCD3 + , data not shown) in the small tumor-bearing small Proliferation and metabolism in mice.
  • the starting point and peak point of rapid expansion of CAR-T cells in different mice are not the same (hCD3 peak point may appear between 14 days and 21 days), on average, on the 14th day of CAR-T injection , the average content of hCD3 + T cells was about 40%, and then gradually decreased. On the 28th day, the average content of hCD3 + T cells decreased to below 5%. T cell proliferation in the Mock T group was not detected at the 4 detection time points.
  • IFN- ⁇ and a small amount of IL-2 cytokines (IL-4, IL-6, IL-10, TNF- ⁇ , IL17A cells were not detected) were detected in the peripheral blood of mice.
  • factor secretion, data not shown) the starting point and peak point of IFN- ⁇ cytokine secretion in different mice are also very different, and combined with A of Figure 10, it can be seen that IFN- ⁇ cytokines The secretion peak was earlier than the CAR-T cell proliferation peak, and its content decreased to below 3 pg/ml on the 21st day. No IFN- ⁇ secretion was detected in the Mock T group at the four detection time points.
  • the secretion level of IL-2 cytokine was significantly lower than that of IFN- ⁇ (Fig. 12), peaked on day 7 of CAR-T injection (mean at 50 pg/ml), and then declined rapidly.

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Abstract

一种靶向BCMA的嵌合抗原受体(CAR)及其应用，所述CAR包含BCMA结合结构域，所述BCMA结合结构域包含特异性结合BCMA的抗体或其抗原结合片段，其中所述抗体包含轻链互补决定区3(HCDR3)，且所述HCDR3包含如SEQ ID NO：25所示的氨基酸序列。包含所述CAR的免疫效应细胞和药物组合物，以及治疗与BCMA表达相关的疾病或病症的方法。

Description

靶向BCMA的嵌合抗原受体及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向BCMA的嵌合抗原受体及其应用。
背景技术
BCMA(B cell maturation antigen)，即B细胞成熟抗原，主要调控B细胞的增殖、存活，以及B细胞向浆细胞(PCs)的成熟分化，在PCs分化过程中，BCMA分子逐渐被诱导表达。由于BCMA分子仅表达于PCs和浆母细胞膜表面，不表达于大多数B细胞、造血干细胞，以及其它正常组织上，使得BCMA分子成为治疗MM的最理想的靶标分子之一。
嵌合抗原受体T(CAR-T)细胞疗法是近年来兴起的一种精准靶向疗法，是目前肿瘤免疫治疗领域最热的研究方向之一。该疗法的基本原理是通过基因工程技术改造T淋巴细胞，成为具有特异性识别能力的CAR-T细胞，经体外扩增培养后回输患者体内攻击并杀死特定肿瘤细胞。CAR-T细胞疗法可以应用于BCMA为靶点的肿瘤免疫疗法。
但是目前还没有一款靶向BCMA的CAR-T产品诞生。鉴于BCMA在B细胞恶性肿瘤，尤其是多发性骨髓瘤中作为治疗靶点的有效，本领域亟待开发新的细胞疗法，以通过作用于BCMA而实现治疗目的。
发明内容
本申请提供了一种靶向BCMA的嵌合抗原受体(CAR)，本申请的CAR能够在免疫效应细胞(例如，T细胞)表面稳定高效地表达，包括该CAR的免疫效应细胞具有下列性质中的一种或多种：(1)杀伤BCMA阳性的靶细胞，杀伤毒性大，(2)在靶细胞刺激下大量分泌细胞因子，(3)较高的靶向增殖能力，和(4)抑制肿瘤生长、消除肿瘤。
一方面，本申请提供了一种嵌合抗原受体(CAR)，其包含BCMA结合结构域，所述BCMA结合结构域包含特异性结合BCMA的抗体或其抗原结合片段，其中所述抗体包含轻链互补决定区3(HCDR3)，且所述HCDR3包含如SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链互补决定区2(HCDR2)，且所述HCDR2包含如SEQ ID NO:41所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的所述HCDR2包含如SEQ ID NO:22-24中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链互补决定区1(HCDR1)，且所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含SEQ ID NO:41所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22-24中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3选自以下任一组氨基酸序列：(1)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:22和HCDR3：SEQ ID NO:25；(2)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:23和HCDR3：SEQ ID NO:25；和(3)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:24和HCDR3：SEQ ID NO:25。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链可变区VH，所述VH包括框架区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:42所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的所述H-FR1包含SEQ ID NO:26-29中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的VH包括框架区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:43所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的H-FR2包含SEQ ID NO:30-32中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的VH包括框架区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:44所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的H-FR3包含SEQ ID NO:33-36中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的VH包括框架区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:37所示的氨基 酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:42所示的氨基酸序列，所述H-FR2包含SEQ ID NO:43所示的氨基酸序列，所述H-FR3包含SEQ ID NO:44所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:37所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的H-FR1包含SEQ ID NO:26-29中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:30-32中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:33-36中任一项所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:37所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的H-FR1、H-FR2、H-FR3和H-FR4包含选自以下任一组氨基酸序列：(1)H-FR1：SEQ ID NO:26，H-FR2：SEQ ID NO:30，H-FR3：SEQ ID NO:33和H-FR4：SEQ ID NO:37；(2)H-FR1：SEQ ID NO:27，H-FR2：SEQ ID NO:31，H-FR3：SEQ ID NO:34和H-FR4：SEQ ID NO:37；(3)H-FR1：SEQ ID NO:28，H-FR2：SEQ ID NO:32，H-FR3：SEQ ID NO:35和H-FR4：SEQ ID NO:37；和(4)H-FR1：SEQ ID NO:29，H-FR2：SEQ ID NO:30，H-FR3：SEQ ID NO:36和H-FR4：SEQ ID NO:37。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链可变区VH，且所述VH包含SEQ ID NO:46所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的VH包含SEQ ID NO:5-9中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链互补决定区3(LCDR3)，且所述LCDR3包含如SEQ ID NO:40所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的LCDR3包含如SEQ ID NO:15-16中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链互补决定区2(LCDR2)，且所述LCDR2包含如SEQ ID NO:39所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的LCDR2包含如SEQ ID NO:13-14中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链互补决定区1(LCDR1)，且所述LCDR1包含如SEQ ID NO:38所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的LCDR1包含如SEQ ID NO:10-12 中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含LCDR1、LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:38所示的氨基酸序列，所述LCDR2包含SEQ ID NO:39所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:40所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含LCDR1、LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:10-12中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:13-14中任一项所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:15-16中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3选自以下任一组氨基酸序列：(1)LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:13和LCDR3：SEQ ID NO:15；(2)LCDR1：SEQ ID NO:11，LCDR2：SEQ ID NO:13和LCDR3：SEQ ID NO:16；(3)LCDR1：SEQ ID NO:12，LCDR2：SEQ ID NO:13和LCDR3：SEQ ID NO:16；和(4)LCDR1：SEQ ID NO:12，LCDR2：SEQ ID NO:14和LCDR3：SEQ ID NO:16。