WO2023024084A1 - 一种嵌合抗原受体及其用途 - Google Patents

一种嵌合抗原受体及其用途 Download PDF

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WO2023024084A1
WO2023024084A1 PCT/CN2021/115057 CN2021115057W WO2023024084A1 WO 2023024084 A1 WO2023024084 A1 WO 2023024084A1 CN 2021115057 W CN2021115057 W CN 2021115057W WO 2023024084 A1 WO2023024084 A1 WO 2023024084A1
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seq
acid sequence
amino acid
car
sequence shown
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PCT/CN2021/115057
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French (fr)
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郭昊
陈思晔
何晓文
李慧姣
凌有国
杨月
徐艳红
杨棋
许志锋
李霄培
杨焕凤
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原启生物科技(上海)有限责任公司
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Priority to PCT/CN2021/115057 priority Critical patent/WO2023024084A1/zh
Priority to CA3229688A priority patent/CA3229688A1/en
Publication of WO2023024084A1 publication Critical patent/WO2023024084A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/53—Liver

Definitions

  • This application relates to the field of biomedicine, in particular to a chimeric antigen receptor and its application.
  • liver cancer is a malignant tumor that has a wide range of hazards.
  • Liver cancer caused by hepatitis B virus has the characteristics of a long incubation period. Once discovered, it is often at an advanced stage, and it progresses rapidly after onset, and the prognosis is poor.
  • Glypican 3 (glypican3, GPC3) is a heparan sulfate proteoglycan on the surface of the cell membrane, which exists in a variety of tumors, especially in liver cancer.
  • CTLs cytotoxic lymphocytes
  • TCRs T lymphocyte receptors
  • the scFv is fused with the T lymphocyte receptor to form a chimeric antigen receptor (CAR), which is genetically modified on the surface of T lymphocytes by means such as lentivirus infection for the treatment of tumors.
  • CAR-T lymphocyte can selectively direct T lymphocytes to tumor cells and specifically kill tumor cells in a non-restricted manner of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • adoptive immunotherapy based on immune effector cells has achieved certain effects in some tumors, but the efficacy in most tumors is still unsatisfactory.
  • the application provides a chimeric antigen receptor (CAR), the CAR comprises a GPC3 binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, and the GPC3 binding domain comprises a specific binding
  • the antibody of GPC3 or its fragment wherein said antibody comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3)
  • said amino acid sequence of said LCDR1 is as SEQ ID NO: 16
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 17
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 18.
  • the antibody comprises a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 3 (HCDR3)
  • HCDR1 is as SEQ ID As shown in NO: 19
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 20
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 21.
  • the antibody comprises a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 29.
  • the antibody comprises a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 25.
  • the antibody is a single chain antibody.
  • the antibody comprises the amino acid sequence shown in SEQ ID NO:30.
  • the transmembrane domain comprises a transmembrane domain derived from a protein 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 comprises the amino acid sequence shown in SEQ ID NO:36.
  • the costimulatory domain comprises a costimulatory domain of a protein selected from the group consisting of CD137, CD28, 4-1BB, OX-40, and ICOS.
  • the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:37.
  • the intracellular signaling domain comprises a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:38.
  • the CAR further comprises a hinge region connecting the GPC3 binding domain and the transmembrane domain.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:39.
  • the CAR is also linked to a signal peptide.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:40.
  • the nucleic acid molecule encoding the CAR is also linked to a promoter.
  • the promoter is a constitutive promoter.
  • the promoter comprises the nucleotide sequence shown in SEQ ID NO:41.
  • the CAR comprises the amino acid sequence shown in any one of SEQ ID NOs: 31-34.
  • the application also provides isolated nucleic acid molecules encoding the CARs described herein.
  • the isolated nucleic acid molecule encoding CAR comprises the nucleic acid sequence shown in any one of SEQ ID NOs: 42-45.
  • the present application also provides a vector comprising the nucleic acid molecule described in the present application.
  • the vector is selected from plasmids, retroviral vectors and lentiviral vectors.
  • the present application also provides an immune effector cell comprising 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 immune effector cells are selected from T lymphocytes.
  • the present application also provides a method for preparing the immune effector cells described in the present application, the method comprising introducing the carrier described in the present application into the immune effector cells described in the present application.
  • the present application also provides a composition comprising the immune effector cells described in the present application.
  • the present application also provides the use of the CAR described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, or the immune effector cells described in the present application for the preparation of medicines, wherein the medicines are used for the treatment of GPC3 Expression-related diseases or conditions.
  • the present application also provides a method for treating a disease or disease related to the expression of GPC3, the method comprising administering the CAR described in the present application, the nucleic acid molecule described in the present application, the nucleic acid molecule described in the present application to a patient or subject.
  • Vector or the immune effector cells described in this application are described in this application.
  • the present application also provides the CAR, the nucleic acid molecule, the carrier or the immune effector cell, which are used to treat diseases or disorders related to the expression of GPC3.
  • the disease or condition associated with expression of GPC3 is cancer or malignancy.
  • Figure 1 shows the ligation sequence of each part of the chimeric antigen receptor in the CAR plasmid.
  • Figure 2 shows the expansion factor of 204A-BBz infected T cells.
  • Figure 3 shows the expansion factor of L1H2-BBz/L1H6-BBz/L2H6-BBz/GC33-BBz infected T cells.
  • Figure 4 shows the killing effect of 204A-CAR-T on HepG2 cells.
  • Figure 5 shows the killing effect of L1H2/L1H6/L2H6-CAR-T on HepG2 cells.
  • Figure 6 shows the amplification factor of 204A-CAR-T after repeated stimulation of Huh7 target cells.
  • Figure 7 shows the amplification factor of L1H2/L1H6/L2H6-CAR-T by repeated stimulation of Huh7 target cells.
  • 8A-8C show the comparison of tumor elimination, tumor suppression and recurrence prevention in L1H2-BBz and GC33-BBz mice.
  • Figures 9A-9C show body weight changes in L1H2-BBz and GC33-BBz mice.
  • Figure 10 shows the changes of cytokines in mice treated with L1H2-BBz and GC33-BBz.
  • Figures 11A-11B show the changes in cell levels in mice treated with L1H2-BBz and GC33-BBz (A is CD8+ cells; B is CD4+ cells).
  • Figure 12 shows the survival curves of L1H2-BBz and GC33-BBz treated mice.
  • chimeric antigen receptor generally refers to the antigen-binding region of an antibody that recognizes a tumor associated antigen (TAA) and the intracellular signaling domain "immunoreceptor tyrosine activation motif (immunoreceptor tyrosine-based activation motifs, ITAM, usually CD3 ⁇ or Fc ⁇ RI ⁇ )" fusion antigen receptor.
  • the basic structure of CAR includes a tumor-associated antigen (tumor-associated antigen, TAA) binding region (usually derived from the scFv of the monoclonal antibody antigen-binding region), an extracellular hinge region (Hinge area), a transmembrane region (Transmembrane region) ) and an intracellular immunoreceptor tyrosine-based activation motif (Immunoreceptor tyrosine-based activation motif, ITAM).
  • TAA tumor-associated antigen binding region
  • Hinge area extracellular hinge region
  • Transmembrane region Transmembrane region
  • ITAM intracellular immunoreceptor tyrosine-based activation motif
  • GPC3 generally refers to glypican 3 (glypican 3, GPC3), which is a heparan sulfate proteoglycan on the surface of cell membranes, present in a variety of tumors, in liver cancer is especially common in .
  • GPC3 protein is highly expressed in fetal liver. Abnormal expression of GPC3 protein after birth is closely related to the occurrence and development of tumors.
  • GPC3 protein is highly expressed in primary liver cancer (PHC), but low or moderately expressed in other tumors or benign liver diseases.
  • PLC primary liver cancer
  • a GPC3 protein described herein can comprise a human GPC3 protein.
  • binding domain generally refers to (specifically) binding to a given target epitope or a given target site on a target molecule (antigen), or The target site interacts, or recognizes said given target epitope or a domain of a given target site.
  • the term "specific binding” generally refers to a measurable and reproducible interaction, such as between a target and an antibody, which can be determined in the presence of a heterogeneous population of molecules, including biomolecules. The presence.
  • an antibody that specifically binds a target is one that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets.
  • an antibody specifically binds an epitope on a protein that is conserved among proteins of different species.
  • specific binding can include, but does not require exclusive binding.
  • CDR generally refers to regions of antibody variable domains, also known as complementarity determining regions, the sequence of which is highly variable and/or forms structurally defined loops.
  • antibodies typically comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • HCDR3 and LCDR3 display most of the diversity of the six CDRs, and HCDR3 in particular is thought to play a unique role in conferring the fine specificity of antibodies.
  • variable region generally refers to the amino-terminal domain of an antibody heavy or light chain.
  • the variable regions of the heavy and light chains may be referred to as the heavy chain variable region (VH) and the light chain variable region (VL), respectively. These regions are usually the most variable parts of the antibody (relative to other antibodies of the same class) and comprise the antigen binding site.
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining 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 , mutated and transplanted antibodies.
  • the term “antibody” also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind GPC3). Typically, such fragments will include the antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 that can be combined with the J chain to form a multivalent A basic 4-chain unit for combinations.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site.
  • immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • the gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl and IgKl.
  • single-chain antibody refers to a single-chain polypeptide containing one or more antigen-binding sites.
  • H and L chains of Fv fragments are encoded by different genes, they can be linked together directly or through peptides, for example, by recombinant methods, synthetic linkers can be used to link the H and L chains into a single protein chain (called a single-chain antibody, sAb; Bird et al. 1988 Science 242: 423-426; and Huston et al. 1988 PNAS 85: 5879-5883).
  • sAb single-chain antibody
  • Bird et al. 1988 Science 242: 423-426 Bird et al. 1988 Science 242: 423-426
  • Huston et al. 1988 PNAS 85: 5879-5883 Such single chain antibodies are also included within the term “antibody” and can be used as binding determinants in the design and manufacture of multispecific binding molecules.
  • transmembrane domain generally refers to a polypeptide or protein encoded at the DNA level by at least one exon comprising an extracellular region, a transmembrane region and an intracellular region.
  • the transmembrane domain generally consists of three distinct structural regions: the N-terminal extracellular region, the middle conserved transmembrane extension region, and the C-terminal cytoplasmic region. Transmembrane domains may also contain intracellular or cytoplasmic regions.
  • co-stimulation generally refers to the source of a secondary signal for lymphocyte activation, usually by co-stimulatory molecules on the surface of immune cells (T cell/B cell or antigen presenting cell/T cell) involved in adaptive immunity produced by interaction with its receptors.
  • T cell/B cell or antigen presenting cell/T cell immune cells
  • the full activation of T cells depends on the action of dual signals 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 co-stimulatory molecules, that is, co-stimulatory molecules on APC and T cell surface corresponding receptor interactions.
  • co-stimulatory domain generally refers to any amino acid sequence capable of interacting with a co-stimulatory molecule to produce co-stimulation.
  • intracellular signaling domain generally refers to the intracellular portion of a molecule.
