WO2023246578A1 - Chimeric antigen receptor specifically binding to gpc3 and use thereof - Google Patents

Chimeric antigen receptor specifically binding to gpc3 and use thereof Download PDF

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Publication number
WO2023246578A1
WO2023246578A1 PCT/CN2023/100146 CN2023100146W WO2023246578A1 WO 2023246578 A1 WO2023246578 A1 WO 2023246578A1 CN 2023100146 W CN2023100146 W CN 2023100146W WO 2023246578 A1 WO2023246578 A1 WO 2023246578A1
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seq
variant
cdr
sequence
whose sequence
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PCT/CN2023/100146
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French (fr)
Chinese (zh)
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宋婷玉
侯攀燕
周阳
蔡珍珍
王江漫
葛均友
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成都科伦精准生物科技有限公司
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Publication of WO2023246578A1 publication Critical patent/WO2023246578A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to single-chain antibodies and chimeric antigen receptors (CAR) that specifically bind to GPC3.
  • the invention also relates to immune cells expressing the CAR, nucleic acid molecules encoding such CAR or co-expressed molecules and methods of preparing the engineered immune cells.
  • the present invention also relates to methods of using these CARs and immune cells to prevent and/or treat GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma.
  • HCC hepatocellular carcinoma
  • melanoma melanoma
  • ovarian clear cell carcinoma GPC3-positive tumors
  • Glypican-3 (Glypican-3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS and SGBS1) is a member of the heparan sulfate proteoglycan family. Phosphatidylinositol is anchored on the cell surface and is one of the representative liver cancer markers in current preclinical research. GPC3 is expressed in many human malignant tumor cells and serum, including hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma, and is rarely expressed in other cancers and normal tissues. GPC3 is a potential biomarker for HCC. It forms a complex with WNT, activates downstream signaling pathways, promotes the proliferation of liver cancer cells, and participates in the regulation of multiple signaling pathways closely related to tumor occurrence and development.
  • HCC hepatocellular carcinoma
  • melanoma melanoma and ovarian clear cell carcinoma
  • liver cancer is the fourth most common malignant tumor and the third leading cause of cancer death in my country, seriously threatening the lives and health of our people.
  • a large number of patients with hepatocellular carcinoma still lack precise and effective clinical treatments.
  • Most liver cancer patients are already in the advanced or late stage when diagnosed. Only 30% of patients have the opportunity for surgical resection.
  • the metastasis and recurrence rate within 5 years after resection is as high as 60% to 70%.
  • the overall 5-year survival rate is low, only 7% to 10%. .
  • Chimeric antigen receptor (CAR)-T cell therapy is considered one of the most promising cancer treatments and has become a new hope for humans to fight cancer. It cultivates immune cells collected from patients in vitro, transduces specific exogenous genes in vitro, amplifies them in vitro and then infuses them back into the patient's body to achieve the purpose of treating tumors in a non-MHC-restricted manner.
  • CAR-T cell therapy has achieved remarkable results in the treatment of hematological malignancies, with a complete remission rate of more than 90% for relapsed and refractory B-cell leukemia. Solid tumors account for approximately 90% of all malignant tumors, and their therapeutic drugs are in high demand. However, the current therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient. The main reason is that solid tumors have complex tumor microenvironments and high tumor heterogeneity.
  • the present invention provides a GPC3-targeting CAR or a modified immune system that co-expresses a GPC3-targeting CAR.
  • Cells and modified immune cells can be used in the treatment of GPC3-positive hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma.
  • HCC hepatocellular carcinoma
  • melanoma melanoma
  • ovarian clear cell carcinoma ovarian clear cell carcinoma
  • the inventor first developed a fully human antibody with low immunogenicity that can specifically recognize/bind GPC3.
  • the present invention further designed and constructed a CAR targeting GPC3.
  • the CAR of the present invention can direct immune effector cells specifically and reactively to GPC3-expressing cells (such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma) in a non-MHC-restricted manner so that they can be eliminated. Therefore, the GPC3-targeting CAR of the present invention has the potential to be used to prevent and/or treat GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma, and has significant clinical value.