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链可变区VL，所述VL包括框架区L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的VL包括框架区L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的VL包括框架区L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的VL包括框架区L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:20所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含L-FR1、L-FR2、L-FR3和L-FR4，其中所述L-FR1包含SEQ ID NO:17所示的氨基酸序列，所述L-FR2包含SEQ ID NO:18所示的氨基酸序列，所述L-FR3包含SEQ ID NO:19所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:20所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含轻链可变区VL，且所述VL包含SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体的VL包含SEQ ID NO:1-4中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含VH和VL，且所述VH包含SEQ ID NO:46所示的氨基酸序列，所述VL包含SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含VH和VL，且所述VH包含SEQ ID NO:5-9中任一项所示的氨基酸序列，所述VL包含SEQ ID NO:1-4中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合BCMA的抗体包含VH和VL，且所述VH和VL选自以下任一组氨基酸序列：(1)VH：SEQ ID NO:5和VL：SEQ ID NO:1；(2)VH：SEQ ID NO:6和VL：SEQ ID NO:2；(3)VH：SEQ ID NO:7和VL：SEQ ID NO:2；(4)VH：SEQ ID NO:8和VL：SEQ ID NO:2；(5)VH：SEQ ID NO:9和VL：SEQ ID NO:3；(6)VH：SEQ ID NO:8和VL：SEQ ID NO:3；(7)VH：SEQ ID NO:9和VL：SEQ ID NO:4；和(8)VH：SEQ ID NO:10和VL：SEQ ID NO:4。
在某些实施方式中，所述BCMA结合结构域包含scFv，且所述scFv包含所述特异性结合BCMA的抗体的所述重链可变区VH和所述轻链可变区VL。
在某些实施方式中，在所述scFv中，所述重链可变区VH的C端和所述轻链可变区VL的N端直接或间接连接。
在某些实施方式中，在所述scFv中，所述重链可变区VL的C端和所述轻链可变区VL的N端直接或间接连接。
在某些实施方式中，所述scFv在VH和VL之间包含连接肽，且所述连接肽包含SEQ ID NO:58所示的氨基酸序列。
在某些实施方式中，所述scFv包含SEQ ID NO:49-56中任一项所示的氨基酸序列。
在某些实施方式中，所述CAR包含跨膜结构域，且所述跨膜结构域包含选自源自下述蛋白的跨膜结构域：CD28、CD3e、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。在某些实施方式中，所述跨膜结构域源自CD8，且所述跨膜结构域包含SEQ ID NO:61所示的氨基酸序列。
在某些实施方式中，所述CAR包含共刺激结构域，且所述共刺激结构域包含选自源自下述蛋白的共刺激结构域：CD137、CD28、4-1BB、OX-40和ICOS。在某些实施方式中，所述 共刺激结构域源自CD137，且所述共刺激结构域包含SEQ ID NO:62所示的氨基酸序列。
在某些实施方式中，所述CAR包含胞内信号传导结构域，且所述胞内信号传导结构域包含源自CD3ζ的胞内信号传导结构域。在某些实施方式中，所述胞内信号传导结构域包含SEQ ID NO:63所示的氨基酸序列。
在某些实施方式中，所述CAR包含铰链区，且所述铰链区包含源自CD8的铰链区。在某些实施方式中，所述铰链区源自CD8，且所述铰链区包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，所述CAR包含信号肽。在某些实施方式中，所述信号肽包含SEQ ID NO:59所示的氨基酸序列。
在某些实施方式中，所述CAR包含L6-Li，所述L6-Li位于所述胞内信号传导结构域的C端。在某些实施方式中，所述L6-Li包含SEQ ID NO:107所示的氨基酸序列。
在某些实施方式中，所述CAR包含SEQ ID NO:64-71和74中任一项所示的氨基酸序列。在某些实施方式中，所述CAR包含SEQ ID NO:95-102和105中任一项所示的核苷酸序列。
另一方面，本申请提供了一种分离的核酸分子，所述核酸分子包含编码本申请所述的CAR的核苷酸序列。
在某些实施方式中，所述分离的核酸分子还包含启动子，所述启动子位于编码所述的CAR的核苷酸序列的5’端。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，本申请提供了编码CAR的核酸分子，所述编码CAR的核酸分子包含SEQ ID NO:95-102和105中任一项所示的核苷酸序列。
另一方面，本申请提供了载体，所述载体包含本申请所述的核酸分子。
在某些实施方式中，所述载体选自质粒、逆转录病毒载体和慢病毒载体。
另一方面，本申请提供了免疫效应细胞，所述免疫效应细胞包含本申请所述CAR，所述核酸分子，和/或所述载体。在某些实施方式中，所述免疫效应细胞选自T淋巴细胞。
另一方面，本申请提供了制备免疫效应细胞的方法，其包括向所述免疫效应细胞中引入本申请所述载体。
另一方面，本申请提供了药物组合物，其包含所述CAR，所述核酸分子，所述载体，和/或所述免疫效应细胞，以及药学上可接受的载剂。
另一方面，本申请提供了所述CAR，所述核酸分子，所述载体，所述免疫效应细胞，和 /或所述药物组合物在制备药物中的用途，其中所述药物用于治疗与BCMA表达相关的疾病或病症。
在某些实施方式中，所述与BCMA表达相关的疾病或病症为癌症或恶性肿瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为BCMA阳性肿瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为实体瘤和/或非实体瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为骨髓瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为多发性骨髓瘤。
另一方面，本申请提供了治疗与BCMA表达相关的疾病或病症的方法，其包括向有需要的受试者使用所述CAR，所述核酸分子，所述载体，所述免疫效应细胞，和/或所述药物组合物。
在某些实施方式中，所述与BCMA表达相关的疾病或病症为癌症或恶性肿瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为BCMA阳性肿瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为实体瘤和/或非实体瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为骨髓瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为多发性骨髓瘤。
另一方面，本申请提供了所述CAR，所述核酸分子，所述载体，所述免疫效应细胞，和/或所述药物组合物，其用于治疗与BCMA表达相关的疾病或病症。
在某些实施方式中，所述与BCMA表达相关的疾病或病症为癌症或恶性肿瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为BCMA阳性肿瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为实体瘤和/或非实体瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为骨髓瘤。在某些实施方式中，所述与BCMA表达相关的疾病或病症为多发性骨髓瘤。
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。对附图简要说明书如下：
图1显示了CAR慢病毒载体中CAR各元件组成及其连接顺序的一种实施方式。
图2显示了包含本申请所述CAR的各组BCMA CAR-T细胞在培养过程中的扩增倍数。
图3显示了包含本申请所述CAR的各组BCMA CAR-T细胞中的CAR阳性细胞比例。
图4显示了包含本申请所述CAR的各组BCMA CAR-T细胞对于靶细胞的杀伤毒性，其中，A：MM1s细胞，B：K562细胞。
图5显示了包含本申请所述CAR的各组BCMA CAR-T细胞与MM1s细胞共孵育后的IFN-γ因子分泌情况。
图6显示了包含本申请所述CAR的各组BCMA CAR-T细胞对于MM1s细胞的靶向扩增能力。
图7显示了L6-Li CAR结构下的BCMA CAR-T细胞对于MM1s细胞的杀伤毒性。
图8显示了L6-Li CAR结构下的BCMA CAR-T细胞与MM1s细胞共孵育后的IFN-γ因子分泌情况。
图9显示了L6-Li CAR结构下的BCMA CAR-T细胞对荷瘤小鼠的抑瘤消瘤能力，其中A为实验组，B为对照组。
图10显示了L6-Li CAR结构下的BCMA CAR-T细胞在荷瘤小鼠体内的代谢情况，其中A为实验组，B为对照组。
图11显示了L6-Li CAR结构下的BCMA CAR-T细胞在荷瘤小鼠体内引起的IFN-γ细胞因子分泌情况，其中A为实验组，B为对照组。
图12显示了L6-Li CAR结构下的BCMA CAR-T细胞在荷瘤小鼠体内引起的IL-2细胞因子分泌情况。
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
在本申请中，术语“BCMA”可与“CD269”、“BCM”“TNFRSF17”互换使用，通常是指B细胞成熟抗原。BCMA蛋白是肿瘤坏死因子受体家族成员。在本申请中，术语 “BCMA”可包括包含突变的蛋白质，例如可包括包含全长野生型BCMA的点突变、片段、插入、缺失和剪接变体的蛋白质。所述BCMA可以包括来自任何脊椎动物，例如，灵长类发动物(例如，人或猴)、啮齿类动物(小鼠或大鼠)、禽和/或畜等。在本申请中，术语“BCMA”还可包括完整BCMA蛋白的一部分，只要保留相关的生物活性即可。在本申请中，所述BCMA可以为人BCMA，其GenBank登录号为BAB60895.1。例如，人BCMA通常是由994个核苷酸长的初级mRNA转录物(NM_001192.2)编码的184个氨基酸长的蛋白质。
在本申请中，术语“结合结构域”通常是指(特异性)结合到靶分子(抗原)上的给定靶表位或给定靶位点，或与所述给定靶表位或给定靶位点相互作用，或识别所述给定靶表位或给定靶位点的结构域。
在本申请中，术语“BCMA结合结构域”通常是指可以与BCMA蛋白特异性结合的结构域。例如，所述BCMA结合结构域可包含能特异性结合B细胞上表达的BCMA多肽的嵌合抗原受体或其片段，抗BCMA抗体或其抗原结合片段。在本申请中使用的术语“结合结构域”、“胞外结构域”、“胞外结合结构域”、“抗原特异性结合结构域”和“胞外抗原特异性结合结构域”可互换使用，并且可以包含具有特异性结合目标靶抗原(例如BCMA)的能力的CAR的结构域或片段。BCMA结合结构域可以为天然来源、合成来源、半合成来源或重组来源。
在本申请中，术语“抗体”通常是指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，术语“抗体”也包括抗体片段，比如抗原结合片段Fab、F(ab')2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合BCMA)的其它抗体片段。通常，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)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和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。