  • the intracellular signaling domain generates signals that promote immune effector functions of CAR-containing cells, such as CART cells. Examples of immune effector functions in, eg, CART cells include cytolytic activity and accessory activities, including secretion of cytokines.
  • the intracellular signaling domain transduces effector function signals and directs the cell to perform specialized functions. While the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain. To the extent that truncated portions of intracellular signaling domains are used, such truncated portions can be used in place of the intact chain, so long as they transduce effector function signals.
  • the term intracellular signaling domain is thus intended to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.
  • CD i.e. Cluster of differentiation, also known as differentiation group
  • CD molecules have many uses, often serving as important receptors or ligands for cells. Some CDs can participate in the signaling cascade of cells to change the behavior of cells, while some CD proteins are not related to cell signaling, but have other functions, such as cell adhesion.
  • CD137 also known as 4-1BB
  • 4-1BB generally refers to a member of the tumor necrosis factor (TNF) receptor family, encoded by the tumor necrosis factor receptor superfamily member 9 (TNFRSF9) gene.
  • Human 4-1BB is located on chromosome 1, with a full length of 255 amino acids, including a signal peptide of 17 amino acids, an extracellular region of 169 amino acids, a transmembrane region of 27 amino acids and an intracellular region of 42 amino acids.
  • Mouse 4-1BB is located on mouse chromosome 4 and has approximately 60% sequence similarity with human 4-1BB.
  • CD137(4-1BB) is an inducible co-stimulator expressed on activated CD4+ and CD8+ T cells, NKTs, NK cells, DCs, macrophages, eosinophils, neutrophils and mast cells, as well as Tregs receptor.
  • CD3 ⁇ also written CD3zeta, generally refers to amino acid residues from the cytoplasmic domain of the ⁇ chain that are sufficient to functionally transmit the initial signal required for T cell activation.
  • the term "hinge region” generally refers to a region located between the scFv and the transmembrane domain in the CAR structure.
  • the hinge region is usually derived from the IgG family, such as IgG1 and IgG4, and some are derived from IgD and CD8.
  • the hinge region has a certain degree of flexibility, which affects the spatial constraints between the CAR molecule and its specific target, thereby affecting the contact between CAR T cells and tumor cells.
  • signal peptide generally refers to the prosegment present on the protein precursor form as an N-terminal peptide as used herein.
  • the function of the signal peptide is to facilitate the translocation of expressed polypeptides linked to the endoplasmic reticulum.
  • the signal peptide is usually cleaved during this process.
  • the signal peptide may be heterologous or homologous to the organism used to produce the polypeptide.
  • promoter generally refers to a deoxyribonucleic acid (DNA) sequence that enables the transcription of a specific gene.
  • the promoter can be recognized by RNA polymerase and start transcription to synthesize RNA.
  • RNA ribonucleic acid
  • promoters can interact with transcription factors that regulate gene transcription, controlling the initiation time and degree of gene expression (transcription).
  • the promoter includes the core promoter region and the regulatory region, and is located in the regulatory sequence controlling gene expression, upstream of the gene transcription start site (5' direction of the DNA antisense strand), and has no translation function itself.
  • constitutive promoters maintain continuous activity in most or all tissues
  • specific promoters tissue specific or developmental stage specific
  • inducible promoters response to External chemical or physical signal regulation
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.
  • 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.
  • the vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 phage and animal viruses.
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector may contain a variety of 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.
  • Vectors may also
  • immune effector cell generally refers to a cell that participates in an immune response, eg, promotes 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.
  • composition generally refers to a composition suitable for administration to a patient, a human patient.
  • the composition described herein may comprise the immune effector cells described herein, and optionally a pharmaceutically acceptable adjuvant.
  • acceptable ingredients of the compositions are nontoxic to recipients at the dosages and concentrations employed.
  • Compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • tumor generally refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • tumors may be solid tumors or hematological tumors.
  • the tumor may be a GPC3-positive tumor, wherein the GPC3-positive tumor may include liver cancer.
  • 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%, above or below the specified value. 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), which comprises a GPC3 binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, and the GPC3 binding domain comprises a specific An antibody or a fragment thereof that sexually binds GPC3, wherein the antibody comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), the amino acid sequence of the HCDR1 As shown in SEQ ID NO: 19, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 20 and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 21.
  • CAR chimeric antigen receptor
  • the antibody described in the present application may also comprise light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3), the amino acid sequence of said LCDR1 is as SEQ As shown in ID NO: 1, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 2 and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 3.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 light chain complementarity determining region 2
  • LCDR3 light chain complementarity determining region 3
  • the GPC3 is human GPC3, and the amino acid sequence of the GPC3 is shown in SEQ ID NO:56.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:19.
  • X 1 YX 2 MH (SEQ ID NO: 19), wherein X 1 can be D or A, and X 2 can be A or E.
  • the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:4 and 12.
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:20.
  • X 1 LX 2 X 3 X 4 X 5 GX 6 X 7 X 8 YX 9 X 10 X 11 X 12 X 13 G (SEQ ID NO:20), wherein X 1 can be G or A, X 2 can be S or D, X3 can be W or P, X4 can be N or K, X5 can be S or T, X6 can be S or Q, X7 can be I or T, X8 can be G or A, X 9 can be A or S, X 10 can be D or Q, X 11 can be S or K, X 12 can be V or F, X 13 can be K or Q.
  • the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:5, 10 and 13.
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 21.
  • X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 (SEQ ID NO:21), wherein X 1 can be D or T, X 2 can be H or R, and X 3 can be T or F, X4 can be I or Y, X5 can be G or S, X6 can be V or Y, X7 can be G or A, X8 can be A, Y or H, X9 can be is F or blank, X 10 can be D or blank, and X 11 can be I or blank.
  • the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:6, 11 and 14.
  • HCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:4, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.
  • HCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:12
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:11.
  • HCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:12
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:13
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16.
  • X 1 X 2 X 3 X 4 SX 5 VX 6 X 7 X 8 X 9 YX 10 X 11 X 12 X 13 (SEQ ID NO:16), wherein X 1 can be T or R, X 2 can be G or S , X3 can be T or S, X4 can be S or Q, X5 can be D or L, X6 can be G or H, X7 can be G or S, X8 can be Y or N, X 9 can be N or G, X 10 can be V or T, X 11 can be S or Y, X 12 can be blank or L, X 13 can be blank or H.
  • the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:1 and 7.
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:17.
  • X 1 X 2 SX 3 RX 4 S (SEQ ID NO: 17), wherein X 1 can be D or K, X 2 can be V or G, X 3 can be N, Y or Q, X 4 can be P or g.
  • the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:2, 8 and 15.
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 (SEQ ID NO:18), wherein X 1 can be S or G, X 2 can be S or Q, X 3 can be Y Or S, X4 can be A or G, X5 can be S or L, X6 can be G or T, X7 can be S or P, X8 can be T or P, X9 can be L or T , X 10 can be V or blank.
  • the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:3 and 9.
  • LCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:1
  • LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.
  • LCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:7
  • LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:8
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9.
  • LCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:7
  • LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:15
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9.
  • LCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:1
  • LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3
  • HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the LCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 7
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9
  • HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:12
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:11.
  • the LCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 7
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9
  • HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:12
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:13
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the LCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:7
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:15
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9
  • HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:12
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:13
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the chimeric antigen receptors described herein may comprise the light chain variable region VL of an antibody and the heavy chain variable region VH of an antibody.
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:25
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the VL of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:22-24.
  • the VH of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NOs: 26-28.
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:22
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:26.
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:23
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:27.
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:23
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:24
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the chimeric antigen receptor may comprise HCDR1 in the VH whose amino acid sequence is shown in SEQ ID NO:29.
  • the chimeric antigen receptor may comprise HCDR2 in the VH whose amino acid sequence is shown in SEQ ID NO:29.
  • the chimeric antigen receptor may comprise HCDR3 in the VH whose amino acid sequence is shown in SEQ ID NO:29.
  • the chimeric antigen receptor may comprise an amino acid sequence such as LCDR1 in VL shown in SEQ ID NO:25.
  • the chimeric antigen receptor may comprise LCDR2 in the VL with the amino acid sequence shown in SEQ ID NO:25.
  • the chimeric antigen receptor may comprise LCDR3 in the VL with the amino acid sequence shown in SEQ ID NO:25.
  • the GPC3-binding domain of the chimeric antigen receptor may include an antibody or an antigen-binding fragment thereof that specifically binds to GPC3.
  • the antibodies specifically binding to GPC3 or antigen-binding fragments thereof described in the present application may include, but are not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single-chain antibodies, Diabodies, triabodies, tetrabodies, Fv fragments, scFv fragments, Fab fragments, Fab' fragments, F(ab')2 fragments and camelized single domain antibodies.
  • the antibody may be a humanized antibody.
  • the antibody specifically binding to GPC3 or an antigen-binding fragment thereof described in the present application may immunospecifically bind to a related antigen (such as human GPC3) and comprise a framework (FR) that essentially has the amino acid sequence of a human antibody Regions and antibodies or variants, derivatives, analogs or fragments thereof having substantially the complementarity determining regions (CDRs) of the amino acid sequence of a non-human antibody.
  • FR framework
  • substantially in the context of a CDR means that the amino acid sequence of the CDR is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR .
  • the humanized antibody may comprise substantially all of at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to non-human
  • the CDR regions and all or substantially all of the framework regions of an immunoglobulin are framework regions having human immunoglobulin consensus sequences.
  • a humanized antibody contains a light chain as well as at least the variable domain of a heavy chain. In certain embodiments, a humanized antibody contains only a humanized heavy chain. In specific embodiments, a humanized antibody contains only the humanized variable domain of a light chain and/or a humanized heavy chain.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the GPC3 binding domain is a single chain antibody.
  • the GPC3 binding domain is a scFv.
  • the scFv may comprise the sequence shown in SEQ ID NO:30.
  • the GPC3 binding domain can comprise the light chain variable region of an antibody, a linker peptide, the heavy chain variable region of an antibody.
  • the light chain variable region of the antibody may comprise the amino acid sequence shown in SEQ ID NO: 25
  • the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 35
  • the heavy chain of the antibody may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the light chain variable region of the antibody may comprise the amino acid sequence shown in SEQ ID NO: 22
  • the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 35
  • the heavy chain variable region of the antibody Can comprise the aminoacid sequence shown in SEQ ID NO:26.
  • the light chain variable region of the antibody may comprise the amino acid sequence shown in SEQ ID NO: 23
  • the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 35
  • the heavy chain variable region of the antibody Can comprise the aminoacid sequence shown in SEQ ID NO:27.
  • the light chain variable region of the antibody may comprise the amino acid sequence shown in SEQ ID NO: 23
  • the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 35
  • the heavy chain variable region of the antibody Can comprise the aminoacid sequence shown in SEQ ID NO:28.
  • the light chain variable region of the antibody may comprise the amino acid sequence shown in SEQ ID NO: 24, the connecting peptide may comprise the amino acid sequence shown in SEQ ID NO: 35, the heavy chain variable region of the antibody Can comprise the aminoacid sequence shown in SEQ ID NO:28.