  • HCC hepatocellular carcinoma
  • melanoma melanoma
  • ovarian clear cell carcinoma a non-MHC-restricted manner
  • the first aspect of the present invention provides an antigen-binding molecule, which includes a binding domain capable of specifically binding to GPC3; the antigen-binding molecule includes the following complementarity determining regions (CDRs):
  • CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:9; and/or, the variable light chain shown in SEQ ID NO:10 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
  • At least one CDR contains a mutation, which is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids).
  • the substitutions are conservative substitutions.
  • the CDRs are defined according to the Kabat, IMGT, Chothia or AbM numbering system.
  • the antigen-binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:
  • the variant described in any one of (1a)-(1f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition); preferably, the substitution is a conservative substitution.
  • the antigen-binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:
  • VH Heavy chain variable region containing the following 3 CDRs: the sequence is SEQ ID NO: 19 or a variant thereof CDR-H1; CDR-H2 whose sequence is SEQ ID NO: 20 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 21 or a variant thereof; and/or, a light chain variable comprising the following 3 CDRs Region (VL): CDR-L1 whose sequence is SEQ ID NO: 22 or its variant; CDR-L2 whose sequence is SEQ ID NO: 23 or its variant; CDR whose sequence is SEQ ID NO: 18 or its variant -L3;
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 34 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 46 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 47 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 56 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 67 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 68 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (2a)-(2f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition); preferably, the substitution is a conservative substitution.
  • the antigen-binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 25 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 51 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof
  • VL light chain variable region
  • a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 whose sequence is SEQ ID NO: 37 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof; The sequence is SEQ ID NO: 52 or a variant thereof CDR-H3 of the body; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 53 or its variant; whose sequence is SEQ ID NO: 54 or its variant CDR-L2 of the variant; CDR-L3 whose sequence is SEQ ID NO: 55 or a variant thereof;
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 72 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (3a)-(3f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition); preferably, the substitution is a conservative substitution.
  • the antibodies of the invention or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 81 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof
  • VL light chain variable region
  • a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 whose sequence is SEQ ID NO: 78 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 29 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or a variant thereof; whose sequence is SEQ ID NO: CDR-L3 of 31 or its variants;
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 97 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 99 or a variant thereof
  • CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof
  • CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (4a)-(4f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition). In certain embodiments, the substitutions are conservative substitutions.
  • the antigen-binding molecules of the invention comprise:
  • VH comprising a sequence as set forth in SEQ ID NO: 1 or a variant thereof and/or a VH comprising a sequence as set forth in SEQ ID NO: 2 VL of the sequence shown or its variant;
  • VH comprising the sequence shown in SEQ ID NO:3 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:4 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:5 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:6 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:7 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:9 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:10 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:11 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:12 or a variant thereof;
  • said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.
  • the antigen-binding molecule described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.
  • the heavy chain of the antigen-binding molecule comprises a heavy chain constant region from or derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).
  • the heavy chain of the antigen-binding molecule comprises a wild-type Fc region, or a mutated or chemically modified Fc region that has altered effector function (e.g., enhanced ADCC activity).
  • the light chain of the antigen-binding molecule comprises a light chain constant region from or derived from a human immunoglobulin (eg, kappa or lambda).
  • a human immunoglobulin eg, kappa or lambda
  • the antigen-binding molecule described in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the antigen-binding molecule is selected from the group consisting of full-length antibodies, Fab fragments, Fab' fragments, F(ab)' 2 fragments, F(ab)' 3 fragments, single chain antibodies (e.g., scFv, di- scFv or (scFv) 2 ), minibodies, disulfide-stabilized Fv proteins (dsFv) and single domain antibodies (sdAb, Nanobodies).
  • the VH and VL of the antigen-binding molecules of the invention are linked by one or more linkers.
  • the linker is typically a peptide linker, for example a flexible and/or soluble peptide linker, for example a glycine, serine and/or threonine rich peptide linker.
  • the linker also includes charged residues (such as lysine and/or glutamic acid), which can improve solubility.
  • the linker further includes one or more prolines.
  • the linker comprises one or several (eg, 1, 2, or 3) sequences represented by (GmS)n, where m is selected from an integer of 1-6 and n is selected from An integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2.
  • the linker has the sequence of SEQ ID NO: 110.