在本申请中，术语“scFv“通常是指单链抗体，是由重链可变区和轻链可变区直接连接或 通过连接分子(例如，连接肽)连接而成的抗体。所述scFv的结构自N端至C端，可以是重链可变区-轻链可变区，轻链可变区-重链可变区，重链可变区-连接肽-轻链可变区，或轻链可变区-连接肽-重链可变区。
在本申请中，术语“嵌合抗原受体(CAR)”通常是指包含能够结合抗原的胞外结构域和至少一个胞内结构域的融合蛋白。CAR可以是识别肿瘤相关抗原(TAA)的抗体的抗原结合区与胞内信号域“免疫受体酪氨酸活化基序(ITAM)”融合形成的抗原受体。CAR的基本结构包括一个肿瘤相关抗原(TAA)结合区(例如，scFv)，一个胞外铰链区，一个跨膜区和一个胞内信号传导结构域。在本申请中，所述CAR可以基于抗体的抗原(例如BCMA)特异性与免疫效应细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR的免疫效应细胞可以特异地识别和消除表达靶抗原的恶性细胞。
在本申请中，术语“信号传导结构域”通常是指位于细胞内部能够转导信号的结构域。在本申请中，所述胞内信号传导结构域可以将信号传导至细胞内。通常，信号传导结构域为用于指导蛋白质找寻靶点的任何一段连续的氨基酸序列。例如，在某些实施方式中，所述胞内信号传导结构域可选自CD3ζ胞内域，CD28胞内域，CD28胞内域，4-1BB胞内域和OX40胞内域。
在本申请中，术语“共刺激”通常指淋巴细胞激活第二信号的来源，通常由参与适应性免疫的免疫细胞(T细胞/B细胞间或抗原提呈细胞/T细胞间)表面共刺激分子及其受体相互作用而产生。例如，T细胞的完全活化有赖于双信号和细胞因子的作用。T细胞活化的第一信号来自其受体TCR与抗原的特异性结合，即T细胞对抗原识别；T细胞活化的第二信号来自共刺激分子，即APC上的共刺激分子与T细胞表面的相应受体的相互作用。
在本申请中，术语“共刺激结构域”通常指能够与共刺激分子相互作用从而产生共刺激的任何氨基酸序列。
在本申请中，术语“铰链区”通常指在CAR结构中位于特异性结合BCMA的抗体或其抗原结合片段(例如，scFv)和跨膜结构域之间的一段区域。铰链区通常来源于IgG家族，例如IgG1和IgG4，还有些来源于IgD和CD8。通常铰链区具有一定程度的灵活性，从而影响CAR分子与其特异性靶点之间的空间约束，进而影响CART细胞与肿瘤细胞之间的接触。
在本申请中，术语“跨膜结构域”通常是指细胞表面蛋白中一段跨越细胞膜的序列，其可以包含疏水性alpha螺旋。跨膜结构域可以与细胞内信号转导结构域相连接，起着传递信号的作用。跨膜结构域一般包含三个不同的结构区：N末端胞外区、中间保守的跨膜伸展 区、以及C末端胞质区。跨膜结构域可能还包含胞内区或胞质区。在本申请中，跨膜结构域可以源自任意的I型、II型或III型跨膜蛋白。
在本申请中，术语“信号肽”通常是指处于新生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。信号肽通常在该过程中被切除。信号肽对于用于产生多肽的生物体可以是异源的或同源的。
在本申请中，术语“L6-Li”通常是指低密度脂蛋白受体相关蛋白和瘦素的融合蛋白，关于L6-Li的相关内容可参见专利申请公开号WO2021057932A1。在本申请中，所述L6-Li可包含SEQ ID NO:107所示的氨基酸序列。在本申请中，编码所述L6-Li的核酸分子可包含SEQ ID NO:106所示的核苷酸序列。
在本申请中，术语“启动子”通常指一段能使特定基因进行转录的脱氧核糖核酸(DNA)序列。启动子可以被RNA聚合酶辨认，并开始转录合成RNA。在核糖核酸(RNA)合成中，启动子可以和调控基因转录的转录因子产生相互作用，控制基因表达(转录)的起始时间和表达的程度。启动子包含核心启动子区域和调控区域，位于控制基因表达的调控序列中、基因转录起始位点的上游(DNA反义链的5’方向)，本身并无编译功能。根据其作用方式及功能分为三类：组成型启动子(在多数或全部组织中保持持续的活性)、特异型启动子(组织特异性或者发育时期的特异性)和诱导型启动子(受外界化学或物理信号调控)。
在本申请中，术语“特异性结合”通常指可测量的和可再现的相互作用，比如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在某些实施方式中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在另一个实施方式中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“核酸分子”通常是指任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。在某些实施方式中，本申请所述的核酸分子可以为从天然环境中分离的。在某些实施方式中，本申请所述的核酸分子可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体或其抗原结合片段的核酸，这些方 法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning,A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor,N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology，Greene Publishing and Wiley-Interscience，New York N.Y.,1993。
在本申请中，术语“多肽”或“蛋白质”可互换地使用，通常是指氨基酸残基的聚合物。该术语也适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的类似物或模拟物的氨基酸聚合物、以及天然存在的氨基酸聚合物、同源物。该术语也可包括修饰的氨基酸聚合物，例如，通过添加糖残基以形成糖蛋白或被磷酸化修饰。多肽和蛋白质可由天然存在的和非重组的细胞或由遗传工程改造的或重组的细胞产生，并且可包含具有天然蛋白质的氨基酸序列的分子、或具有天然序列的一个或多个氨基酸的缺失、添加和/或取代的分子。术语“多肽”和“蛋白质”特别包括本申请所述的抗原结合蛋白的一个或多个氨基酸的缺失、添加和/或取代的序列。
在提到蛋白质的氨基酸序列或核酸分子的核苷酸序列时，本申请还包括这些序列的同源物。在本申请中，术语“同源物”通常是指与野生型氨基酸序列和野生型核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、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得以表达。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅 仅只有这些物质。
在本申请中，术语“免疫效应细胞”通常指参与免疫应答，例如，促进免疫效应子应答的细胞。免疫效应细胞的实例包括T细胞，例如，α/β的T细胞和γ/δT细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。
在本申请中，术语“药物组合物”通常是指适合施用于患者、人患者的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的免疫效应细胞、CAR、核酸分子或载体，以及任选地药学上可接受的载剂。药物组合物包含有效生物活性的活性成分形式，并且不含有对所述制剂待施用的受试者有不可接受的毒性的另外成分。
在本申请中，术语“药学上可接受的载剂”通常是指不干扰活性成分的生物活性的有效性的一种或多种非毒性材料。这类制剂常规地可以含有盐、缓冲剂、防腐剂、相容的载体、以及任选地其他治疗剂。
在本申请中，术语“与BCMA表达相关的疾病或病症”包括，但不限于与BCMA表达相关的疾病、或与表达BCMA的细胞(包括各种癌症的肿瘤细胞)相关的病症。与正常机体或正常细胞相比，具有与BCMA表达相关的疾病或病症的个体的BCMA表达异常(例如，BCMA过表达)，或细胞的BCMA表达异常(例如，BCMA过表达)。
在本申请中，术语“癌症或恶性肿瘤”通常是指由异常细胞生长形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或非实体瘤(例如，血液瘤)。
在本申请中，术语“BCMA阳性肿瘤”通常是指与正常细胞相比，BCMA蛋白过量表达的肿瘤。例如，BCMA阳性肿瘤可包括非实体瘤。例如，BCMA阳性肿瘤可包括骨髓瘤。例如，BCMA阳性肿瘤可包括多发性骨髓瘤。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
一方面，本申请提供了一种嵌合抗原受体(CAR)，其包含BCMA结合结构域，所述BCMA结合结构域包含特异性结合BCMA的抗体或其抗原结合片段。
BCMA结合结构域
在本申请中，所述CAR中的特异性结合BCMA的抗体或其抗原结合片段可包含重链互补决定区3(HCDR3)，且所述HCDR3可包含如SEQ ID NO:25所示的氨基酸序列： DIRYVMDY(SEQ ID NO:25)。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段可包含重链互补决定区2(HCDR2)，且所述HCDR2可包含如SEQ ID NO:41所示的氨基酸序列：WINTETREPX 10YAYDFRX 17(SEQ ID NO:41)，其中，X 10＝A或T，X 17＝G或S。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述HCDR2可包含如SEQ ID NO:22-24中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含重链互补决定区1(HCDR1)，且所述HCDR1可包含如SEQ ID NO:21所示的氨基酸序列：DYTIN(SEQ ID NO:21)。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:41和SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2和HCDR3，其中所述HCDR1可包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:22-24中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:24和SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述CAR中的特异性结合BCMA的抗体或其抗原结合片段可包含轻链互补决定区3(LCDR3)，且所述LCDR3可包含如SEQ ID NO:40所示的氨基酸序列：LQSRX 5FPRX 9(SEQ ID NO:40)，其中，X 5＝I或R，X 9＝H或T。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述LCDR3可包含如SEQ ID NO:15-16中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含轻链互补决定区2(LCDR2)，且所述LCDR2可包含如SEQ ID NO:39所示的氨基酸序列：LGX 3NRX 6X 7(SEQ ID NO:39)，其中， X 3＝R或S，X 6＝A或P，且X 7＝A或S。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述LCDR2可包含如SEQ ID NO:13-14中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含轻链互补决定区1(LCDR1)，且所述LCDR1可包含如SEQ ID NO:38所示的氨基酸序列：RASESVSVX 9GX 11X 12X 13X 14H(SEQ ID NO:38)，其中，X 9＝I、P或S，X 11＝A或I，X 12＝H或S，X 13＝F或L，且X 14＝I或L。