  • the GPC3 binding domain may comprise the amino acid sequence shown in SEQ ID NO:31.
  • the GPC3 binding domain may comprise the amino acid sequence shown in SEQ ID NO:32.
  • the GPC3 binding domain may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the GPC3 binding domain may comprise the amino acid sequence shown in SEQ ID NO:34.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO: 31-34.
  • Transmembrane domains Transmembrane domains, co-stimulatory domains, and intracellular signaling domains
  • the transmembrane domain may comprise a transmembrane domain selected from the following proteins: CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the co-stimulatory domain may comprise a co-stimulatory domain selected from the following proteins: CD137, CD28, 4-1BB, OX-40 and ICOS.
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the intracellular signaling domain may comprise a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the chimeric antigen receptor may sequentially comprise a GPC3 binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the GPC3 binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:31-34
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36
  • the structural domain may comprise the amino acid sequence shown in SEQ ID NO:37
  • the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the chimeric antigen receptor may sequentially comprise a GPC3 binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the GPC3 binding domain may comprise the amino acid sequence shown in SEQ ID NO:31
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the amino acid sequence shown in NO:37, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the chimeric antigen receptor may sequentially comprise a GPC3 binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the GPC3 binding domain may comprise the amino acid sequence shown in SEQ ID NO:32
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the amino acid sequence shown in NO:37, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the chimeric antigen receptor may sequentially comprise a GPC3 binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the GPC3 binding domain may comprise the amino acid sequence shown in SEQ ID NO:33
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the amino acid sequence shown in NO:37, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the chimeric antigen receptor may sequentially comprise a GPC3 binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the GPC3 binding domain may comprise the amino acid sequence shown in SEQ ID NO:34
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:36
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the amino acid sequence shown in NO:37, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the chimeric antigen receptor may further comprise a hinge region connecting the GPC3 binding domain and the transmembrane domain.
  • the hinge region is derived from the IgG family, eg, the hinge region is derived from IgG1.
  • the hinge region is derived from IgG4.
  • the hinge region is derived from IgD.
  • the hinge region is derived from CD8.
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO:39.
  • the chimeric antigen receptor may sequentially comprise a GPC3 binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus to the C-terminus.
  • the GPC3 binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:31-34
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO:39
  • the transmembrane domain Can comprise the amino acid sequence shown in SEQ ID NO:36
  • described co-stimulatory domain can comprise the amino acid sequence shown in SEQ ID NO:37
  • described intracellular signaling domain can comprise the aminoacid sequence shown in SEQ ID NO:38 amino acid sequence.
  • the chimeric antigen receptor can also be linked with a signal peptide.
  • the signal peptide is derived from CD8.
  • the signal peptide may comprise the amino acid sequence shown in SEQ ID NO:40.
  • the chimeric antigen receptor may sequentially comprise a signal peptide, a GPC3 binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from N-terminus to C-terminus.
  • the signal peptide may comprise the amino acid sequence shown in SEQ ID NO:40
  • the GPC3 binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:31-34
  • the hinge region may comprise The amino acid sequence shown in SEQ ID NO:39
  • the transmembrane domain can include the amino acid sequence shown in SEQ ID NO:36
  • the co-stimulatory domain can include the amino acid sequence shown in SEQ ID NO:37
  • the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:66.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:68.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:70.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:72.
  • the said promoter can also be linked.
  • the promoter is the constitutive promoter.
  • the promoter is the elongation factor-1 alpha (EF-1 alpha) promoter.
  • the promoter may comprise the nucleotide sequence shown in SEQ ID NO:41.
  • Nucleic acid molecule Nucleic acid molecule, vector, cell, preparation method and pharmaceutical composition
  • the present application also provides one or more isolated nucleic acid molecules, which can encode the chimeric antigen receptor (CAR) described in the present application.
  • the isolated nucleic acid molecule(s) described herein can be nucleotides of any length in isolated form, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized , but may encode a chimeric antigen receptor (CAR) as described herein.
  • the present application also provides a vector, which may comprise the nucleic acid molecule described in the present application.
  • the vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors can include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage and Animal viruses, etc.
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • the vector may contain various expression-controlling elements, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain an origin of replication.
  • the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.
  • the present application also provides immune effector cells, which may comprise the nucleic acid molecule or the vector described in the present application.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • the cells may also include cells transfected with the vectors of the present invention in vitro.
  • the cells may be mammalian cells.
  • the immune effector cells include T lymphocytes, for example, ⁇ / ⁇ T lymphocytes and ⁇ / ⁇ T lymphocytes; in some 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 present application also provides a method for preparing the immune effector cells described in the present application, and 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 a composition, which may comprise the immune effector cells described in the present application.
  • the composition may also include, optionally, a pharmaceutically acceptable adjuvant.
  • compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, isotonic agents and absorption delaying agents compatible with the immune effector cells, generally safe, non-toxic, and is neither biologically nor otherwise undesirable.
  • the composition may comprise parenteral, transdermal, intracavity, 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 different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. Such uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.
  • the present application also provides the chimeric antigen receptor described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, the immune effector cell described in the present application and/or the nucleic acid molecule described in the present application.
  • the composition of the invention is used in the preparation of medicines, and the medicines can be used to treat diseases or diseases related to the expression of GPC3.
  • the disease or disease related to the expression of GPC3 is cancer or malignant tumor, for example, the disease or disease related to the expression of GPC3 may include liver cancer. .
  • the present application also provides a method for preventing, alleviating or treating tumors, which may include administering the immune effector cells described in the present application to a subject in need.
  • the administration can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the chimeric antigen receptor described in the present application the nucleic acid molecule described in the present application, the carrier described in the present application, the immune effector cell described in the present application and/or the composition described in the present application, which It can be used to prevent, relieve or treat tumors.
  • the tumor may be a solid tumor or a hematological tumor.
  • the subject may include humans and non-human animals.
  • the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the following examples are only for explaining the chimeric antigen receptor of the present application, the preparation method and use, etc., and are not intended to limit the scope of the present invention.
  • the Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, to insert genes encoding proteins into such vectors and plasmids, or to introduce plasmids into host cells.
  • Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press.
  • the nucleic acid sequence of the light chain variable region of the 204A antibody and the nucleic acid sequence of the heavy chain variable region are connected with a connecting peptide nucleic acid sequence to obtain the 204AscFv nucleic acid sequence and amino acid sequence, and the nucleic acid sequences of the L1H2, L1H6, and L2H6 antibodies are processed in the same way to obtain The nucleic acid sequence and amino acid sequence of L1H2scFv, the nucleic acid sequence and amino acid sequence of L1H6scFv, and the nucleic acid sequence and amino acid sequence of L2H6scFv were obtained.
  • SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:60, SEQ ID NO:61 respectively show the nucleic acid sequences of the light chain variable regions of 204A, L1H2, L1H6, and L2H6 antibodies
  • SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:64 show the 204A, L1H2, L1H6, L2H6 antibody heavy chain variable region nucleic acid sequence respectively
  • SEQ ID NO:46 or SEQ ID NO:47 shows the connecting peptide nucleic acid Sequence
  • SEQ ID NO:35 shows the connecting peptide amino acid sequence
  • SEQ ID NO:42, SEQ ID NO:31 shows the 204AscFv nucleic acid sequence and amino acid sequence, respectively
  • SEQ ID NO:43, SEQ ID NO:32 shows the L1H2scFv Nucleic acid sequence and amino acid sequence
  • SEQ ID NO:44, SEQ ID NO:33 show the L1H6s
  • the scFv sequence is connected to the digested BBz platform plasmid in Escherichia coli by homologous recombination through its front and rear homology arms (SEQ ID NO:57-58). Single clone colonies were picked, plasmids were shaken and sequenced to verify the correctness of the plasmids.
  • the connection sequence of each part of the CAR structure in the complete plasmid and the sequence number in this application are shown in Table 1.
  • Table 1 shows the connection sequence of each part of the chimeric antigen receptor in the CAR plasmid obtained in this example, and the connection can also be referred to in Figure 2 of the specification.
  • SEQ ID NO:48 shows the nucleic acid sequence of the CAR structural region of the BBz platform plasmid
  • SEQ ID NO:49 shows the signal peptide nucleic acid sequence of the CAR structural region of the BBz platform plasmid
  • SEQ ID NO:40 shows the amino acid sequence of the signal peptide of the CAR structural region of the BBz platform plasmid Sequence
  • SEQ ID NO:50 shows the nucleotide sequence of the hinge region of the CAR structural region of the BBz platform plasmid
  • SEQ ID NO:39 shows the amino acid sequence of the hinge region of the CAR structural region of the BBz platform plasmid
  • SEQ ID NO:51 shows the CAR structure of the BBz platform plasmid Region transmembrane region nucleic acid sequence
  • SEQ ID NO:36 has shown the transmembrane region amino acid sequence of BBz platform plasmid CAR structure region
  • SEQ ID NO:52 has shown BBz platform plasm
  • the vector system used to construct the lentiviral plasmid vector of the present invention belongs to the third generation lentiviral vector system, which has three plasmids in total, namely the packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein (gifted by the laboratory)
  • the PMD2.G plasmid (gifted by the laboratory) of encoding envelope protein VSV-G, the core plasmid (i.e. 204A-BBz, L1H2-BBz, L1H6-BBz, L2H6-BBz, L2H6- BBz or GC33-BBz).
  • the gene encoding CAR in the core plasmid based on the BBz platform plasmid is regulated by the elongation factor-1 ⁇ (EF-1 ⁇ ) promoter.
  • EF-1 ⁇ elongation factor-1 ⁇
  • the packaging process of the virus is as follows:
  • SEQ ID NO: 41 shows the nucleic acid sequence of elongation factor-1 ⁇ .
  • the detection of virus titer is carried out as follows:
  • the cell culture plate was placed in a 37° C., 5% CO 2 incubator for static culture for 72 hours;
  • Virus titer [number of plated cells (cell count value) ⁇ (positive rate of test tube - positive rate of control tube)] / volume of inoculated virus solution (converted to the volume before dilution)
  • the viral supernatant packaging titers are shown in Table 2, and the titer ranges from 9 to 30x10e6/ml, which can be directly used for T cell infection.
  • the method for generating CAR-T cells containing scFv sequences based on the GPC3 antibody is as follows:
  • Human peripheral blood mononuclear cells were obtained by density gradient centrifugation (provided by volunteers of the Ark Project of Shanghai Yuanneng Cell Medical Technology Co., Ltd.);
  • Peripheral blood mononuclear cells were resuspended in X-VIVO (Lonza Company) medium containing 200 U/ml interleukin 2 (Singer) to a cell density of 2 ⁇ 10 6 /m, at a ratio of 1:3 (cell:magnetic beads) adding CD3/CD28 magnetic beads (Thermo Fisher) to activate T cells;
  • Example 3 (4) Add the virus supernatant obtained in Example 3 at a ratio of 5 at the multiplicity of infection (MOI) of the virus, add polybrene to a final concentration of 5ug/ml, place the cell suspension in an orifice plate and centrifuge at 1200rpm in a horizontal centrifuge for 1 hour ;
  • MOI multiplicity of infection
  • the positive rate of CAR-T cells cultured for 9 to 14 days the virus used to infect the cells carries GFP, and the positive rate of CAR expression is obtained by detecting the positive rate of GFP by flow cytometry after the virus infects the cells.