  • the antigen-binding molecules of the invention are single chain antibodies, such as scFv, di-scFv, or (scFv) 2 .
  • the single-chain antibody sequentially includes from its N-terminus to its C-terminus:
  • a VH-linker comprising the sequence shown in SEQ ID NO: 1 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO: 2 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO: 2 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO: 1 or a variant thereof; VL-linker of the sequence shown in 2 or its variant - VH containing the sequence shown in SEQ ID NO:1 or its variant;
  • a VH-linker comprising the sequence shown in SEQ ID NO:3 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:3 or a variant thereof; VL-linker of the sequence shown in 4 or a variant thereof - VH containing the sequence shown in SEQ ID NO:3 or a variant thereof;
  • a VH-linker comprising the sequence shown in SEQ ID NO:5 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:6 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:6 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:5 or a variant thereof;
  • VH-linker comprising the sequence shown in SEQ ID NO:7 or a variant thereof - a VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof; or, comprising a VL such as SEQ ID NO: VL-linker of the sequence shown in 8 or its variant- A VH comprising the sequence shown in SEQ ID NO:7 or a variant thereof;
  • a VH-linker comprising the sequence shown in SEQ ID NO:9 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:10 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:10 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:
  • a VH-linker comprising the sequence shown in SEQ ID NO:11 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:11 or a variant thereof;
  • said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, substitutions, deletions, or additions of 3, 4, or 5 amino acids); in certain embodiments, the substitutions are conservative substitutions.
  • the single-chain antibody comprises the sequence shown in any one of SEQ ID NO:86, 88, 90, 92, 94, 82 or a variant thereof, which variant is consistent with SEQ ID NO:86 , 88, 90, 92, 94 or 82 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least A sequence that is 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); in certain embodiments, the substitutions are conservative substitutions.
  • the antigen-binding molecules of the invention further comprise a constant region derived from a human immunoglobulin.
  • the heavy chain of the antigen-binding molecule comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4)
  • the light chain of the antigen-binding molecule comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).
  • the light chain constant region of an immunoglobulin eg kappa or lambda).
  • the heavy chain of the antigen-binding molecule comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof that has an or Substitution, deletion or addition of multiple amino acids (e.g., substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., 1, 2, 3, 4 or 5 substitution, deletion or addition of amino acids); and/or,
  • CH heavy chain constant region
  • the light chain of the antigen-binding molecule includes the light chain constant region (CL) of a human immunoglobulin or a variant thereof, which variant
  • the entity has one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived; For example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids).
  • the heavy chain constant region is an IgG, IgM, IgE, IgD or IgA heavy chain constant region.
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the light chain constant region is a kappa or lambda light chain constant region. In certain preferred embodiments, the light chain constant region is a human kappa light chain constant region.
  • the antigen-binding molecules of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology. For example, a DNA molecule encoding the antigen-binding molecule is obtained through chemical synthesis or PCR amplification, the obtained DNA molecule is inserted into an expression vector, and then the host cell is transfected. Then, the transfected host cells are cultured under specific conditions and express the antigen-binding molecule of the present invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly from recombinant host cells (Reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000 )).
  • Fab’ fragments can be obtained directly from host cells; Fab’ fragments can be chemically coupled to form F(ab’)2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab’)2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • a second aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antigen-binding molecule of the invention.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 100, (ii) a sequence substantially identical to SEQ ID NO: 100 (e.g., the same sequence as SEQ ID NO: 100) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 100, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
  • the nucleic acid molecule encoding the variable region of the antibody light chain includes: (iv) the nucleotide sequence shown in SEQ ID NO: 101, (v) A sequence that is substantially identical to SEQ ID NO:101 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO:101, or sequences with one or more nucleotide substitutions), or (vi) degenerate sequences of (iv) or (v) above.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 102, (ii) a sequence substantially identical to SEQ ID NO: 102 (e.g., the same sequence as SEQ ID NO: 102). A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 102, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
  • the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 103, (v) a sequence substantially identical to SEQ ID NO: 103 (e.g., the same sequence as SEQ ID NO: 103) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 103, or a sequence having one or more nucleotide substitutions), or (vi) the above ( Degenerate sequence of iv) or (v).