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述LCDR1可包含如SEQ ID NO:10-12中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含LCDR1、LCDR2和LCDR3，其中所述LCDR1可包含SEQ ID NO:10-12中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:13-14中任一项所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:15-16中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:10、SEQ ID NO:13和SEQ ID NO:15所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:11、SEQ ID NO:13和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:12、SEQ ID NO:14和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:41、SEQ ID NO:25、SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，，其中所述HCDR1可包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:22-24中任一项所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:25所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:10-12中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:13-14中任一项所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:15-16中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:25、SEQ ID NO:10、SEQ ID NO:13和SEQ ID NO:15所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:11、SEQ ID NO:13和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:11、SEQ ID NO:13和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:25、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:25、SEQ ID NO:12、SEQ ID NO:14和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:21、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:12、SEQ ID NO:14和SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含框架区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:42所示的氨基酸序列：QVQLVQSGX 9EX 11KX 13PGASVKVSCKASGYX 28FA(SEQ ID NO:42),其中，所述X 9＝A或S，X 11＝L或V，X 13＝K或Q，且X 28＝S或T。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段的H-FR1可包含SEQ ID NO:26-29中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含框架区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:43所示的氨基酸序列：WVRQAX 6GQGLEWX 13G(SEQ ID NO:43),其中，所述X 6＝P或T，且X 13＝I或M。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段的H-FR2可包含SEQ ID NO:30-32中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含框架区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:44所示的氨基酸序列：RFX 3FX 5LX 7TSX 10STAYLX 16X 17SSLX 21X 22EDTAVYYCAR(SEQ ID NO:44),其中，所述X 3＝T或V，X 5＝S或T，X 7＝D或N，X 10＝A、I或V，X 16＝E或Q，X 17＝I或L，X 21＝K或R，且X 22＝A或S。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段的H-FR3可包含SEQ ID NO:33-36中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含框架区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:37所示的氨基酸序列：WGQGTLVTVSS(SEQ ID NO:37)。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段可包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1、H-FR2、H-FR3和H-FR4可分别依次包含SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44和SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含H-FR1、H-FR2、H-FR3和H-FR4，且 所述H-FR1可包含SEQ ID NO:26-29中任一项所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:30-32中任一项所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:33-36中任一项所示的氨基酸序列且所述H-FR4可包含SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1、H-FR2、H-FR3和H-FR4包含选自以下任一组氨基酸序列：(1)H-FR1：SEQ ID NO:26，H-FR2：SEQ ID NO:30，H-FR3：SEQ ID NO:33和H-FR4：SEQ ID NO:37；(2)H-FR1：SEQ ID NO:27，H-FR2：SEQ ID NO:31，H-FR3：SEQ ID NO:34和H-FR4：SEQ ID NO:37；(3)H-FR1：SEQ ID NO:28，H-FR2：SEQ ID NO:32，H-FR3：SEQ ID NO:35和H-FR4：SEQ ID NO:37；和(4)H-FR1：SEQ ID NO:29，H-FR2：SEQ ID NO:30，H-FR3：SEQ ID NO:36和H-FR4：SEQ ID NO:37。
在本申请中，所述抗体或其抗原结合片段可包含抗体重链可变区VH，且所述VH可包含SEQ ID NO:46所示的氨基酸序列：QVQLVQSGX 9EX 11KX 13PGASVKVSCKASGYX 28FADYTINWVRQAX 41GQGLEWX 48GWINT ETREPX 59YAYDFRX 66RFX 69FX 71LX 73TSX 76STAYLX 82X 83SSLX 87X 88EDTAVYYCARDIRYVM DYWGQGTLVTVSS(SEQ ID NO:46)，其中，X 9＝A或S，X 11＝L或V，X 13＝K或Q，X 28＝S或T，X 41＝P或T，X 48＝I或M，X 59＝A或T，X 66＝G或S，X 69＝T或V，X 71＝S或T，X 73＝D或N，X 76＝A、I或V，X 82＝E或Q，X 83＝I或L，X 87＝K或R且X 88＝A或S。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段的VH可包含SEQ ID NO:5-9中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含框架区L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1可包含SEQ ID NO:17所示的氨基酸序列：EIVLTQSPATLSLSPGERATLSC(SEQ ID NO:17)。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段可包含框架区L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2可包含SEQ ID NO:18所示的氨基酸序列：WYQQKPGQAPRLLIY(SEQ ID NO:18)。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段可包含框架区L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3可包含SEQ ID NO:19所示的氨基酸序列：GIPARFSGSGSGTDFTLTISSLEPEDAAIYYC(SEQ ID NO:19)。例如，所述序列可以根据Kabat 规则划分。
在本申请中，所述抗体或其抗原结合片段可包含框架区L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4可包含SEQ ID NO:20所示的氨基酸序列：FGQGTKLEIK(SEQ ID NO:20)。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段可包含L-FR1、L-FR2、L-FR3和L-FR4，且所述L-FR1、L-FR2、L-FR3和L-FR4可分别依次包含SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含抗体轻链可变区VL，且所述VL可包含SEQ ID NO:45所示的氨基酸序列：EIVLTQSPATLSLSPGERATLSCRASESVSVX 32GX 34X 35X 36X 37HWYQQKPGQAPRLLIYLGX 56NRX 59X 60GIPARFSGSGSGTDFTLTISSLEPEDAAIYYCLQSRX 97FPRX 101FGQGTKLEIK(SEQ ID NO:45)，其中，X 32＝I、P或S，X 34＝A或I，X 35＝H或S，X 36＝F或L，X 37＝I或L，X 56＝R或S，X 59＝A或P，X 60＝A或S，X 97＝I或R，且X 101＝H或T。例如，所述序列可以根据Kabat规则划分。
在本申请中，所述抗体或其抗原结合片段的VL可包含SEQ ID NO:1-4中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:46所示的氨基酸序列，所述VL可包含SEQ ID NO:45所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:5-9中任一项所示的氨基酸序列，所述VL可包含SEQ ID NO:1-4中任一项所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:5所示的氨基酸序列，所述VL可包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:6所示的氨基酸序列，所述VL可包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:7所示的氨基酸序列，所述VL可包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:8所示的氨基酸序列，所述VL可包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID  NO:9所示的氨基酸序列，所述VL可包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:8所示的氨基酸序列，所述VL可包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:9所示的氨基酸序列，所述VL可包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可包含VH和VL，且所述VH可包含SEQ ID NO:8所示的氨基酸序列，所述VL可包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述抗体或其抗原结合片段可以是scFv，且所述scFv可包含所述VH和所述VL。在所述scFv中，所述VH的C端和所述VL的N端可以直接连接，例如，框内连接。在所述scFv中，所述VH的C端和所述VL的N端可以间接连接，例如，通过连接子(如连接肽)连接。在所述scFv中，所述VL的C端和所述VH的N端可以直接连接，例如，框内连接。在所述scFv中，所述VL的C端和所述VH的N端可以间接连接，例如，通过连接子(如连接肽)连接。