  • the positive rate > 20% of the cells can be used for tumor killing experiments;
  • the expansion times of T cells activated and expanded for 9-12 days were between 20-100 times, and the infection positive rates were between 30% and 90% (Table 3), It can be used in cytological function experiments.
  • HepG2 is a liver cancer cell with high expression of GPC3 protein, which is used as a positive target cell in this embodiment.
  • Cells infected with CAR-T virus are used as effector cells, and uninfected T cells can be used as control effector cells.
  • the specific experimental process is:
  • effect-target ratio 0.3:1, 1:1, 3:1, mix the effector cells and target cells in 200 ⁇ l X-VIVO medium, and the number of target cells is 1x10e4 /well, as the experimental group;
  • step (5) Centrifuge the cell culture well plate obtained in step (5) at 300g for 5 minutes respectively, and collect 50 ⁇ l of supernatant for detecting the release amount of lactate dehydrogenase LDH.
  • the detection method refers to the CytoTox96 non-radioactive cytotoxicity kit (Promega Company) manual.
  • LDH is a stable cytoplasmic enzyme that is released upon cell lysis in much the same way as 51Cr is released in radioactive assays.
  • the released LDH in the medium supernatant can be detected by a coupled enzyme reaction. In the enzyme reaction, LDH can convert a tetrazolium salt (INT) into red formazan. proportional to the number of cells.
  • Killing toxicity % 100x(experimental group-effector cell self-release-target cell self-release+culture base value)/(target cell maximum release-target cell self-release)
  • CAR-T based on 204A/L1H2/L1H6/L2H6scFv can effectively kill liver cancer target cells (HepG2 cells) with high expression of GPC3.
  • Example 7 Evaluation of the cellular function of CAR-T based on CAR-T repeated stimulation experiments in vitro
  • Example 5 (1) Prepare CAR-T according to the infection and amplification methods mentioned in Example 5. When the expansion reaches the 9th-12th day, the positive rate is detected according to the method in Example 5, and the CAR-T cells are used without Serum x-vivo 15 (lonza) medium was resuspended to a density of 4x10e5/ml as effector cells;
  • the cells were counted to calculate the amplification factor.
  • 2x10e5 CAR-T effector cells were taken, mixed with 2x10e5 Huh7 cells treated with new UV irradiation again, cultured statically in a 5% CO2 incubator at 37°C, and the culture medium was observed every 2 days. Add 1 volume of medium when the medium turns from orange to yellow.
  • CAR-T cells expressing GC33-BBz, L1H2-41BBz and T cells not infected with CAR-T were prepared according to the method of Example 5, and the dose of 8 ⁇ 10e5 CAR-positive cells was used to treat 6-week-old subcutaneous tumor-bearing Huh7 tumors (1 ⁇ 10e7/body) ) to 80-150mm 3 size NSG mice were injected with CAR-T by single tail vein, 11 mice in each group. Body weight (Fig. 9A-C) and tumor size measurements (Fig. 8A-C) were performed twice a week.
  • mice in each group were randomly selected from the tail vein to obtain anticoagulated blood (anticoagulated with sodium heparin) for IFNg detection in the blood (BD human th1/th2CBA kit) ( Figure 10).
  • anticoagulated blood anticoagulated with sodium heparin
  • mice in each group were randomly selected from the tail vein to take anticoagulated blood (anticoagulated with sodium heparin) to measure the content of human CD8 cells ( Figure 11A) and human CD4 cells ( Figure 11B) in the blood. flow detection.

Abstract

本申请涉及一种嵌合抗原受体(CAR)，其包含GPC3结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，所述GPC3结合结构域包含特异性结合GPC3的抗体或其片段，所述抗体包含轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，所述LCDR1的氨基酸序列如SEQ ID NO：16所示，所述LCDR2的氨基酸序列如SEQ ID NO：17所示且所述LCDR3的氨基酸序列如SEQ ID NO：18所示。本申请还涉及编码所述CAR的分离的核酸、包含所述核酸的载体、包含所述核酸或载体的免疫效应细胞及其制备方法以及所述CAR的用途。

Description

一种嵌合抗原受体及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种嵌合抗原受体及其用途。
背景技术
肿瘤是一种严重威胁人类健康的疾病，其中肝癌是危害很广泛的一种恶性肿瘤。由乙肝病毒引起的肝癌具有潜伏期长的特点，一旦发现往往已至晚期，而且发病后进展快速、治疗预后差。
磷脂酰肌醇蛋白聚糖3(glypican3,GPC3)是一种细胞膜表面的硫酸乙酰肝素蛋白多糖，存在于多种肿瘤之中，在肝癌中尤为常见。近年来，随着分子生物学、基因组学及蛋白质组学的进展，陆续问世了一系列的分子靶向药物，用于肝癌的救治。而随着免疫学的快速发展，根据细胞毒性淋巴细胞(CTL)对靶细胞的识别特异性依赖于T淋巴细胞受体(TCR)这一发现，进一步研究了将针对肿瘤细胞相关抗原的抗体的scFv与T淋巴细胞受体融合成嵌合抗原受体(CAR)，并将其通过如慢病毒感染等方式基因修饰在T淋巴细胞表面用于肿瘤的治疗。这种CAR-T淋巴细胞能够以主要组织兼容性复合物(MHC)非限制性方式选择性地将T淋巴细胞定向到肿瘤细胞并特异性地杀伤肿瘤细胞。然而基于免疫效应细胞的过继性免疫治疗在部分肿瘤中取得了一定的效果，但在大多数肿瘤的疗效仍不能令人满意。
发明内容
本申请提供一种嵌合抗原受体(CAR)，所述CAR包含GPC3结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，所述GPC3结合结构域包含特异性结合GPC3的抗体或其片段，其中所述抗体包含轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，所述LCDR1的氨基酸序列如SEQ ID NO：16所示，所述LCDR2的氨基酸序列如SEQ ID NO：17所示且所述LCDR3的氨基酸序列如SEQ ID NO：18所示。
在某些实施方式中，所述抗体包含重链互补决定区1(HCDR1)，重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，所述HCDR1的氨基酸序列如SEQ ID NO：19所示，所述HCDR2的氨基酸序列如SEQ ID NO：20所示且所述HCDR3的氨基酸序列如SEQ ID NO：21所示。
在某些实施方式中，所述抗体包含重链可变区，所述重链可变区的氨基酸序列如SEQ ID  NO：29所示。
在某些实施方式中，所述抗体包含轻链可变区，所述轻链可变区的氨基酸序列如SEQ ID NO：25所示。
在某些实施方式中，所述抗体为单链抗体。
在某些实施方式中，所述抗体包含SEQ ID NO:30所示的氨基酸序列。
在某些实施方式中，所述跨膜结构域包含源自选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，所述跨膜结构域包含SEQ ID NO:36所示的氨基酸序列。
在某些实施方式中，所述共刺激结构域包含选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，所述共刺激结构域包含SEQ ID NO:37所示的氨基酸序列。
在某些实施方式中，所述胞内信号传导结构域包含源自CD3ζ的信号传导结构域。
在某些实施方式中，所述胞内信号传导结构域包含SEQ ID NO:38所示的氨基酸序列。
在某些实施方式中，所述CAR还包含铰链区，所述铰链区连接所述GPC3结合结构域和所述跨膜结构域。
在某些实施方式中，所述铰链区包含SEQ ID NO:39所示的氨基酸序列。
在某些实施方式中，所述CAR还连接信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:40所示的氨基酸序列。
在某些实施方式中，编码所述CAR的核酸分子还连接启动子。
在某些实施方式中，所述启动子为组成型启动子。
在某些实施方式中，所述启动子包含SEQ ID NO:41所示的核苷酸序列。
在某些实施方式中，所述CAR包含SEQ ID NO:31-34中任一项所示的氨基酸序列。
本申请还提供分离的核酸分子，其编码本申请所述的CAR。
在某些实施方式中，编码CAR的分离的核酸分子包含SEQ ID NO:42-45中任一项所示的核酸序列。
本申请还提供一种载体，其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。
本申请还提供一种免疫效应细胞，其包含本申请所述的CAR，本申请所述的核酸分子，或本申请所述的载体。
在某些实施方式中，所述免疫效应细胞选自T淋巴细胞。
本申请还提供制备本申请所述的免疫效应细胞的方法，所述方法包括向本申请所述的免疫效应细胞中引入本申请所述的载体。
本申请还提供一种组合物，其包含本申请所述的所述的免疫效应细胞。