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 104, (ii) a sequence substantially identical to SEQ ID NO: 104 (e.g., the same sequence as SEQ ID NO: 104). A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 104, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
  • the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 105, (v) a sequence substantially identical to SEQ ID NO: 105 (e.g., the same sequence as SEQ ID NO: 105) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 105, or a sequence having one or more nucleotide substitutions), or (vi) the above ( Degenerate sequence of iv) or (v).
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 106, (ii) a sequence that is substantially the same as SEQ ID NO: 106 (e.g., the same sequence as SEQ ID NO: 106). A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 106, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
  • the nucleic acid molecule encoding the variable region of the antibody light chain includes: (iv) the nucleotide sequence shown in SEQ ID NO: 107, (v) A sequence that is substantially identical to SEQ ID NO:107 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO:107, or sequences with one or more nucleotide substitutions), or (vi) degenerate sequences of (iv) or (v) above.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 108, (ii) a sequence substantially identical to SEQ ID NO: 108 (e.g., the same sequence as SEQ ID NO: 108). A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity as compared to NO: 108, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
  • the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 109, (v) a sequence substantially identical to SEQ ID NO: 109 (e.g., the same sequence as SEQ ID NO: 109) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 109, or a sequence having one or more nucleotide substitutions), or (vi) the above ( Degenerate sequence of iv) or (v).
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO:84, (ii) a sequence substantially identical to SEQ ID NO:84 (e.g., the same sequence as SEQ ID NO:84). A sequence with at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO:84, or a sequence with one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
  • the nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO:85, (v) a sequence substantially identical to SEQ ID NO:85 (e.g., the same sequence as SEQ ID NO:85) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO:85, or a sequence having one or more nucleotide substitutions), or (vi) the above ( Degenerate sequence of iv) or (v).
  • the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO:87, (ii) a sequence substantially identical to SEQ ID NO:87 (e.g., a sequence similar to SEQ ID NO:87) A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO:87, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
  • the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO:89, (ii) a sequence substantially identical to SEQ ID NO:89 (e.g., a sequence similar to SEQ ID NO:89) Sequences having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO:89, or having one or more nucleotide substitutions sequence), or (iii) a degenerate sequence of (i) or (ii) above.
  • the isolated nucleic acid molecule comprises: (i) the nucleotide sequence set forth in SEQ ID NO: 91, (ii) a sequence substantially identical to SEQ ID NO: 91 (e.g., the same sequence as SEQ ID NO: 91 A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO: 91, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
  • the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO: 93, (ii) a sequence substantially identical to SEQ ID NO: 93 (e.g., a sequence similar to SEQ ID NO: 93) A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO: 93, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
  • the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO:95, (ii) a sequence substantially identical to SEQ ID NO:95 (e.g., a sequence similar to SEQ ID NO:95) A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO: 95, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
  • the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO:83, (ii) a sequence substantially identical to SEQ ID NO:83 (e.g., a sequence similar to SEQ ID NO:83) A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO:83, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
  • a third aspect of the invention provides a vector (eg a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above.
  • the vector of the invention is, for example, a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector or a viral vector; preferably, the vector is an expression vector; preferably, the vector It is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.
  • a fourth aspect of the invention provides a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention.
  • the host cell may be a eukaryotic cell (eg, mammalian cell, insect cell, yeast cell) or prokaryotic cell (eg, E. coli).
  • Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • host cells of the invention are mammalian cells, such as CHO (eg, CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).
  • host cells of the invention may be chimeric antigen receptor T cells (CAR-T).
  • the isolated nucleic acid molecule comprised by the host cell may comprise a nucleotide sequence encoding a chimeric antigen receptor further comprising a nucleotide sequence encoding a chimeric antigen receptor of the invention.
  • the nucleotide sequence of an antigen-binding molecule e.g., ScFv.
  • the host cell contains an isolated nucleic acid molecule encoding a chimeric antigen receptor comprising an antigen-binding molecule of the invention (eg, scFv).
  • the present invention also relates to a method for preparing an antigen-binding molecule of the present invention, comprising culturing a host cell as described above under conditions that allow protein expression, and recovering said host cell culture from the cultured host cell culture. Antigen-binding molecules.