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，所述VH可包含SEQ ID NO:5所示的氨基酸序列，所述VL可包含SEQ ID NO:1所示的氨基酸序列，所述连接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，且所述VH可包含SEQ ID NO:6所示的氨基酸序列，所述VL可包含SEQ ID NO:2所示的氨基酸序列，所述连接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，且所述VH可包含SEQ ID NO:7所示的氨基酸序列，所述VL可包含SEQ ID NO:2所示的氨基酸序列，所述连接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，且所述VH可包含SEQ ID NO:8所示的氨基酸序列，所述VL可包含SEQ ID NO:2所示的氨基酸序列，所述连接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，且所述VH可包含SEQ ID NO:9所示的氨基酸序列，所述VL可包含SEQ ID NO:3所示的氨基酸序列，所述连接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，且所述VH可包含SEQ ID NO:8所示的氨基酸序列，所述VL可包含SEQ ID NO:3所示的氨基酸序列，所述连 接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，且所述VH可包含SEQ ID NO:9所示的氨基酸序列，所述VL可包含SEQ ID NO:4所示的氨基酸序列，所述连接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，且所述VH可包含SEQ ID NO:8所示的氨基酸序列，所述VL可包含SEQ ID NO:4所示的氨基酸序列，所述连接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含VH、VL和连接肽，所述VL的C端可以与所述VH的N端通过连接肽连接，且所述连接肽可包含SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述CAR的BCMA结构域可包含scFv，且所述scFv可包含39-49中任一项所示的氨基酸序列。在本申请中，所述scFv可包含与SEQ ID NO:39-49中任一项所示的氨基酸序列具有至少80％(例如，至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高)序列同源性的氨基酸序列。
嵌合抗原受体(CAR)
在本申请中，所述CAR包含胞外的BCMA结构域外，还可包含胞内结构域。
在某些情形中，所述CAR可包含胞内共刺激信号传导结构域，其可提供刺激信号。所述共刺激信号传导结构域可以包括但不限于下组：CD137、CD28、4-1BB、OX-40和ICOS中的共刺激信号传导区及其组合组成的共刺激分子。例如，所述共刺激结构域可以是来自CD137的共刺激结构域。例如，所述共刺激结构域可包含SEQ ID NO:62所示的氨基酸序列
在某些情形中，所述CAR可包含胞内信号传导结构域，其可以包含至少有一个ITAM基序的结构域。所述胞内信号传导结构域可以将激活信号传输到细胞内部。示例性的信号传导结构域可源自选自下组的信号传导结构域，包括但不限于CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30激活区、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和DAP-12，及上述的变体。例如，所述胞内信号传导结构域可以是来自CD3ζ的信号转导结构域。例如，所述胞内信号传导结构域可包含SEQ ID NO:63所示的氨基酸序列。
在某些情形中，所述CAR可包含跨膜域，所述跨膜域是细胞表面蛋白中一段跨越细胞膜的序列，其可以包含疏水性alpha螺旋。跨膜域可以源自CD28，具有良好的稳定性。跨膜域可以源自任意的I型跨膜蛋白。跨膜域可以是预测为形成疏水螺旋的合成序列。所述跨膜域 可包含源自选自下组中的一种或多种蛋白的跨膜域：CD28、CD3e、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。例如，所述跨膜域可以是来自CD8的跨膜域。例如，所述跨膜结构域可包含SEQ ID NO:61所示的氨基酸序列。
在某些情形中，所述CAR可包含铰链区，所述铰链区可以位于所述胞外的靶向部分和所述跨膜域之间。所述铰链区可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、CD8alpha、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。例如，所述所述铰链区可包含SEQ ID NO:60所示的氨基酸序列。
在本申请中，所述CAR在所述BCMA结合结构域的N端还可包含信号肽，例如，人CD8信号肽。例如，所述信号肽可包含SEQ ID NO:59所示的氨基酸序列。
在本申请中，自N端至C端，所述CAR可依次包含BCMA靶向部分(例如，所述抗原结合蛋白，又例如，所述scFv)、所述铰链区、所述跨膜域、所述共刺激信号传导区和所述信号传导结构域。
例如，自N端至C端，所述CAR可依次包含所述scFv、所述CD8铰链区、所述CD8跨膜域、所述CD137共刺激信号传导区和所述CD3ζ信号传导结构域。例如，所述CAR可包含SEQ ID NO:64-71中任一项所示的氨基酸序列。
在本申请中，所述CAR的C端可连接瘦素和/或其功能性片段，和/或低密度脂蛋白受体相关蛋白或其片段。例如，所述瘦素可包括分泌型瘦素。例如，低密度脂蛋白受体相关蛋白6。
在本申请中，所述CAR，所述瘦素和/或其功能性片段，和/或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。
在本申请中，在所述CAR的C端，可连接L6-Li。例如，所述L6-Li可包含SEQ ID NO:107所示的氨基酸序列。例如，编码所述L6-Li的核酸分子可包含SEQ ID NO:106所示的核苷酸序列。
在本申请中，自N端至C端，所述CAR可依次包含BCMA靶向部分(例如，所述抗原结合蛋白，又例如，所述scFv)、所述铰链区、所述跨膜域、所述共刺激信号传导区、所述信号传导结构域和所述L6-Li。
例如，自N端至C端，所述CAR可依次包含所述scFv、所述CD8铰链区、所述CD8跨膜域、所述CD137共刺激信号传导区、所述CD3ζ信号传导结构域和所述L6-Li。例如， 所述CAR可包含SEQ ID NO:74所示的氨基酸序列。
核酸分子、载体、细胞、制备方法和药物组合物
另一方面，本申请还提供了分离的一种或多种核酸分子，其可以编码本申请所述的嵌合抗原受体(CAR)。本申请所述的分离的一种或多种核酸分子可以为任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物，但可以编码本申请所述的嵌合抗原受体(CAR)。例如，所述核酸分子可包含SEQ ID NO:95-102和105中任一项所示的核苷酸序列。
另一方面，本申请还提供了载体，其可以包含本申请所述的核酸分子。所述载体可通过转化、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得以表达。例如，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。又例如，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
另一方面，本申请还提供了免疫效应细胞，其可以包含本申请所述的CAR、所述的核酸分子或本申请所述的载体。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)。在某些实施方式中，所述细胞还可以包括用本发明所述的载体在体外转染的细胞。在某些实施方式中，所述细胞可以为哺乳动物细胞。在某些实施方式中，所述免疫效应细胞包括T淋巴细胞，例如，α/β的T淋巴细胞和γ/δT淋巴细胞；在某些实施方式中，所述免疫效应细胞可以包括自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。
一方面，本申请提供一种经修饰的免疫效应细胞，其包含嵌合抗原受体和/或其编码元件，或者包含T细胞受体和/或其编码元件，且所述经修饰的免疫细胞还包含：瘦素和/或其功能性片段；和/或，瘦素受体和/或其功能性片段，且与未经相应经修饰的免疫细胞相比，所述经修饰的免疫细胞中所述瘦素受体和/或其功能性片段的表达量增加。在本申请中，所述免疫效应细胞可包含瘦素和/或其功能性片段，和/或，瘦素受体和/或其功能性片段，以及低密度脂蛋白受体相关蛋白或其片段。关于对免疫效应细胞的修饰的内容可参见WO2021057932A1， WO2021057932A1中的描述可以同样应用于本申请。
另一方面，本申请还提供了制备本申请所述的免疫效应细胞的方法，所述方法可以包括向免疫效应细胞中引入本申请所述的分离的核酸分子或本申请所述的载体。
另一方面，本申请还提供了组合物，其可以包含本申请所述的免疫效应细胞。在某些实施方式中，所述组合物还可以包括任选地药学上可接受的载剂。在某些实施方式中，所述组合物的可接受成分在所用剂量和浓度下对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。在某些实施方式中，所述药学上可接受的佐剂可以包括与所述免疫效应细胞相容的任何和所有的溶剂、分散介质、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。
在某些实施方式中，所述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。在某些实施方式中，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。
治疗方法
另一方面，本申请还提供了本申请所述的嵌合抗原受体、本申请所述的核酸分子、本申请所述的载体、本申请所述的免疫效应细胞和/或本申请所述的组合物在制备药物中的用途，所述药物可以用于治疗与BCMA的表达相关的疾病或病症。
在本申请中，所述与BCMA的表达相关的疾病或病症为癌症或恶性肿瘤，例如，所述与BCMA的表达相关的疾病或病症可以包括BCMA阳性肿瘤。例如，所述癌症或恶性肿瘤可包含实体瘤或非实体瘤。例如，例如，癌症或恶性肿瘤可以为非实体瘤。例如，所述癌症或恶性肿瘤可包含骨髓瘤，例如，所述骨髓瘤可以为多发性骨髓瘤。
另一方面，本申请还提供了预防、缓解或治疗肿瘤的方法，所述方法可以包括向有需要的受试者施用本申请所述的CAR、核酸分子、载体、免疫效应细胞和/或药物组合物。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
在本申请中，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。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 scFv片段的产生
利用重组人BCMA-hFc和BCMA-His蛋白(原启生物科技(上海)有限公司制备，其中人BCMA-hFc表示由人的BCMA与人的Fc连接形成的蛋白，人BCMA蛋白的氨基酸序列见NCBI官网GenBank ID：BAB60895.1，人的Fc的氨基酸如SEQ ID NO:57所示，进行噬菌体天然人源性抗体文库(原启生物科技(上海)有限公司构建)交替分选，共进行四轮分选，四轮淘选之后，观察到显著的富集。利用ELISA对淘选得到的噬菌体抗体克隆进行鉴定。再把得到的阳性抗体序列做亲和力检测和测序鉴定。把经过筛选后的候选抗体经过1-2轮亲和力进化，及再次的亲和力检测和流式结合活性鉴定，共获得了8株能特异性结合人BCMA的噬菌体抗体克隆，测序后获得轻链可变区(VL)核酸序列和重链可变区(VH)核酸序列，VL和VH核酸序列间以连接肽核酸序列(其核酸序列见SEQ ID NO:58、其氨基酸序列见SEQ ID NO:85)进行连接形成单链抗体(scFv)形式，并分别命名为hBCMA-01、hBCMA-02、hBCMA-15、hBCMA-17、hBCMA-19、hBCMA-20、hBCMA-22、hBCMA-23；阳性对照的scFv序列来自专利US 2017/0226216A1中的huBCMA-10。将所有上述9个(包括huBCMA-10)scFv的核酸序列交付南京金斯瑞生物科技有限公司进行基因合成，并分别在scFv核酸序列的5’端加正向引物(核酸序列SEQ ID NO:87)，在scFv核酸序列3’端加反向引物(核酸序列SEQ ID NO:88)，用于下一步同源重组的分子构建。这8个抗体克隆和阳性对照的VH和VL基因序列如表1所示。
表1. 8株噬菌体抗体克隆和阳性对照的VH和VL序列
Figure PCTCN2022089287-appb-000001
Figure PCTCN2022089287-appb-000002
实施例2 CAR慢病毒载体的构建
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，以下简称CAR慢病毒空载体)或CAR慢病毒载体(包含L6-Li元件，见专利申请公开号WO2021057932A1，L6-Li的氨基酸序列如SEQ ID NO:107所示)(原启生物科技(上海)有限责任公司自构，以下简称CAR-L6-Li慢病毒载体)进行双酶切，前者产生7749bp线性化片段，后者产生9605bp线性化片段。
割胶回收后，使用产生的7749bp线性化片段与实施例1所得的scFv的核酸序列片段(含两端的同源臂)，按1:3的摩尔比例混合(体积不超过10μl)，进行同源重组后，转化大肠杆菌DH5α感受态细胞。挑单克隆菌落，摇菌、质粒抽提并送测序验证插入的scFv序列是否正确。