本申请还提供本申请所述的CAR，本申请所述的核酸分子，本申请所述的载体或本申请所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗与GPC3的表达相关的疾病或病症。
本申请还提供一种治疗与GPC3的表达相关的疾病或病症的方法，所述方法包括向患者或受试者施用本申请所述的CAR，本申请所述的核酸分子，本申请所述的载体或本申请所述的免疫效应细胞。
本申请还提供所述CAR，所述核酸分子，所述载体或所述免疫效应细胞，其用于治疗与GPC3的表达相关的疾病或病症。
在某些实施方式中，所述与GPC3的表达相关的疾病或病症为癌症或恶性肿瘤。
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质粒中嵌合抗原受体各部分的连接顺序。
图2显示了204A-BBz感染T细胞的扩增倍数。
图3显示了L1H2-BBz/L1H6-BBz/L2H6-BBz/GC33-BBz感染T细胞的扩增倍数。
图4显示了204A-CAR-T对于HepG2细胞的杀伤效果。
图5显示了L1H2/L1H6/L2H6-CAR-T对于HepG2细胞的杀伤效果。
图6显示了204A-CAR-T经Huh7靶细胞的反复刺激的扩增倍数。
图7显示了L1H2/L1H6/L2H6-CAR-T的经Huh7靶细胞的反复刺激的扩增倍数。
图8A-8C显示了L1H2-BBz和GC33-BBz小鼠体内消瘤抑瘤及防止复发的比较。
图9A-9C显示了L1H2-BBz和GC33-BBz小鼠体重变化。
图10显示了L1H2-BBz和GC33-BBz治疗的小鼠体内细胞因子变化。
图11A-11B显示了L1H2-BBz和GC33-BBz治疗的小鼠体内细胞水平变化(A为CD8+细胞；B为CD4+细胞)。
图12显示了L1H2-BBz和GC33-BBz治疗小鼠的生存曲线。
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，熟悉此技术的人士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。
以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，术语“嵌合抗原受体(CAR)”通常指识别肿瘤相关抗原(tumor associated antigen，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(immunoreceptor tyrosine-based activation motifs,ITAM,通常为CD3ζ或FcεRIγ)”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(tumor-associated antigen,TAA)结合区(通常来源于单克隆抗体抗原结合区域的scFv)，一个胞外铰链区(Hinge area)，一个跨膜区(Transmembrane region)和一个胞内免疫受体酪氨酸活化基序(Immunoreceptor tyrosine-based activation motif,ITAM)。
在本申请中，术语“GPC3”通常指通常指磷脂酰肌醇蛋白聚糖3(glypican 3,GPC3)，是一种细胞膜表面的硫酸乙酰肝素蛋白多糖，存在于多种肿瘤之中，在肝癌中尤为常见。GPC3蛋白在胎儿时期的肝脏高表达。出生后GPC3蛋白异常表达与肿瘤发生发展关系密切，GPC3蛋白在原发性肝癌(PHC)中呈高表达，而在其他肿瘤或良性肝病中呈低表达或中表达。例如，本申请所述的GPC3蛋白可以包含人GPC3蛋白。
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“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“CDR”通常指抗体可变结构域的区域，也称作互补决定区，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在天然抗体中，HCDR3和LCDR3显示所述六个CDR的大多数多样性，并且特别地HCDR3被认为在赋予抗体的精细特异性方面起独特作用。参见，例如Xu et al,Immunity 13:37-45(2000)；Johnson and Wu,in Methods in Molecular Biology 248:1-25(Lo,ed.,Human Press,Totowa,N.J.,2003)。实际上，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况功能正常且稳定。参见，例如，Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。
在本申请中，术语“可变区”通常指抗体重链或轻链的氨基末端结构域。重链和轻链的可变区可以分别称为重链可变区(VH)和轻链可变区(VL)。这些区域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。
在本申请中，术语“抗体”通常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，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab') 2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合GPC3)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994，第71页和第6章。来自任何脊椎 动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。取决于其重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。基于CH序列和功能方面的相对小的差异，将γ和α类进一步分成亚类，例如，人表达下述亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgK1。
在本申请中，术语“单链抗体”指含有一个或多个抗原结合部位的单链多肽。另外，尽管Fv片段的H和L链是由不同的基因编码的，它们可直接或通过肽而连接在一起，例如，通过重组的方法，可用合成的衔接物(linker)将H和L链连接成单一蛋白链(称为单链抗体，sAb；Bird et al.1988Science242：423-426；and Huston et al.1988PNAS 85：5879-5883)。该单链抗体也被包括在术语“抗体”之中，可在设计和制造多特异性结合分子中用作结合决定簇。
在本申请中，术语“跨膜结构域”通常指多肽或蛋白质，所述多肽或蛋白质在DNA水平上由至少一个包含胞外区、跨膜区和胞内区的外显子编码。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，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“胞内信号传导结构域”通常指分子的细胞内部分。胞内信号传导结构域产生促进含有CAR的细胞例如CART细胞的免疫效应子功能的信号。在例如CART细胞中免疫效应子功能的实例包括细胞裂解活性和辅助活性，包括细胞因子的分泌。在某些实施方式中，细胞内信号结构域转导效应子功能信号并指导细胞进行特化功能。虽然可以使用整个胞内信号传导结构域，但在许多情况下，不必使用整个链。就使用胞内信号传导结构域的截短部分而言，此类截短部分可用于代替完整链，只要其转导效应子功能信号即可。术语胞内信号传导结构域因此意在包括足以转导效应子功能信号的胞内信号传导结构域的任一截短部分。
在本申请中，术语“CD”，即分化簇(Cluster of differentiation)，也称作分化群，通常指 的是用来识别用作免疫抗原标识的细胞表面分子。CD分子有许多用途，通常用作细胞的重要受体或配体。部分CD可参于细胞的讯号级联从而此改变细胞的行为，有些CD蛋白则与细胞信号传导无关，但有着其他功能，例如细胞黏附。截至2016年4月21日人类的CD分子总数为371，例如本申请中作为跨膜结构域来源的CD28、CD3e、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154，作为共刺激结构域来源的CD137、CD28、CD134(OX-40)和CD278(ICOS)。
在本申请中，术语“CD137”，也称作4-1BB，通常指一种肿瘤坏死因子(TNF)受体家族的成员，由肿瘤坏死因子受体超家族成员9(TNFRSF9)基因编码。人4-1BB位于1号染色体，全长255氨基酸，包含17氨基酸信号肽、169氨基酸的胞外区、27氨基酸跨膜区和42氨基酸的胞内区。小鼠4-1BB位于小鼠4号染色体，与人4-1BB大约有60％的序列相似性。CD137(4-1BB)是在活化的CD4+和CD8+T细胞、NKT、NK细胞、DC、巨噬细胞、嗜酸性粒细胞、嗜中性粒细胞和肥大细胞以及Tregs上表达的诱导型共刺激受体。
在本申请中，术语“CD3ζ”，也写作CD3zeta，通常指来自ζ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。
在本申请中，术语“铰链区”通常指在CAR结构中位于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CAR T细胞与肿瘤细胞之间的接触。
在本申请中，术语“信号肽”通常指如本文中所使用的，术语“信号肽”是指作为N-端肽存在于蛋白前体形式上的前台。信号肽的功能是促进连接至内质网的表达多肽的易位。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“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“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多 聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“分离的核酸分子”通常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，或从其天然环境分离的或人工合成的类似物。
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***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、***瘤病毒、***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“组合物”通常指涉及适合施用于患者、人患者的组合物。例如，本申请所述的组合物，其可以包含本申请所述的免疫效应细胞，以及任选地药学上可接受的佐剂。在某些实施方式中，组合物的可接受成分在所用剂量和浓度下对接受者无毒。本申请的组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“肿瘤”通常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血液瘤。例如，在本申请中，肿瘤可以是GPC3阳性的肿瘤，其中所述GPC3阳性的肿瘤可以包括肝癌。
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。
嵌合抗原受体(CAR)
一方面本申请提供了一种嵌合抗原受体(CAR)，其包含包含GPC3结合结构域、跨膜 结构域、共刺激结构域和胞内信号传导结构域，所述GPC3结合结构域包含特异性结合GPC3的抗体或其片段，其中所述抗体包含重链互补决定区1(HCDR1)，重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，所述HCDR1的氨基酸序列如SEQ ID NO：19所示，所述HCDR2的氨基酸序列如SEQ ID NO：20所示且所述HCDR3的氨基酸序列如SEQ ID NO：21所示。
例如，本申请所述的抗体还可以包含包含轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，所述LCDR1的氨基酸序列如SEQ ID NO：1所示，所述LCDR2的氨基酸序列如SEQ ID NO：2所示且所述LCDR3的氨基酸序列如SEQ ID NO：3所示。
例如，所述GPC3为人GPC3，所述GPC3的氨基酸序列如SEQ ID NO:56所示。
CDR
在本申请中，所述HCDR1可以包含SEQ ID NO:19所示的氨基酸序列。
X 1YX 2MH(SEQ ID NO:19)，其中X 1可以为D或A，X 2可以为A或E。
在本申请中，所述HCDR1可以包含SEQ ID NO:4和12中任一项所示的氨基酸序列。
在本申请中，所述HCDR2可以包含SEQ ID NO:20所示的氨基酸序列。
X 1LX 2X 3X 4X 5GX 6X 7X 8YX 9X 10X 11X 12X 13G(SEQ ID NO:20)，其中X 1可以为G或A，X 2可以为S或D，X 3可以为W或P，X 4可以为N或K，X 5可以为S或T，X 6可以为S或Q，X 7可以为I或T，X 8可以为G或A，X 9可以为A或S，X 10可以为D或Q，X 11可以为S或K，X 12可以为V或F，X 13可以为K或Q。
在本申请中，所述HCDR2可以包含SEQ ID NO:5、10和13中任一项所示的氨基酸序列。