  • the present invention relates to a CAR targeting GPC3, the characteristics of which include non-MHC restricted GPC3 recognition ability, which confer an immune cell (for example, T cell, NK cell, monocyte, macrophage or dendritic cell) expressing the CAR The ability to recognize GPC3-expressing cells (such as tumor cells) independent of antigen processing and presentation.
  • an immune cell for example, T cell, NK cell, monocyte, macrophage or dendritic cell
  • the fifth aspect of the present invention provides a chimeric antigen receptor comprising an antigen-binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain.
  • the antigen-binding domain contained in the chimeric antigen receptor of the present invention confers the ability of the CAR to recognize GPC3.
  • the antigen binding domain comprises the antigen binding molecule of the first aspect.
  • the antigen binding domain comprises the antigen binding molecule as a first antigen binding domain and further comprises a second antigen binding domain that does not bind GPC3.
  • the second antigen binding domain binds an antigen selected from: PD-1, PD-L1, CTLA-, CD3, ASGPR1, CD19, MSLN, PSMA, MUCl, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
  • the antigen binding domain is a single chain antibody.
  • the first antigen-binding domain comprises the sequence shown in any one of SEQ ID NO: 86, 88, 90, 92, 94, 82 or a variant thereof, which variant is identical to SEQ ID NO. NO: 86, 88, 90, 92, 94, 82 any one has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% , a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, or has one or several amino acid substitutions, deletions, or additions (e.g., 1 , substitution, deletion or addition of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids); preferred Specifically, the substitutions are conservative substitutions.
  • the transmembrane domain contained in the chimeric antigen receptor of the present invention can be any protein structure known in the art, as long as it can be thermodynamically stable in the cell membrane (especially the eukaryotic cell membrane).
  • the transmembrane domains of CARs suitable for use in the present invention can be derived from natural sources.
  • the transmembrane domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain may be a synthetic non-naturally occurring protein segment, such as a protein segment containing primarily hydrophobic residues such as leucine and valine.
  • the transmembrane domain is a transmembrane region selected from the following proteins: alpha, beta or zeta chain of T cell receptor, CD28, CD45, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1 and any combination thereof.
  • the transmembrane domain is a transmembrane region selected from the group consisting of CD8, CD28, CD4, PD-1, CD152 and CD154.
  • the transmembrane domain comprises a CD8 transmembrane region with the sequence set forth in SEQ ID NO: 111.
  • the chimeric antigen receptor of the present invention includes a spacer domain located between the extracellular antigen-binding domain and the transmembrane domain.
  • the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (eg, IgG1 or IgG4).
  • an immunoglobulin eg, IgG1 or IgG4
  • CH2 and CH3 extend the antigen-binding domain of the CAR away from the membrane of the CAR-expressing cell and more accurately mimic the size and domain structure of the native TCR structure.
  • the spacer domain comprises a hinge domain.
  • a hinge domain may be a stretch of amino acids typically found between two domains of a protein that may allow flexibility of the protein and movement of one or both domains relative to each other. Therefore, the hinge domain may be any amino acid sequence that provides the flexibility of the extracellular antigen-binding domain and its mobility relative to the transmembrane domain.
  • the hinge domain is the hinge region of a naturally occurring protein, or a portion thereof.
  • the spacer domain is selected from the hinge domain and/or the CH2 and CH3 regions of an immunoglobulin (eg, IgG1 or IgG4).
  • the hinge domain comprises the hinge region of CD8, IgG4, PD-1, CD152 or CD154.
  • the hinge domain comprises a CD8 hinge region with the sequence set forth in SEQ ID NO: 112.
  • the CAR of the invention may further comprise a signal peptide at its N-terminus.
  • a signal peptide is a polypeptide sequence that targets the sequence to which it is linked to a desired site.
  • the signal peptide can target the CAR to which it is linked to the secretory pathway of the cell and allow further integration and anchoring of the CAR into the lipid bilayer.
  • Signal peptides useful for CARs are known to those skilled in the art.
  • the signal peptide comprises a heavy chain signal peptide (eg, a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or CD8 ⁇ signal peptide.
  • the signal peptide is selected from the group consisting of CD8 ⁇ signal peptides.
  • the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 116.
  • the CARs of the invention can also be co-expressed with additional biologically active molecules.