割胶回收后，使用产生的9605bp线性化片段与实施例1中的hBCMA-22和huBCMA-10的scFv的核酸序列片段(含两端的同源臂)，按1:3的摩尔比例混合(体积不超过10μl)，进行同源重组后，转化大肠杆菌DH5α感受态细胞。挑单克隆菌落，摇菌、质粒抽提并送测序鉴定。
CAR慢病毒空载体和h(u)BCMA-XX核心质粒(以下简称h(u)BCMA-XX质粒)中CAR和CAR-L6-Li各部分元件及连接顺序见图1。各部分元件的氨基酸序列和核酸序列见表2。其中CAR慢病毒空载体的CAR部分依次包含信号肽-CD8铰链区-CD8跨膜区-CD137共刺激结构域-CD3ζ胞内信号传导结构域。CAR载体在CAR慢病毒空载体的基础上，在信号肽和CD8铰链区之间插入靶向BCMA的scFv，CAR-L6-Li载体在CAR载体的基础上，在CD3ζ的3’端连接L6-Li序列。各载体的5’端与EF1α启动子连接。各质粒载体中CAR部分的序列见表3。
表2 CAR和CAR-L6-Li中各部分元件的序列
元件 核苷酸序列(SEQ ID NO) 氨基酸序列(SEQ ID NO)
信号肽 90 59
CD8铰链区 91 60
CD8跨膜结构域 92 61
CD137共刺激结构域 93 62
CD3ζ胞内信号传导结构域 94 63
L6-Li SEQ ID NO:106 SEQ ID NO:107
表3 各质粒载体中CAR部分的序列
  核苷酸序列(SEQ ID NO) 氨基酸序列(SEQ ID NO)
hBCMA-01 CAR 95 64
hBCMA-02 CAR 96 65
hBCMA-15 CAR 97 66
hBCMA-17 CAR 98 67
hBCMA-19 CAR 99 68
hBCMA-20 CAR 100 69
hBCMA-22 CAR 101 70
hBCMA-23 CAR 102 71
huBCMA-10 CAR 103 72
huBCMA-10-L6-Li CAR 104 73
huBCMA-22-L6-Li CAR 105 74
实施例3 慢病毒的包装
作为示例，用于构建本发明的慢病毒载体系统属于第三代，该系统共有三个质粒组成，即编码Gag-Pol蛋白和Rev蛋白的包装质粒psPAX2(Addgene，质粒号#12260)、编码包膜蛋白VSV-G的PMD2.G质粒(Addgene，质粒号#12259)，以及核心质粒，即上述实施例1-2中的含scFv序列的各个CAR慢病毒质粒(即hBCMA-01、hBCMA-02、hBCMA-15、hBCMA-17、hBCMA-19、hBCMA-20、hBCMA-22、hBCMA-23、huBCMA-10)组成。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。
慢病毒的包装过程如下：
(1)取生长状态良好(一般选取20代以内的细胞)、约2.5×10 6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％CO 2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6μg的三种质粒(按质量比CAR慢病毒核心质粒：psPAX2：PMD2.G＝4:3:2的比例加入)，混匀后加入18uL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，于37℃、5％CO 2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℃待用。
实施例4 病毒滴度测定
慢病毒滴度的检测按以下方法进行：
(1)快速解冻实施例3中的病毒液。
(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(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，然后将细胞密度调整为2.0×10 5/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％CO 2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5×10 5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0.5μg BCMA-Fc-Avi蛋白(Acro，BC7-H82FO)，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0.5μl Native Streptavidin蛋白(
Figure PCTCN2022089287-appb-000003
650)(Abcam，ab134341)(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，荧光为Dylight 650。
(14)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。
慢病毒滴度(IU/mL)＝铺板细胞个数×(测试管阳性率-对照管阳性率)/接种病毒液体 积(mL)。
经检测，上述各个含scFv的CAR病毒滴度范围在5-15×10 6IU/mL
实施例5 T细胞的感染和BCMA CAR-T细胞体外扩增
含有BCMA scFv序列的CAR-T细胞的制备方法如下：
(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)(单采血由原启生物科技(上海)有限责任公司方舟计划志愿者提供)。
(2)PBMC经CD3阳选磁珠分选后得到纯度>90％的CD3 +T细胞，T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-VIVO 15(Lonza)培养基中加入5％的FBS，再加入300IU/mL的白介素2(欣吉尔))重悬CD3 +T细胞，再按3倍细胞量加入已经洗涤的CD3/CD28Dynabeads(Gibco，40203D)，然后补加T细胞完全培养基，调整细胞密度为1.0-1.2×10 6/mL，并将细胞置于37℃，5％CO 2培养箱中激活培养(记录为D0)。
(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，按照病毒复染指数(MOI)为3-6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0.6-1.0×10 6/mL，再将细胞置于37℃，5％CO 2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0.5-0.7×10 6/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0.5-1.0×10 6/mL。
(8)一般情况下，在培养D7时去除Dynabeads，总培养周期为12天左右。
如图2和图3所示，经激活培养12天的BCMA CAR-T细胞的总扩增倍数介于100-1600倍之间，感染后的CAR阳性率均介于65％至90％之间，均可以用于细胞学功能实验。
实施例6 BCMA CAR-T细胞的体外靶向杀伤活性评估
MM1s细胞是表达BCMA的骨髓瘤细胞，K562细胞是不表达BCMA的肿瘤细胞。在本实验中，我们首先构建稳定表达荧光素酶(Luciferase)的这两种肿瘤细胞，再将肿瘤细胞与CAR-T细胞共孵育一段时间后，通过底物来间接检测Luciferase酶的活性(原理详见Promega, E2510)，进而确定CAR-T细胞的靶向杀伤能力。
过表达Luciferase的MM1s-Luci和K562-Luci方法如下：
(1)从Promega公司购买pGL4.30[luc2P/NFAT-RE/Hygro]Vector，将其中的luc2P reporter基因(228-2003)克隆到不含CAR结构的慢病毒核心空载质粒上(该空载含GFP报告基因)，并定义为Luciferase-GFP慢病毒核心质粒。
(2)按照实施例3的方法包装Luciferase-GFP(简写为Luci-GFP)慢病毒，并参照实施例4的方法，通过GFP荧光通道检测出Luciferase-GFP慢病毒的滴度。
(3)按照MOI＝5-10，分别感染MM1s细胞和K562细胞，在稳定培养3-4周后，通过流式分选仪，分选出GFP +的细胞群，构建得到MM1s-Luci-GFP细胞和K562-Luci-GFP细胞。
在培养D8-D12期间进行CAR-T细胞的体外杀伤活性检测，需提前测定各组的CAR阳性率，取0.5-1.0×10 6个细胞，检测方法可参考实施例4中的(6)-(13)。
感染了CAR慢病毒的细胞为效应细胞，未做慢病毒感染的T细胞为空白效应细胞，MM1s-Luci-GFP细胞和K562-Luci-GFP为靶细胞。
CAR-T细胞的体外杀伤活性实验过程如下：
(1)提前一天用流式法检测各组的CAR阳性率。
(2)杀伤实验当天，通过补加空白效应细胞的方式，将各组的CAR阳性率调成一致，培养基为不含细胞因子的CAR-T培养基。
(3)在96孔板中进行杀伤活性实验，按照效应细胞和靶细胞的比例为3:1、1:1和0.3:1三种(或3:1和1:1两种)，固定每个96孔的靶细胞数2×10 4个，依次加入靶细胞和效应细胞，每组每个效靶比下设置3个重复孔，另需设置3个单独靶细胞孔作为阴性对照。
(4)一般在杀伤5-6小时后进行杀伤活性检测。即先将96孔板中细胞悬液吹打混匀，然后均匀取出1/2体积的细胞悬液至96孔全白板中，再加入等体积的荧光素酶的底物，室温避光10-15min后，于酶标仪上读取相应的荧光值。具体检测方法参见
Figure PCTCN2022089287-appb-000004
萤光素酶检测系统(Promega,E2510)。
(5)由于仅活的MM1s细胞能被检测到，CAR-T细胞对靶细胞的杀伤毒性可以用以下公式进行计算：
Figure PCTCN2022089287-appb-000005
如图4的A和B所示，和空白效应细胞T细胞相比，各组BCMA CAR-T细胞对于表达BCMA的MM1s细胞均有较强的杀伤活性，且呈现明显的效靶比依赖性，而对于不表达BCMA 的K562细胞，则没有任何杀伤作用，证明CAR-T细胞的肿瘤细胞杀伤作用是具有特异性的，此外从图4中可以看出，在三种效靶比下，本申请的hBCMA-17、hBCMA-19、hBCMA-20、hBCMA-22、hBCMA-23均强于阳性对照huBCMA-10，在效靶比为1：1下，对靶细胞的杀伤毒性大于20％，在效靶比为3：1下，对靶细胞的杀伤毒性大于70％。
实施例7 BCMA CAR-T细胞体外杀伤后的IFN-γ分泌情况
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1:3，然后固定每个96孔的效应细胞数2×10 4个，依次加入靶细胞和效应细胞，每组每个效靶比下设置3个重复孔，另外每组均需设置3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24h左右，将96孔板离心，收集各个孔的上层清液(若不立即检测，需将上层清液保存于-80℃冰箱中)。
Elisa法检测IFN-γ细胞因子分泌(R&D，DY285)，方法如下：
(1)提前将试剂盒中的IFN-γ捕获抗体加入96孔中室温包被过夜；
(2)包被后的96孔板用缓冲液清洗后，加入封闭液，室温封闭1h，然后用缓冲液清洗后待用。
(3)按照说明书，将试剂盒中的标准品做梯度稀释，同时将收集的待测上层清液做5-30倍的稀释，然后将稀释后的标准品和待检样本加入到上述处理后的96孔板中，室温孵育2h。
(4)用缓冲液清洗各孔后，加入试剂盒中的IFN-γ检测抗体，室温孵育2h。
(5)用缓冲液清洗各孔后，加入试剂盒中Streptavidin-HRP，室温避光孵育20min。
(6)用缓冲液清洗各孔后，加入底物液TMB(配方见R&D，DY285)，室温避光孵育20min。
(7)加入终止液(配方见R&D，DY285)，于酶标仪上读取OD450对应的数值。
各试剂，包括IFN-γ标准品、捕获抗体、检测抗体、Streptavidin-HRP等的工作浓度，详见说明书(R&D，DY285)，其余试剂，包括缓冲液、封闭液、稀释液、底物液、终止液等的配方，详见R&D，DY285。
最后根据四参数拟合法得到标准曲线和相应的拟合公式，由拟合公式和待测样本的稀释倍数，计算出待测样本中IFN-γ的浓度。
如图5所示，在没有MM1s细胞刺激下，各组CAR-T细胞均没有IFN-γ分泌，而在MM1s细胞刺激下，与本申请选取的阳性对照huBCMA-10(专利号：US 2017/0226216A1)相比，本专利申请的hBCMA-17、hBCMA-19、hBCMA-20、hBCMA-22、hBCMA-23在杀伤 靶细胞时能产生更多的IFN-γ分泌，分泌量均在1500pg/mL以上，甚至达到2000pg/mL。
实施例8 BCMA CAR-T细胞的靶向增殖能力
由于MM1s细胞经γ射线辐照后存活率极低，所以本实施例CAR-T细胞的靶向增殖能力检测选用未经处理的MM1s细胞作为靶细胞。
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-VIVO 15培养基)。
将CAR-T细胞和MM1s细胞，以效靶比1:1的比例进行第一轮靶向刺激，在共孵育4-5天后，收集所有细胞，取样计数，再按照0.5:1(总CAR-T细胞:新的MM1s细胞)的比例，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-VIVO 15培养基。