在本申请中，所述HCDR3可以包含SEQ ID NO:21所示的氨基酸序列。
X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11(SEQ ID NO:21)，其中X 1可以为D或T，X 2可以为H或R，X 3可以为T或F，X 4可以为I或Y，X 5可以为G或S，X 6可以为V或Y，X 7可以为G或A，X 8可以为A、Y或H，X 9可以为F或空白，X 10可以为D或空白，X 11可以为I或空白。
在本申请中，所述HCDR3可以包含SEQ ID NO:6、11和14中任一项所示的氨基酸序列。
例如，本申请所述的HCDR1可包含SEQ ID NO:4所示的氨基酸序列，HCDR2可包含SEQ ID NO:5所示的氨基酸序列，HCDR3可包含SEQ ID NO:6所示的氨基酸序列。
例如，本申请所述的HCDR1可包含SEQ ID NO:12所示的氨基酸序列，HCDR2可包含 SEQ ID NO:10所示的氨基酸序列，HCDR3可包含SEQ ID NO:11所示的氨基酸序列。
例如，本申请所述的HCDR1可包含SEQ ID NO:12所示的氨基酸序列，HCDR2可包含SEQ ID NO:13所示的氨基酸序列，HCDR3可包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述LCDR1可以包含SEQ ID NO:16所示的氨基酸序列。
X 1X 2X 3X 4SX 5VX 6X 7X 8X 9YX 10X 11X 12X 13(SEQ ID NO:16)，其中X 1可以为T或R，X 2可以为G或S，X 3可以为T或S，X 4可以为S或Q，X 5可以为D或L，X 6可以为G或H，X 7可以为G或S，X 8可以为Y或N，X 9可以为N或G，X 10可以为V或T，X 11可以为S或Y，X 12可以为空白或L，X 13可以为空白或H。
在本申请中，所述LCDR1可以包含SEQ ID NO:1和7中任一项所示的氨基酸序列。
在本申请中，所述LCDR2可以包含SEQ ID NO:17所示的氨基酸序列。
X 1X 2SX 3RX 4S(SEQ ID NO:17)，其中X 1可以为D或K，X 2可以为V或G，X 3可以为N、Y或Q，X 4可以为P或G。
在本申请中，所述LCDR2可以包含SEQ ID NO:2、8和15中任一项所示的氨基酸序列。
在本申请中，所述LCDR3可以包含SEQ ID NO:18所示的氨基酸序列。
X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10(SEQ ID NO:18)，其中X 1可以为S或G，X 2可以为S或Q，X 3可以为Y或S，X 4可以为A或G，X 5可以为S或L，X 6可以为G或T，X 7可以为S或P，X 8可以为T或P，X 9可以为L或T，X 10可以为V或空白。
在本申请中，所述LCDR3可以包含SEQ ID NO:3和9中任一项所示的氨基酸序列。
例如，本申请所述的LCDR1可包含SEQ ID NO:1所示的氨基酸序列，LCDR2可包含SEQ ID NO:2所示的氨基酸序列，LCDR3可包含SEQ ID NO:3所示的氨基酸序列。
例如，本申请所述的LCDR1可包含SEQ ID NO:7所示的氨基酸序列，LCDR2可包含SEQ ID NO:8所示的氨基酸序列，LCDR3可包含SEQ ID NO:9所示的氨基酸序列。
例如，本申请所述的LCDR1可包含SEQ ID NO:7所示的氨基酸序列，LCDR2可包含SEQ ID NO:15所示的氨基酸序列，LCDR3可包含SEQ ID NO:9所示的氨基酸序列。
又例如，本申请所述的LCDR1可包含SEQ ID NO:1所示的氨基酸序列，LCDR2可包含SEQ ID NO:2所示的氨基酸序列，LCDR3可包含SEQ ID NO:3所示的氨基酸序列，且HCDR1可包含SEQ ID NO:4所示的氨基酸序列，HCDR2可包含SEQ ID NO:5所示的氨基酸序列，HCDR3可包含SEQ ID NO:6所示的氨基酸序列。
又例如，本申请所述的LCDR1可包含SEQ ID NO:7所示的氨基酸序列，LCDR2可包含SEQ ID NO:8所示的氨基酸序列，LCDR3可包含SEQ ID NO:9所示的氨基酸序列，且HCDR1可包含SEQ ID NO:12所示的氨基酸序列，HCDR2可包含SEQ ID NO:10所示的氨基酸序列， HCDR3可包含SEQ ID NO:11所示的氨基酸序列。
又例如，本申请所述的LCDR1可包含SEQ ID NO:7所示的氨基酸序列，LCDR2可包含SEQ ID NO:8所示的氨基酸序列，LCDR3可包含SEQ ID NO:9所示的氨基酸序列，且HCDR1可包含SEQ ID NO:12所示的氨基酸序列，HCDR2可包含SEQ ID NO:13所示的氨基酸序列，HCDR3可包含SEQ ID NO:14所示的氨基酸序列。
又例如，本申请所述的LCDR1可包含SEQ ID NO:7所示的氨基酸序列，LCDR2可包含SEQ ID NO:15所示的氨基酸序列，LCDR3可包含SEQ ID NO:9所示的氨基酸序列，且HCDR1可包含SEQ ID NO:12所示的氨基酸序列，HCDR2可包含SEQ ID NO:13所示的氨基酸序列，HCDR3可包含SEQ ID NO:14所示的氨基酸序列。
VL和VH
本申请所述的嵌合抗原受体可包含抗体的轻链可变区VL和抗体的重链可变区VH。例如，所述VL可包含SEQ ID NO:25所示的氨基酸序列，所述VH可包含SEQ ID NO:29所示的氨基酸序列。又例如，所述嵌合抗原受体的VL可以包含SEQ ID NO:22-24中任一项所示的氨基酸序列。所述嵌合抗原受体的所述VH可以包含SEQ ID NO:26-28中任一项所示的氨基酸序列。
例如，所述VL可包含SEQ ID NO:22所示的氨基酸序列，所述VH可包含SEQ ID NO:26所示的氨基酸序列。
例如，所述VL可包含SEQ ID NO:23所示的氨基酸序列，所述VH可包含SEQ ID NO:27所示的氨基酸序列。
例如，所述VL可包含SEQ ID NO:23所示的氨基酸序列，所述VH可包含SEQ ID NO:28所示的氨基酸序列。
例如，所述VL可包含SEQ ID NO:24所示的氨基酸序列，所述VH可包含SEQ ID NO:28所示的氨基酸序列。
例如，在本申请中，所述嵌合抗原受体可以包含氨基酸序列如SEQ ID NO:29所示的VH中的HCDR1。例如，在本申请中，所述嵌合抗原受体可以包含氨基酸序列如SEQ ID NO:29所示的VH中的HCDR2。例如，在本申请中，所述嵌合抗原受体可以包含氨基酸序列如SEQ ID NO:29所示的VH中的HCDR3。又例如，在本申请中，所述嵌合抗原受体可以包含氨基酸序列如SEQ ID NO:25所示的VL中的LCDR1。例如，在本申请中，所述嵌合抗原受体可以包含氨基酸序列如SEQ ID NO:25所示的VL中的LCDR2。例如，在本申请中，所述嵌合抗原受体可以包含氨基酸序列如SEQ ID NO:25所示的VL中的LCDR3。
GPC3结合结构域
在本申请中，所述嵌合抗原受体的GPC3结合结构域可以包括特异性结合GPC3抗体或其抗原结合片段。例如，本申请所述的特异性结合GPC3抗体或其抗原结合片段可以包括但不限于重组抗体、单克隆抗体、人抗体、人源化抗体、嵌合抗体、双特异性抗体、单链抗体、双抗体、三抗体、四抗体、Fv片段、scFv片段、Fab片段、Fab'片段、F(ab')2片段和骆驼化单结构域抗体。
在本申请中，所述抗体可以为人源化抗体。换句话说，本申请所述的特异性结合GPC3抗体或其抗原结合片段，其可以为免疫特异性结合至相关抗原(例如人类GPC3)且包含基本上具有人类抗体的氨基酸序列的框架(FR)区及基本上具有非人类抗体的氨基酸序列的互补决定区(CDR)的抗体或其变异体、衍生物、类似物或片段。此处的“基本上”在CDR的情况下是指CDR的氨基酸序列与非人类抗体CDR的氨基酸序列至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一。所述人源化抗体基本上可以包含所有至少一个且通常两个可变域(Fab、Fab′、F(ab′)2、FabC、Fv)，其中所有或基本上所有CDR区对应于非人类免疫球蛋白(即抗体)的CDR区且所有或基本上所有框架区为具有人类免疫球蛋白共有序列的框架区。在某些实施方式中，人源化抗体含有轻链以及重链的至少可变域。在某些实施方式中，人源化抗体仅含人源化重链。在特定实施例中，人源化抗体仅含轻链和/或人源化重链的人源化可变域。
在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
在本申请中，所述GPC3结合结构域为单链抗体。例如，所述GPC3结合结构域为scFv。scFv可以包含SEQ ID NO:30所示的序列。例如，GPC3结合结构域可以包含抗体的轻链可变区、连接肽、抗体的重链可变区。
在本申请中，所述抗体的轻链可变区可以包含SEQ ID NO:25所示的氨基酸序列，所述连接肽可以包含SEQ ID NO:35所示的氨基酸序列，所述抗体的重链可变区可以包含SEQ ID NO:29所示的氨基酸序列。
例如，所述抗体的轻链可变区可以包含SEQ ID NO:22所示的氨基酸序列，所述连接肽可以包含SEQ ID NO:35所示的氨基酸序列，所述抗体的重链可变区可以包含SEQ ID NO:26所示的氨基酸序列。
例如，所述抗体的轻链可变区可以包含SEQ ID NO:23所示的氨基酸序列，所述连接肽可以包含SEQ ID NO:35所示的氨基酸序列，所述抗体的重链可变区可以包含SEQ ID NO:27所示的氨基酸序列。
例如，所述抗体的轻链可变区可以包含SEQ ID NO:23所示的氨基酸序列，所述连接肽可以包含SEQ ID NO:35所示的氨基酸序列，所述抗体的重链可变区可以包含SEQ ID NO:28所示的氨基酸序列。
例如，所述抗体的轻链可变区可以包含SEQ ID NO:24所示的氨基酸序列，所述连接肽可以包含SEQ ID NO:35所示的氨基酸序列，所述抗体的重链可变区可以包含SEQ ID NO:28所示的氨基酸序列。
例如，所述GPC3结合结构域可以包含SEQ ID NO:31所示的氨基酸序列。
例如，所述GPC3结合结构域可以包含SEQ ID NO:32所示的氨基酸序列。
例如，所述GPC3结合结构域可以包含SEQ ID NO:33所示的氨基酸序列。
例如，所述GPC3结合结构域可以包含SEQ ID NO:34所示的氨基酸序列。
在本申请中，所述嵌合抗原受体(CAR)可以包含SEQ ID NO:31-34中任一项所示的氨基酸序列。
跨膜结构域、共刺激结构域和胞内信号传导结构域
在本申请中，所述跨膜结构域可以包含选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，所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述共刺激结构域可以包含选自下述蛋白的共刺激结构域：CD137、CD28、4-1BB、OX-40和ICOS。例如，所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述胞内信号传导结构域可以包含源自CD3ζ的信号传导结构域。例如，所述胞内信号传导结构域可以包含SEQ ID NO:38所示的氨基酸序列。
在本申请中，所述嵌合抗原受体(CAR)可以从N端到C端依次包含GPC3结合结构域、跨膜结构域、共刺激结构域、以及胞内信号传导结构域。例如，所述GPC3结合结构域可以包含SEQ ID NO:31-34中任一项所示的氨基酸序列、所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列、所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列、所述胞内信号传导结构域可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以从N端到C端依次包含GPC3结合结构域、跨膜结构域、共刺激结构域、以及胞内信号传导结构域。例如，所述GPC3结合结构域可以包含SEQ ID NO:31所示的氨基酸序列、所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列、所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列、所述胞内信号传导结构域可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以从N端到C端依次包含GPC3结合结构域、跨膜结构域、共刺激结构域、以及胞内信号传导结构域。例如，所述GPC3结合结构域可以包含SEQ ID NO:32所示的氨基酸序列、所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列、所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列、所述胞内信号传导结构域可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以从N端到C端依次包含GPC3结合结构域、跨膜结构域、共刺激结构域、以及胞内信号传导结构域。例如，所述GPC3结合结构域可以包含SEQ ID NO:33所示的氨基酸序列、所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列、所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列、所述胞内信号传导结构域可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以从N端到C端依次包含GPC3结合结构域、跨膜结构域、共刺激结构域、以及胞内信号传导结构域。例如，所述GPC3结合结构域可以包含SEQ ID NO:34所示的氨基酸序列、所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列、所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列、所述胞内信号传导结构域可以包含SEQ ID NO:38所示的氨基酸序列。