  • the additional bioactive molecule may have its own proprietary signal peptide, which is named signal peptide-2 to distinguish it from the signal peptide in the previous paragraph.
  • the signal peptide-2 guides the transport of additional bioactive molecules to specific sites within the cell or outside the cell membrane.
  • the signal peptide-2 may be the same as or different from the signal peptide described in the previous paragraph.
  • the signal peptide-2 may be different from the signal peptide described in the previous paragraph.
  • the signal peptide-2 is an IL2 signal peptide (e.g., the amino acid sequence is set forth in SEQ ID NO: 129).
  • the intracellular signaling domain contained in the CAR of the present invention is involved in transmitting the signal generated by the combination of the CAR of the present invention and GPC3 into the immune effector cells, activating at least one normal effector function of the immune effector cells expressing the CAR , or enhance the secretion of at least one cytokine (e.g., IL-2, IFN- ⁇ ) by CAR-expressing immune effector cells.
  • cytokine e.g., IL-2, IFN- ⁇
  • the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain.
  • the primary signaling domain can be any intracellular signaling domain comprising an immunoreceptor tyrosine activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of a protein selected from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the primary signaling domain comprises the intracellular signaling domain of CD3 ⁇ .
  • the costimulatory signaling domain can be an intracellular signaling domain from a costimulatory molecule.
  • the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137(4-1BB), CD150(SLAMF1), CD270(HVEM), CD278(ICOS) or DAP10.
  • the costimulatory signaling domain is selected from the intracellular signaling domain of CD28, or the intracellular signaling domain of CD137(4-1BB), or a combination of fragments thereof.
  • the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains. In such embodiments, the two or more costimulatory signaling domains may be the same or different.
  • the intracellular signaling domain includes a primary signaling domain and at least one costimulatory signaling domain.
  • the primary signaling domain and the at least one costimulatory signaling domain can be concatenated in any order to the carboxyl terminus of the transmembrane domain.
  • the intracellular signaling domain may comprise the intracellular signaling domain of CD3 ⁇ and the intracellular signaling domain of CD137(4-1BB).
  • the intracellular signaling domain of CD3 ⁇ comprises the amino acid sequence set forth in SEQ ID NO: 113.
  • the intracellular signaling domain of CD137(4-1BB) comprises the amino acid sequence set forth in SEQ ID NO: 114.
  • the intracellular signaling domain of the chimeric antigen receptor has the sequence set forth in SEQ ID NO: 115.
  • the invention provides a chimeric antigen receptor that can specifically bind to GPC3.
  • the chimeric antigen receptor sequentially includes an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal from its N-terminus to its C-terminus. conductive domain.
  • the intracellular signaling domain is a costimulatory signaling domain and a primary signaling domain from N-terminus to C-terminus.
  • the spacer domain comprises a hinge region of CD8 (e.g., CD8 ⁇ ) (e.g., a hinge region with the sequence set forth in SEQ ID NO: 112).
  • the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8 ⁇ ) (e.g., a transmembrane region with the sequence set forth in SEQ ID NO: 111).
  • the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain
  • the primary signaling domain comprises an intracellular signaling domain of CD3 ⁇ (e.g., as Sequence shown in SEQ ID NO: 113)
  • the costimulatory signaling domain includes the intracellular signaling domain of CD137 (4-1BB) (for example, sequence shown in SEQ ID NO: 114); More preferably, The intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 115.
  • the chimeric antigen receptor includes the signal peptide, antigen-binding domain, spacer domain, transmembrane domain, and intracellular signaling domain in order from its N-terminus to its C-terminus. (From N-terminus to C-terminus are the costimulatory signaling domain and the primary signaling domain).
  • the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8 ⁇ signal peptide (e.g., a signal peptide with the sequence shown in SEQ ID NO: 116).
  • the CAR of the present invention includes the sequence shown in any one of SEQ ID NO: 117, 119, 121, 123, 125, 127 or a variant thereof, which variant is the same as the sequence from which it is derived.
  • Sequence comparison has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5) compared to the sequence from which it is derived substitution, deletion or addition of amino acids); preferably, the substitution is a conservative substitution.
  • the CAR described in the fifth aspect of the present invention can also be co-expressed with other biologically active molecules.