如图6所示，BCMA CAR-T细胞经三轮靶向刺激后，本申请的hBCMA-17、hBCMA-19、hBCMA-20、hBCMA-22和hBCMA-23靶向增殖倍数均较高。
实施例9 L6-Li CAR结构下的BCMA CAR-T细胞的体外靶向杀伤活性和杀伤后的IFN-γ分泌情况
如实施例2所述，将hBCMA-22和hu-BCMA-10的scFv的核酸序列构建到含L6-Li的CAR慢病毒载体上，得到hBCMA-22-L6-Li和hu-BCMA-10-L6-Li的慢病毒载体。经慢病毒包装、病毒滴度测定，CAR-T细胞体外扩增后、于D8-D12期间进行CAR-T细胞的体外杀伤活性和杀伤后的IFN-γ分泌量的检测，具体操作方法参见实施例3-7。
如图7所示，hBCMA-22-L6-Li和hu-BCMA-10-L6-Li的CAR-T细胞对于MM1s细胞，在三个效靶比下的特异性杀伤能力基本一致。在效靶比为3：1下，CAR-T细胞对于靶细胞的杀伤毒性约为90％；在效靶比为1：1下，CAR-T细胞的杀伤毒性约为40％。同时如图8所示，这两组CAR-T细胞在与MM1s细胞共培养后的IFN-γ分泌能力，也基本趋于一致。在效靶比为1：1下，CAR-T细胞与靶细胞共培养48h后，可刺激IFN-γ分泌量约为5000pg/mL；在效靶比为1：6下，CAR-T细胞与靶细胞共培养48h后，可刺激IFN-γ分泌量约为17500pg/mL。
实施例10 hBCMA-22-L6-Li CAR结构下的BCMA CAR-T细胞对荷瘤小鼠的抑制消瘤能力和在小鼠体内的代谢情况
取6周龄的NSG小鼠，每只皮下接种1×10 7MM1s细胞，14天后小鼠皮下瘤体积大小 约150-250mm 3，将小鼠分成2组(每组8只小鼠)，分别接种新鲜制备hBCMA-22-L6-Li组的CAR-T细胞(2×10 6CAR +/只)和阴性对照T细胞(T细胞组每只小鼠接种细胞数与hBCMA-22-L6-Li组中2×10 6CAR +对应的总细胞数一致)，并定义为CAR-T注射后第0天。
接种完CAR-T细胞后，每周2次游标卡尺测量肿瘤大小，称量小鼠体重，并观察小鼠毛色、排泄物、饮食进水、躯体运动以及小鼠死亡情况。在CAR-T注射后第7天、14天、21天对各组的5只小鼠进行尾静脉取血，分别检测细胞因子分泌(Th1/2/17CBA检测IFN-γ和IL-2细胞因子)和人T细胞比例(hCD3和hCD8)，当肿瘤体积大于3000mm 3后处死小鼠，在第90天时处死余下所有小鼠，结束实验。
10.1流式检测方法检测小鼠外周血中的hCD3和hCD8分子标志物
(1)用10ml人用10ml人EDTA.K2采血管(江苏康捷医疗器械有限公司)，500ul PBS注入，混匀后，从中取出20ul至相应的1.5ml EP管内；
(2)小鼠尾静脉采血30ul，即总共50ul；
(3)3000rpm，离心10分钟；取上清15ul测CBA(若不当即测，则一定要保存于-80℃冰箱中)；
(4)在剩余的35ul里，补加40ul PBS，再次充分混匀，并转移至流式管内；
(5)加抗体APC-Anti-Human-CD3抗体(BD，555335)和Percp-cy5.5-Anti-Human-CD8抗体(BD，341051)；
(6)涡旋仪低速混匀后，4℃冰箱孵育30分钟；
(7)无需洗涤直接加2ml红细胞裂解液(BD，34902)，涡旋仪低速混匀后，室温避光孵育10分钟；
(8)500g，离心4分钟，倒扣去上清；
(9)加入实施例4中的NBS溶液2ml，涡旋仪低速混匀；
(10)500g，离心4分钟；
(11)用200ul NBS溶液重悬细胞沉淀，混匀准备流式上机检测。
10.2 TH1/2/17 CBA(BD，560484)检测细胞因子(IFN-γ和IL-2)方法
(1)根据样本数，计算所需要的beads数；
(2)剧烈涡旋每一个捕获磁珠悬液管(A1-A7)至少1分钟；
(3)取1.5ml EP管，标记为TH1/2/17 beads；
(4)取所需的A1-A7 beads数至EP管内；
(5)混合好的beads悬液涡旋1分钟，充分混匀；
(6)300g，离心5分钟；
(7)去除上清，用等体积的Serum Enhancement Buffer(试剂盒自带)重悬TH1/2/17 beads；
(8)涡旋1分钟后，室温避光孵育30分钟；
(9)取新的EP管，加入50ul小鼠血浆样本，再加入50ul的TH1/2/17 beads，再加入50ul PE-detection Regent(试剂盒自带)；
(10)涡旋30s后，室温避光孵育3小时；
(11)加500ul PBS，涡旋混匀后，300g，离心5分钟；
(12)去上清，用200ul PBS重悬，充分涡旋后，转至流式管，等待上机检测。
如图9的A和B所示，hBCMA-22-L6-Li组的CAR-T细胞注射荷瘤小鼠后，肿瘤体积继续增大，并在D8时达到最大，与此时Mock T组的平均肿瘤体积接近。在大约2周时间，hBCMA-22-L6-Li组开始消瘤，在4周左右肿瘤完全消失，而Mock T组在第三周时，肿瘤体积已升至3000mm 3以上。
图10的A和B显示了hBCMA-22-L6-Li组的CAR-T细胞(以hCD3 +细胞为例，hCD8 +细胞比例变化趋势与hCD3 +的基本一致，数据未显示)在荷瘤小鼠体内的增殖代谢情况。不同小鼠体内CAR-T细胞的快速扩增起始点和峰值点并不相同(hCD3峰值点可能出现在14天到21天之间)，从平均水平来看，在CAR-T注射第14天时，hCD3 +T细胞含量均值在40％左右，之后逐步下降，在第28天时，hCD3 +T细胞含量均值降低至5％以下。在4个检测时间点下没有检测到Mock T组的T细胞增殖。
在抑瘤消瘤同时，小鼠外周血中检测到大量的IFN-γ和少量的IL-2细胞因子(未能检测到IL-4、IL-6、IL-10、TNF-α、IL17A细胞因子分泌，数据未显示)，如图11的A所示，不同小鼠体内的IFN-γ细胞因子分泌起始点和峰值点也很不相同，并且结合图10的A可知，IFN-γ细胞因子分泌高峰均提前于CAR-T细胞增殖高峰，在第21天时，其含量降低至3pg/ml以下。在4个检测时间点下没有检测到Mock T组的IFN-γ分泌。
IL-2细胞因子的分泌水平明显低于IFN-γ(图12)，在CAR-T注射第7天时达到峰值(均值在50pg/ml)，之后迅速下降。
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 (86)

  1. 嵌合抗原受体(CAR)，其包含BCMA结合结构域，所述BCMA结合结构域包含特异性结合BCMA的抗体或其抗原结合片段，其中所述抗体包含重链互补决定区3(HCDR3)，且所述HCDR3包含如SEQ ID NO:25所示的氨基酸序列。
  2. 根据权利要求1所述的CAR，其中所述抗体包含重链互补决定区2(HCDR2)，且所述HCDR2包含如SEQ ID NO:41所示的氨基酸序列。
  3. 根据权利要求2所述的CAR，其中所述HCDR2包含如SEQ ID NO:22-24中任一项所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的CAR，其中所述抗体包含重链互补决定区1(HCDR1)，且所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的CAR，其中所述抗体包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含SEQ ID NO:41所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:25所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的CAR，其中所述抗体包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22-24中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:25所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的CAR，其中所述抗体包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3可包含选自以下任一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:22和HCDR3：SEQ ID NO:25；
    (2)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:23和HCDR3：SEQ ID NO:25；和，
    (3)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:24和HCDR3：SEQ ID NO:25。
  8. 根据权利要求1-7中任一项所述的CAR，其中所述抗体包含重链可变区VH，所述VH包括框架区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:42所示的氨基酸序列。
  9. 根据权利要求8所述的CAR，其中所述H-FR1包含SEQ ID NO:26-29中任一项所示的氨基酸序列。
  10. 根据权利要求8-9中任一项所述的CAR，其中所述VH包括框架区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:43所示的氨基酸序列。
  11. 根据权利要求10所述的CAR，其中所述H-FR2包含SEQ ID NO:30-32中任一项所示的氨基酸序列。
  12. 根据权利要求8-11中任一项所述的CAR，其中所述VH包括框架区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:44所示的氨基酸序列。
  13. 根据权利要求12所述的CAR，其中所述H-FR3包含SEQ ID NO:33-36中任一项所示的氨基酸序列。
  14. 根据权利要求8-13中任一项所述的CAR，其中所述VH包括框架区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:37所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的CAR，其中所述抗体包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:42所示的氨基酸序列，所述H-FR2包含SEQ ID NO:43所示的氨基酸序列，所述H-FR3包含SEQ ID NO:44所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:37所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的CAR，其中所述H-FR1包含SEQ ID NO:26-29中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:30-32中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:33-36中任一项所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:37所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的CAR，其中所述H-FR1、H-FR2、H-FR3和H-FR4包含选自以下任一组氨基酸序列：
    (1)H-FR1：SEQ ID NO:26，H-FR2：SEQ ID NO:30，H-FR3：SEQ ID NO:33和H-FR4：SEQ ID NO:37；
    (2)H-FR1：SEQ ID NO:27，H-FR2：SEQ ID NO:31，H-FR3：SEQ ID NO:34和H-FR4：SEQ ID NO:37；
    (3)H-FR1：SEQ ID NO:28，H-FR2：SEQ ID NO:32，H-FR3：SEQ ID NO:35和H-FR4：SEQ ID NO:37；和
    (4)H-FR1：SEQ ID NO:29，H-FR2：SEQ ID NO:30，H-FR3：SEQ ID NO:36和H-FR4：SEQ ID NO:37。
  18. 根据权利要求1-17中任一项所述的CAR，其中所述抗体包含重链可变区VH，且所述VH包含SEQ ID NO:46所示的氨基酸序列。
  19. 根据权利要求18中任一项所述的CAR，其中所述VH包含SEQ ID NO:5-9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CAR，其中所述抗体包含轻链互补决定区3(LCDR3)，且所述LCDR3包含如SEQ ID NO:40所示的氨基酸序列。
  21. 根据权利要求20所述的CAR，其中所述LCDR3包含如SEQ ID NO:15-16中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的CAR，其中所述抗体包含轻链互补决定区2(LCDR2)，且所述LCDR2包含如SEQ ID NO:39所示的氨基酸序列。