在本申请中，所述嵌合抗原受体(CAR)还可以包含铰链区，所述铰链区连接所述GPC3结合结构域和所述跨膜结构域。例如，所述铰链区来源于IgG家族，例如，所述铰链区来源于IgG1。例如，所述铰链区来源于IgG4。例如，所述铰链区来源于IgD。例如，所述铰链区来源于CD8。例如，所述铰链区可以包含SEQ ID NO:39所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以从N端到C端依次包含GPC3结合结构域、铰链区、跨膜结构域、共刺激结构域、以及胞内信号传导结构域。例如，所述GPC3结合结构域可以包含SEQ ID NO:31-34中任一项所示的氨基酸序列、所述铰链区可以包含SEQ ID NO:39所示的氨基酸序列、所述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列、所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列、所述胞内信号传导结构域可以包含SEQ ID NO:38所示的氨基酸序列。
在本申请中，所述嵌合抗原受体(CAR)还可以连接信号肽。例如，所述信号肽来源于CD8。例如，所述信号肽可以包含SEQ ID NO:40所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以从N端到C端依次包含信号肽、GPC3结合结构域、铰链区、跨膜结构域、共刺激结构域、以及胞内信号传导结构域。例如，所述信号肽可以包含SEQ ID NO:40所示的氨基酸序列、所述GPC3结合结构域可以包含SEQ ID NO:31-34中任一项所示的氨基酸序列、所述铰链区可以包含SEQ ID NO:39所示的氨基酸序列、所 述跨膜结构域可以包含SEQ ID NO:36所示的氨基酸序列、所述共刺激结构域可以包含SEQ ID NO:37所示的氨基酸序列、所述胞内信号传导结构域可以包含SEQ ID NO:38所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以包含SEQ ID NO:66所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以包含SEQ ID NO:68所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以包含SEQ ID NO:70所示的氨基酸序列。
例如，所述嵌合抗原受体(CAR)可以包含SEQ ID NO:72所示的氨基酸序列。
在本申请中，所述还可以连接启动子。例如，所述启动子为所述组成型启动子。例如，所述启动子为延长因子-1α(EF-1α)启动子。例如，所述启动子可以包含SEQ ID NO:41所示的核苷酸序列。
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)。
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、***瘤病毒、***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。
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佐剂。
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。在某些实施方式中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵***来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。
用途和应用
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GPC3的表达相关的疾病或病症。
在本申请中，所述与GPC3的表达相关的疾病或病症为癌症或恶性肿瘤，例如，所述与GPC3的表达相关的疾病或病症可以包括肝癌。。
另一方面，本申请还提供了预防、缓解或***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免疫效应细胞。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。
另一方面，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，其可以用于预防、缓解或***。
在本申请中，所述肿瘤可以是实体瘤或血液瘤。
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的嵌合抗原受体、制备方 法和用途等，而不用于限制本申请发明的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因***到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例
实施例1.scFv片段的制备
将204A抗体轻链可变区核酸序列和重链可变区核酸序列用连接肽核酸序列进行连接，从而得到204AscFv核酸序列和氨基酸序列，以同样的方式处理L1H2、L1H6、L2H6抗体核酸序列，从而得到L1H2scFv核酸序列和氨基酸序列，L1H6scFv核酸序列和氨基酸序列、L2H6scFv核酸序列和氨基酸序列。将所有上述scFv(204AscFv、L1H2scFv、L1H6scFv、L2H6scFv)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’端加核酸序列SEQ ID NO:57，在scFv核酸序列3’端加核酸序列SEQ ID NO:58，用于分子构建(同源重组)。
SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:60、SEQ ID NO:61分别显示了204A、L1H2、L1H6、L2H6抗体轻链可变区核酸序列，SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:64分别显示了204A、L1H2、L1H6、L2H6抗体重链可变区核酸序列，SEQ ID NO:46或SEQ ID NO:47显示了连接肽核酸序列，SEQ ID NO:35显示了连接肽氨基酸序列，SEQ ID NO:42、SEQ ID NO:31分别显示了204AscFv核酸序列和氨基酸序列，SEQ ID NO:43、SEQ ID NO:32分别显示了L1H2scFv核酸序列和氨基酸序列，SEQ ID NO:44、SEQ ID NO:33分别显示了L1H6scFv核酸序列和氨基酸序列，SEQ ID NO:45、SEQ ID NO:34分别显示了L2H6scFv核酸序列和氨基酸序列。
实施例2.CAR质粒的构建
用MluI和NheI内切酶(购自NEB)对BBz平台质粒(原能自构)进行双酶切产生7924bp线性化片段，割胶回收后与实施例1制备所得的的scFv片段分别按100ng：30ng的比例混合(体积不超过10μl)，之后转化大肠杆菌DH5α感受态细胞，热激45s后涂布在含氨苄抗性的LB固体培养基上，37℃过夜培养。scFv序列通过其前后的同源臂(SEQ ID NO:57-58)以同源重组的方式与酶切后的BBz平台质粒在大肠杆菌内连接。挑单克隆菌落，摇菌抽质粒并 测序验证质粒正确性。构建完整的质粒中CAR结构各部分元件的连接顺序及在本申请中的序列号如表1所示。
表1 CAR质粒中嵌合抗原受体各部分的连接顺序
Figure PCTCN2021115057-appb-000001
表1中展示了本实施例所得CAR质粒中嵌合抗原受体各部分的连接顺序，该连接还可参见说明书附图2中所示。
SEQ ID NO:48显示了BBz平台质粒CAR结构区核酸序列，SEQ ID NO:49显示了BBz平台质粒CAR结构区信号肽核酸序列，SEQ ID NO:40显示了BBz平台质粒CAR结构区信号肽氨基酸序列，SEQ ID NO:50显示了BBz平台质粒CAR结构区铰链区核酸序列，SEQ ID NO:39显示了BBz平台质粒CAR结构区铰链区氨基酸序列，SEQ ID NO:51显示了BBz平台质粒CAR结构区跨膜区核酸序列，SEQ ID NO:36显示了BBz平台质粒CAR结构区跨膜区氨基酸序列，SEQ ID NO:52显示了BBz平台质粒CAR结构区胞内CD137共刺激结构域核酸序列，SEQ ID NO:37显示了BBz平台质粒CAR结构区胞内CD137共刺激域氨基酸序列，SEQ ID NO:53显示了BBz平台质粒CAR结构区胞内信号转导结构域CD3ZETA核酸序列，SEQ ID NO:38显示了BBz平台质粒CAR结构区胞内信号转导结构域CD3ZETA氨基酸序列，SEQ ID NO:65显示了204A-BBz质粒CAR结构区核酸序列，SEQ ID NO:66显示了204A-BBz质粒CAR结构区氨基酸序列，SEQ ID NO:67显示了L1H2-BBz质粒CAR结构区核酸序列，SEQ ID NO:68显示了L1H2-BBz质粒CAR结构区氨基酸序列，SEQ ID NO:69显示了L1H6-BBz质粒CAR结构区核酸序列，SEQ ID NO:70显示了L1H6-BBz质粒CAR结构区氨基酸序列，SEQ ID NO:71显示了L2H6-BBz质粒CAR结构区核酸序列，SEQ ID NO:72显示了L2H6-BBz质粒CAR结构区氨基酸序列，SEQ ID NO:54显示了GC33-BBz质粒CAR结构区核酸序列，SEQ ID NO:55显示了GC33-BBz质粒CAR结构区氨基酸序列。
实施例3.病毒的包装
作为示例，用于构建本发明的慢病毒质粒载体的载体***属于第三代慢病毒载体***，该***共有三个质粒即编码Gag-Pol蛋白和Rev蛋白的包装质粒psPAX2(实验室赠予)；编码包膜蛋白VSV-G的PMD2.G质粒(实验室赠予)，上述表1中构建的含有编码目的基因CAR的核心质粒(即204A-BBz、L1H2-BBz、L1H6-BBz、L2H6-BBz或GC33-BBz)。基于BBz平台质粒的核心质粒中编码CAR的基因由延长因子-1α(EF-1α)启动子调控表达。
病毒的包装过程如下：
(1)10e6的293T细胞悬于2ml 10％FBS的DMEM培养基中铺于6孔板的单个孔中，过夜培养；
(2)吸去1ml培养基，混入1ml含有4.5ug包装质粒(psPAX2：PMD2.G：核心质粒＝3:2:4)和13μl FuGENEHD转染试剂(Promega公司)的Opti-MEM培养基，轻轻混匀后在CO2培养箱中37℃培养12小时；
(3)去除含质粒的培养基，PBS(Hyclone公司)洗涤一次后更换为2ml含5％FBS的DMEM培养基，培养30小时；
(4)收集1ml病毒上清，该上清3000rpm离心5分钟后置4℃暂存，补加1ml新鲜含5％FBS的DMEM培养基至孔板中继续培养24小时；
(5)收取全部2ml病毒上清，3000rpm离心5分钟后与原收集1ml上清混匀后分装冻存-80℃待用。
SEQ ID NO:41显示了延长因子-1α的核酸序列。
实施例4.病毒滴度测定
病毒滴度的检测按以下方法进行：
(1)配置含8ug/ml polybrene的DMEM完全培养基；
(2)取细胞状态良好的293T细胞，去上清，用PBS洗涤后用0.25％的胰酶(GIBICO)37℃消化3分钟后用少量本实施例步骤(1)所得的完全培养基重悬并计数，用本实施例步骤(1)所得的完全培养基将细胞浓度调整至1x10e5cells/ml；
(3)每管加1ml细胞悬液(1x10e5细胞)，然后分别加入实施例3所得的病毒液2μl和2倍稀释的病毒液4μl，混匀，加入到12孔板中，每孔1ml。额外在4个孔中分别加入1ml不含病毒的细胞悬液(1x10e5细胞)，用于流式检测时做空白对；
(4)额外在4个孔中分别加入1ml不含病毒的细胞悬液(1x10e5细胞)，贴壁6小时后消化细胞并进行计数，取平均值作为最后的铺板细胞数；
(5)细胞培养板置于37℃，5％CO 2培养箱中静置培养72小时；
(6)取接种病毒72小时后的细胞，小心吸去孔内培养基，每孔贴壁缓慢加入1ml PBS，轻轻漂洗后吸去(注意防止细胞过多脱落)；
(7)去上清，用PBS洗涤后用0.25％的胰酶(GIBICO)37℃消化，轻轻拍打孔板，显微镜下观察到大部分细胞不再贴壁时终止消化；
(8)每孔加入1ml本实施例步骤(1)所得的完全培养基终止消化，移液枪轻轻吹打孔壁至细胞全部脱落，移至1.5ml无菌离心管，细胞计数；
(9)每管取等量细胞(2～5x10e5)，标号(管号与加入的病毒管号一致)，用NBS溶液(含1％新生牛血清(GIBICO)的PBS溶液)补齐至1.2ml，4℃，2000rpm，离心5min，弃上清；
(10)每管加入1mlNBS，轻轻吹打混匀，4℃，2000rpm，离心5min，充分吸去上清；