  23. 根据权利要求22所述的CAR，其中所述LCDR2包含如SEQ ID NO:13-14中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的CAR，其中所述抗体包含轻链互补决定区1(LCDR1)，且所述LCDR1包含如SEQ ID NO:38所示的氨基酸序列。
  25. 根据权利要求24所述的CAR，其中所述LCDR1包含如SEQ ID NO:10-12中任一项所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的CAR，其中所述抗体包含LCDR1、LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:38所示的氨基酸序列，所述LCDR2包含SEQ ID NO:39所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:40所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的CAR，其中所述抗体包含LCDR1、LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:10-12中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:13-14中任一项所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:15-16中任一项所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的CAR，其中所述抗体包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3选自以下任一组氨基酸序列：
    (1)LCDR1：SEQ ID NO:10，LCDR2：SEQ ID NO:13和LCDR3：SEQ ID NO:15；
    (2)LCDR1：SEQ ID NO:11，LCDR2：SEQ ID NO:13和LCDR3：SEQ ID NO:16；
    (3)LCDR1：SEQ ID NO:12，LCDR2：SEQ ID NO:13和LCDR3：SEQ ID NO:16；和
    (4)LCDR1：SEQ ID NO:12，LCDR2：SEQ ID NO:14和LCDR3：SEQ ID NO:16。
  29. 根据权利要求1-28中任一项所述的CAR，其中所述抗体包含轻链可变区VL，所述VL包括框架区L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:17所示的氨基酸序列。
  30. 根据权利要求29所述的CAR，其中所述VL包括框架区L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:18所示的氨基酸序列。
  31. 根据权利要求29-30中任一项所述的CAR，其中所述VL包括框架区L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:19所示的氨基酸序列。
  32. 根据权利要求29-31中任一项所述的CAR，其中所述VL包括框架区L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:20所示的氨基酸序列。
  33. 根据权利要求1-32中任一项所述的CAR，其中所述抗体包含L-FR1、L-FR2、L-FR3和L-FR4，其中所述L-FR1包含SEQ ID NO:17所示的氨基酸序列，所述L-FR2包含SEQ ID NO:18所示的氨基酸序列，所述L-FR3包含SEQ ID NO:19所示的氨基酸序列，且
    所述L-FR4包含SEQ ID NO:20所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的CAR，其中所述抗体包含轻链可变区VL，且所述VL包含SEQ ID NO:45所示的氨基酸序列。
  35. 根据权利要求34中所述的CAR，其中所述VL包含SEQ ID NO:1-4中任一项所示的氨基酸序列。
  36. 根据权利要求1-35中任一项所述的CAR，其中所述抗体包含VH和VL，且所述VH包含SEQ ID NO:46所示的氨基酸序列，所述VL包含SEQ ID NO:45所示的氨基酸序列。
  37. 根据权利要求1-36中任一项所述的CAR，其中所述抗体包含VH和VL，且所述VH包含SEQ ID NO:5-9中任一项所示的氨基酸序列，所述VL包含SEQ ID NO:1-4中任一项所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的CAR，其中所述抗体包含VH和VL，且所述VH和VL选自以下任一组氨基酸序列：
    (1)VH：SEQ ID NO:5和VL：SEQ ID NO:1；
    (2)VH：SEQ ID NO:6和VL：SEQ ID NO:2；
    (3)VH：SEQ ID NO:7和VL：SEQ ID NO:2；
    (4)VH：SEQ ID NO:8和VL：SEQ ID NO:2；
    (5)VH：SEQ ID NO:9和VL：SEQ ID NO:3；
    (6)VH：SEQ ID NO:8和VL：SEQ ID NO:3；
    (7)VH：SEQ ID NO:9和VL：SEQ ID NO:4；和
    (8)VH：SEQ ID NO:8和VL：SEQ ID NO:4。
  39. 根据权利要求1-38中任一项所述的CAR，其中所述BCMA结合结构域包含scFv，且所述scFv包含所述特异性结合BCMA的抗体的重链可变区VH和轻链可变区VL。
  40. 根据权利要求39所述的CAR，其中所述重链可变区VH的C端和所述轻链可变区VL的N端直接或间接连接。
  41. 根据权利要求39-40中任一项所述的CAR，其中所述重链可变区VL的C端和所述轻链可变区VL的N端直接或间接连接。
  42. 根据权利要求39-41中任一项所述的CAR，其中所述scFv在VH和VL之间包含连接肽，且所述连接肽包含SEQ ID NO:58所示的氨基酸序列。
  43. 根据权利要求39-42中任一项所述的CAR，其中所述scFv包含SEQ ID NO:49-56中任一项所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的CAR，其包含跨膜结构域，且所述跨膜结构域包含选自源自下述蛋白的跨膜结构域：CD28、CD3e、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  45. 根据权利要求44所述的CAR，其中所述跨膜结构域源自CD8，且所述跨膜结构域包含SEQ ID NO:61所示的氨基酸序列。
  46. 根据权利要求1-45中任一项所述的CAR，其包含共刺激结构域，且所述共刺激结构域包含选自源自下述蛋白的共刺激结构域：CD137、CD28、4-1BB、OX-40和ICOS。
  47. 根据权利要求46所述的CAR，其中所述共刺激结构域源自CD137，且所述共刺激结构域包含SEQ ID NO:62所示的氨基酸序列。
  48. 根据权利要求1-47中任一项所述的CAR，其包含胞内信号传导结构域，且所述胞内信号传导结构域包含源自CD3ζ的胞内信号传导结构域。
  49. 根据权利要求48所述的CAR，其中所述胞内信号传导结构域包含SEQ ID NO:63所示的氨基酸序列。
  50. 根据权利要求1-49中任一项所述的CAR，其包含铰链区，且所述铰链区包含源自CD8的铰链区。
  51. 根据权利要求50所述的CAR，其中所述铰链区源自CD8，且所述铰链区包含SEQ ID NO:60所示的氨基酸序列。
  52. 根据权利要求1-51中任一项所述的CAR，其包含信号肽。
  53. 根据权利要求52所述的CAR，其中所述信号肽包含SEQ ID NO:59所示的氨基酸序列。
  54. 根据权利要求1-53中任一项所述的CAR，其包含L6-Li，所述L6-Li位于所述胞内信号传导结构域的C端。
  55. 根据权利要求54所述的CAR，其中所述L6-Li包含SEQ ID NO:107所示的氨基酸序列。
  56. 根据权利要求1-55中任一项所述的CAR，其包含SEQ ID NO:64-71和74中任一项所示的氨基酸序列。
  57. 根据权利要求1-56x中任一项所述的CAR，其包含SEQ ID NO:95-102和105中任一项所示的核苷酸序列。
  58. 分离的核酸分子，其包含编码权利要求1-57中任一项所述的CAR的核苷酸序列。
  59. 根据权利要求58所述的分离的核酸分子，其还包含启动子，所述启动子位于编码所述的CAR的核苷酸序列的5’端。
  60. 根据权利要求58-59中任一项所述的分离的核酸分子，其中所述启动子为组成型启动子。
  61. 根据权利要求58-60中任一项所述的分离的核酸分子，其中所述启动子为EF1α启动子。
  62. 编码CAR的核酸分子，其包含SEQ ID NO:95-102和105中任一项所示的核苷酸序列。
  63. 载体，其包含权利要求58-62中任一项所述的核酸分子。
  64. 根据权利要求63所述的载体，其中所述载体选自质粒、逆转录病毒载体和慢病毒载体。
  65. 免疫效应细胞，其包含权利要求1-57中任一项所述的CAR，权利要求58-62中任一项所述的核酸分子，和/或权利要求63-64中任一项所述的载体。
  66. 根据权利要求65所述的细胞，其中所述免疫效应细胞选自T淋巴细胞。
  67. 制备免疫效应细胞的方法，其包括向所述免疫效应细胞中引入权利要求63-64中任一项所述的载体。
  68. 药物组合物，其包含权利要求1-57中任一项所述的CAR，权利要求58-62中任一项所述的核酸分子，权利要求63-64中任一项所述的载体，和/或权利要求65-66中任一项所述的免疫效应细胞，以及药学上可接受的载剂。
  69. 权利要求1-57中任一项所述的CAR，权利要求58-62中任一项所述的核酸分子，权利要求63-64中任一项所述的载体，权利要求65-66中任一项所述的免疫效应细胞，和/或权利要求68所述的药物组合物在制备药物中的用途，其中所述药物用于治疗与BCMA表达相关的疾病或病症。
  70. 根据权利要求69所述的用途，其中所述与BCMA表达相关的疾病或病症为癌症或恶性肿瘤。
  71. 根据权利要求69-70中任一项所述的用途，其中所述与BCMA表达相关的疾病或病症为BCMA阳性肿瘤。
  72. 根据权利要求69-71中任一项所述的用途，其中所述与BCMA表达相关的疾病或病症为实体瘤和/或非实体瘤。
  73. 根据权利要求69-72中任一项所述的用途，其中所述与BCMA表达相关的疾病或病症为骨髓瘤。
  74. 根据权利要求69-73中任一项所述的用途，其中所述与BCMA表达相关的疾病或病症为多发性骨髓瘤。
  75. 治疗与BCMA表达相关的疾病或病症的方法，其包括向有需要的受试者使用权利要求1-57中任一项所述的CAR，权利要求58-62中任一项所述的核酸分子，权利要求63-64中任一项所述的载体，权利要求65-66中任一项所述的免疫效应细胞，和/或权利要求68所述的药物组合物。
  76. 根据权利要求75所述的方法，其中所述与BCMA表达相关的疾病或病症为癌症或恶性肿瘤。
  77. 根据权利要求75-76中任一项所述的方法，其中所述与BCMA表达相关的疾病或病症为BCMA阳性肿瘤。
  78. 根据权利要求75-77中任一项所述的方法，其中所述与BCMA表达相关的疾病或病症为实体瘤和/或非实体瘤。
  79. 根据权利要求75-78中任一项所述的方法，其中所述与BCMA表达相关的疾病或病症为骨髓瘤。
  80. 根据权利要求75-79中任一项所述的方法，其中所述与BCMA表达相关的疾病或病症为多发性骨髓瘤。
  81. 权利要求1-57中任一项所述的CAR，权利要求58-62中任一项所述的核酸分子，权利要求63-64中任一项所述的载体，权利要求65-66中任一项所述的免疫效应细胞，和/或权利要求68所述的药物组合物，其用于治疗与BCMA表达相关的疾病或病症。
  82. 根据权利要求81所述的CAR，核酸分子，载体，免疫效应细胞，和/或药物组合物，其中所述与BCMA表达相关的疾病或病症为癌症或恶性肿瘤。
  83. 根据权利要求81-82中任一项所述的CAR，核酸分子，载体，免疫效应细胞，和/或药物组合物，其中所述与BCMA表达相关的疾病或病症为BCMA阳性肿瘤。
  84. 根据权利要求81-83中任一项所述的CAR，核酸分子，载体，免疫效应细胞，和/或药物组合物，其中所述与BCMA表达相关的疾病或病症为实体瘤和/或非实体瘤。
  85. 根据权利要求81-84中任一项所述的CAR，核酸分子，载体，免疫效应细胞，和/或药物组合物，其中所述与BCMA表达相关的疾病或病症为骨髓瘤。
  86. 根据权利要求81-85中任一项所述的CAR，核酸分子，载体，免疫效应细胞，和/或药物组合物，其中所述与BCMA表达相关的疾病或病症为多发性骨髓瘤。
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