(11)每管加入50μl NBS和0.5μg CAR检测一抗，移液枪轻轻吹打混匀(空白孔不加)，4℃孵育60min；
(12)直接加1mlNBS重悬，4℃，2000rpm，离心5min，吸去上清；
(13)加入1mlNBS重悬，4℃，2000rpm，离心5min，充分吸去上清；
(14)每管加入50μl NBS和0.5μl二抗(二抗浓度为1mg/ml)(空白孔不加二抗)，4℃孵育30min；
(15)重复步骤(13)-(14)；
(16)每管加入200μlNBS重悬；
(17)BDFACSCantoII流式仪上检测荧光表达百分比,荧光为Dylight 650；
(18)数据处理：
病毒滴度＝[铺板细胞个数(细胞计数值)×(测试管阳性率-对照管阳性率)]/接种病毒液体积(换算为稀释前的体积)
质控点：阳性管阳性率-对照管阳性率为5％-20％时所得结果可用。
经检测，病毒上清包装滴度如表2，滴度范围在9～30x10e6/ml之间，可直接用于T细胞感染。
表2病毒滴度
病毒名称 滴度
204A-BBz 1.325*10 7IU/ml
L1H2-BBz 9.1*10 6IU/ml
L1H6-BBz 1.31*10 7IU/ml
L2H6-BBz 1.0*10 7IU/ml
GC33-BBz 2.68*10 7IU/ml
实施例5.T细胞的感染和扩增
含有基于GPC3抗体scFv序列的CAR-T细胞的产生方法如下：
(1)通过密度梯度离心法获得人外周血单核细胞(上海原能细胞医学技术有限公司方舟计划志愿者提供)；
(2)用含有200U/ml白介素2(欣吉尔)的X-VIVO(Lonza公司)培养基重悬外周血单核细胞至细胞密度为2x10 6/m，按1:3的比例(细胞:磁珠)加入CD3/CD28磁珠(赛默飞)对T细胞进行激活；
(3)激活的外周血单核细胞静置在37℃CO 2培养箱中培养24小时；
(4)按病毒感染复数(MOI)为5的比例加入实施例3所得病毒上清，加polybrene至终浓度为5ug/ml，将细胞悬液置于孔板中用水平离心机1200rpm离心1小时；
(5)将孔板放回37℃CO 2培养箱中培养24小时；
(6)300g离心5分钟，去上清，并用新鲜的含500U/ml白介素2的X-VIVO培养基重悬细胞至细胞密度为0.6x10e6/ml，置于37℃CO 2培养箱中培养；
(7)每2天对细胞进行计数，并补加新鲜的含500U/ml白介素2的X-VIVO培养基将细胞密度调回0.6x10e6/ml；
(8)培养9～14天的CAR-T细胞检测细胞阳性率：用于感染细胞的病毒携带GFP，病毒感染细胞后通过流式细胞仪检测GFP阳性率从而得到CAR表达阳性率，阳性率>20％的细胞可以用于进行肿瘤杀伤实验；
如图2和图3所示，经激活扩增9-12天的T细胞扩增倍数介于20-100倍之间，感染阳性率均介于30％至90％之间(表3)，可以用于细胞学功能实验。
表3 CAR感染阳性率
CAR名称 感染阳性率
204A-BBz 65.8％
L1H2-BBz 79.1％
L1H6-BBz 74.5％
L2H6-BBz 80.6％
GC33-BBz 82.7％
实施例6.CAR-T体外肿瘤杀伤活性实验评估
HepG2是GPC3蛋白高表达的肝癌细胞，在本实施例中作为阳性靶细胞。感染了CAR-T病毒的细胞作为效应细胞，未感染的T细胞可作为对照效应细胞。具体实验过程为：
(1)检测CAR的感染效率，用未感染的T细胞调整感染CAR比例至每组一致；
(2)按效应细胞：靶细胞(效靶比)＝0.3:1、1:1、3:1的比例将效应细胞和靶细胞混匀在200μl X-VIVO培养基中，靶细胞数量为1x10e4/孔，作为实验组；
(3)只含有与实验组等量的效应细胞的孔作为效应细胞自释放背景组；
(4)只含有与实验组等量的靶细胞的孔作为靶细胞自释放背景组；
(5)将步骤(2)-(4)得到的细胞在37℃CO 2培养箱中培养18小时；
(6)在部分仅含靶细胞的孔中加入20μl 10x的裂解液(CytoTox96非放射性细胞毒性试剂盒，Promega公司提供)，反应45分钟，作为靶细胞最大释放。
(7)将步骤(5)所得细胞培养孔板分别300g离心5分钟，收集50μl上清用于检测乳酸脱氢酶LDH的释放量，检测方法参照CytoTox96非放射性细胞毒性试剂盒(Promega公司)的说明书。LDH是一种稳定的胞质酶，在细胞裂解时会释放出来，其释放方式与51Cr在放射性分析中的释放方式基本相同。释放的LDH在培养基上清中，可通过偶联的酶反应来检测，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜，其生成的红色产物量与裂解细胞数成正比。
(8)细胞杀伤活性计算公式为：
杀伤毒性％＝100x(实验组-效应细胞自释放-靶细胞自释放+培养基本底值)/(靶细胞最大释放-靶细胞自释放)
如图4和图5所示，相较于未感染病毒的T细胞，基于204A/L1H2/L1H6/L2H6scFv的CAR-T均能够有效杀伤GPC3高表达的肝癌靶细胞(HepG2细胞)。
实施例7.基于CAR-T体外反复刺激实验评价CAR-T的细胞学功能
(1)根据实施例5中提到的感染和扩增方式制备CAR-T，在扩增到第9-12天时，根据实施例5中的方法进行阳性率检测，取CAR-T细胞用无血清x-vivo 15(lonza)培养基重悬 到4x10e5/ml的密度，作为效应细胞；
(2)用含有10％血清的DMEM培养中等GPC3表达的Huh7肝癌细胞，用胰酶消化并用实施例4第(1)步配制的完全培养基终止胰酶反应后1000rpm离心5分钟，去上清后用2ml PBS重悬，之后铺于润湿过的10cm培养皿中，在安全柜中紫外照射10分钟，照射后的细胞经洗涤离心后用无血清x-vivo 15(lonza)培养基重悬到4x10e5/ml的密度，作为靶细胞；
将500μl效应细胞与500μl靶细胞混合，在37℃，5％CO 2培养箱中静置培养，每2天观察一次培养基颜色，培养基由橙色转黄色时补加1倍体积的培养基。
第四至五天时进行细胞计数，计算扩增倍数。
计数后取2x10e5CAR-T效应细胞，再次与新紫外照射处理过的2x10e5的Huh7细胞混合，在37℃，5％CO2培养箱中静置培养，每2天观察一次培养基。培养基由橙色转黄色时补加1倍体积的培养基。
再次培养四至五天后再次进行细胞计数，计算扩增倍数。
如图6和图7所示的反复刺激的结果，204A-CAR-T反复刺激的扩增倍数高于GC33-CAR-T，而L1H2/L1H6/L2H6-CAR-T的经过Huh7靶细胞的反复刺激扩增倍数明显高于基于GC33scFv的CAR-T的扩增倍数，表明基于L1H2/L1H6/L2H6/204A的CAR-T有优于GC33-CAR-T的临床治疗效果。
实施例8.CAR-T抑瘤消瘤实验(GPC3靶向)
按实施例5的方法制备表达GC33-BBz、L1H2-41BBz的CAR-T细胞以及未感染CAR-T的T细胞，按8x10e5CAR阳性细胞的剂量对6周龄的皮下荷瘤Huh7肿瘤(1x10e7/只)至80-150mm 3大小的NSG小鼠进行单次尾静脉注射CAR-T，每组11只。每周进行2次体重(图9A-C)和瘤体大小测量(图8A-C)。CAR-T注射后第11天，各组随机选4只小鼠尾静脉取抗凝血(肝素钠抗凝)进行血液内IFNg检测(BD human th1/th2CBA试剂盒)(图10)。CAR-T注射后第17天，各组随机选4只小鼠尾静脉取抗凝血(肝素钠抗凝)进行血液内人CD8细胞含量(图11A)和人CD4细胞含量(图11B)的流式检测。CAR-T注射后85天(肿瘤注射后98天)对消瘤后无复发的小鼠另侧背部进行二次荷瘤Huh7(1x10e7/只)，模拟肿瘤复发，观察肿瘤形成情况(图8A-C)。
实验结果显示，与GC33-BBz对照组相比，L1H2-BBz组具有更显著的抑瘤消瘤效果和抗复发能力(图8A-8C)，同时各组均未表现出明显的毒副作用(图9A-9C，体重未出现明显下降)；体内细胞因子检测结果显示L1H2-BBz组具有更高的因子分泌，有利于促进CAR-T在体内的扩增(图10)；体内细胞增殖检测结果显示L1H2-BBz组具有更高的体内细胞增殖能力，与更优的消瘤抑瘤效果成正相关(图11A-11B)。小鼠的存活曲线也显示出L1H2-BBz 组小鼠与GC33-BBz对照组相比有更长的存活周期。
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 (30)

  1. 嵌合抗原受体(CAR)，其中所述CAR包含GPC3结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，所述GPC3结合结构域包含特异性结合GPC3的抗体或其片段，其中所述抗体包含轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，所述LCDR1的氨基酸序列如SEQ ID NO：16所示，所述LCDR2的氨基酸序列如SEQ ID NO：17所示且所述LCDR3的氨基酸序列如SEQ ID NO：18所示。
  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1(HCDR1)，重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，所述HCDR1的氨基酸序列如SEQ ID NO：19所示，所述HCDR2的氨基酸序列如SEQ ID NO：20所示且所述HCDR3的氨基酸序列如SEQ ID NO：21所示。
  3. 根据权利要求1-2中任一项所述的CAR，其中所述抗体包含重链可变区，所述重链可变区的氨基酸序列如SEQ ID NO：29所示。
  4. 根据权利要求1-3中任一项所述的CAR，其中所述抗体包含轻链可变区，所述轻链可变区的氨基酸序列如SEQ ID NO：25所示。
  5. 根据权利要求1-4中任一项所述的CAR，其中所述抗体为单链抗体。
  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SEQ ID NO:30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，其中所述跨膜结构域包含源自选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。
  8. 根据权利要求1-7中任一项所述的CAR，其中所述跨膜结构域包含SEQ ID NO:36所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的CAR，其中所述共刺激结构域包含选自下述蛋白的共刺激结构域：CD137、CD28、4-1BB、OX-40和ICOS。
  10. 根据权利要求1-9中任一项所述的CAR，其中所述共刺激结构域包含SEQ ID NO:37所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的CAR，其中所述胞内信号传导结构域包含源自CD3ζ的信号传导结构域。
  12. 根据权利要求1-11中任一项所述的CAR，其中所述胞内信号传导结构域包含SEQ ID NO:38所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的CAR，其中所述CAR还包含铰链区，所述铰链区连 接所述GPC3结合结构域和所述跨膜结构域。
  14. 根据权利要求13所述的CAR，其中所述铰链区包含SEQ ID NO:39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CAR还包含连接信号肽。
  16. 根据权利要求15所述的CAR，其中所述信号肽包含SEQ ID NO:40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，其中编码所述CAR的核酸分子还连接启动子。
  18. 根据权利要求17所述的CAR，其中所述启动子为组成型启动子。
  19. 根据权利要求17-18中任一项所述的CAR，其中所述启动子包含SEQ ID NO:41所示的核苷酸序列。
  20. 根据权利要求1-19中任一项所述的CAR，其包含SEQ ID NO:31-34中任一项所示的氨基酸序列。
  21. 分离的核酸分子，其编码权利要求1-20中任一项所述的CAR。
  22. 编码CAR的分离的核酸分子，其包含SEQ ID NO:42-45中任一项所示的核酸序列。
  23. 一种载体，其包含权利要求21-22中任一项所述的核酸分子。
  24. 根据权利要求23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。
  25. 一种免疫效应细胞，其包含权利要求1-20中任一项所述的CAR，权利要求21-22中任一项所述的核酸分子，或权利要求23-24中任一项所述的载体。
  26. 根据权利要求25所述的细胞，其中所述免疫效应细胞选自T淋巴细胞。
  27. 一种制备免疫效应细胞的方法，其包括向免疫效应细胞中引入权利要求23所述的载体。
  28. 组合物，其包含权利要求25-26中任一项所述的免疫效应细胞。
  29. 权利要求1-20中任一项所述的CAR，权利要求21-22中任一项所述的核酸分子，权利要求23-24所述的载体，或权利要求25-26中任一项所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗与GPC3的表达相关的疾病或病症。
  30. 根据权利要求29所述的用途，其中所述与GPC3的表达相关的疾病或病症为癌症或恶性肿瘤。
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