  • the self-cleaving peptide can prevent amino acids from forming covalent bonds during translation and maintain translation to continue. In this way, the translation product is "self-cleaved", thereby separating the chimeric antigen receptor of the present invention from other biologically active molecules. Therefore, when the CAR described in the fifth aspect of the present invention can also be co-expressed with other biologically active molecules, the chimeric antigen receptor that can specifically bind to GPC3 becomes an independent extracellular antigen-binding domain and a spacer domain.
  • transmembrane domain and intracellular signaling domain of CAR while other bioactive molecules can be secreted extracellularly or expressed into membrane chimeric polypeptides or proteins.
  • CAR-expressing immune cells expand and enrich in the tumor microenvironment, additional bioactive molecules are enriched in the tumor microenvironment and cooperate with CAR to exert anti-tumor effects.
  • a nucleic acid sequence encoding a CAR is linked to a nucleic acid sequence of another biologically active molecule through a nucleic acid sequence of a self-cleaving peptide.
  • the CAR can be at the N-terminus or C-terminus of another biologically active molecule.
  • the CAR is 5' to another biologically active molecule. Any self-cleaving peptide capable of causing the cleavage of the fusion protein into two independent proteins can be used in the present invention.
  • the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 130, and its nucleotide sequence can be optimized according to the needs of genetic recombination.
  • a fusion protein comprising a CAR and an additional biologically active molecule has the following structure:
  • N'-Signal peptide--Extracellular antigen-binding domain that specifically binds GPC3--Spacer domain-Transmembrane domain-Intracellular signaling domain-Self-cleaving peptide-Signal peptide-2--Additional biological activities Molecule-C'.
  • the signal peptide-2 is the same as or different from the N-terminal signal peptide.
  • the signal peptide-2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (e.g., as set forth in SEQ ID NO: 129).
  • Methods of generating chimeric antigen receptors and immune effector cells comprising the chimeric antigen receptors are known in the art and may include transfecting the cells with at least one polynucleotide encoding a CAR and in Expression of polynucleotides in cells.
  • a nucleic acid molecule encoding a CAR of the invention can be included in an expression vector (eg, a lentiviral vector) capable of expression in a host cell, such as a T cell, to produce the CAR.
  • the sixth aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor according to the fifth aspect.
  • nucleotide sequence encoding a chimeric antigen receptor of the invention can have a variety of different sequences. Therefore, unless otherwise stated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.
  • the nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect is selected from: (1) any of SEQ ID NO: 118, 120, 122, 124, 126, 128 The sequence shown in one item or a degenerate variant thereof; (2) A sequence that is substantially the same as the sequence described in (1), for example, at least 50%, at least 55% compared with the sequence described in (1) , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least A sequence that has 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or a sequence that has one or more nucleotide substitutions compared to the sequence described in (1); and The sequence substantially retains at least one biological activity of the nucleotide sequence from which it is derived (e.g., is capable of
  • the CAR of the present invention can also be co-expressed with other bioactive molecules to synergistically exert anti-tumor effects.
  • the seventh aspect of the present invention also provides a nucleic acid construct comprising a first nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect, and further comprising a second nucleic acid sequence encoding another biologically active molecule. .
  • the second nucleic acid sequence encodes an additional biologically active molecule that has anti-tumor activity.
  • the second nucleotide sequence encodes an additional biologically active molecule at its N-terminus.
  • One step contains signal peptide-2.
  • the first nucleotide sequence is located upstream of the second nucleotide sequence.
  • the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (eg, P2A, E2A, F2A, T2A, or any combination thereof).
  • a self-cleaving peptide eg, P2A, E2A, F2A, T2A, or any combination thereof.
  • the sequence encoding a self-cleaving peptide is linked to the 3' end of the first nucleotide sequence and to the 5' end of the second nucleotide sequence.
  • the self-cleaving peptide is P2A (e.g., as set forth in SEQ ID NO: 130).
  • the nucleic acid construct described in the seventh aspect sequentially includes from its 5' end to its 3' end: a nucleotide sequence encoding the signal peptide, a nucleic acid sequence encoding the antigen-binding domain Nucleotide sequence, nucleotide sequence encoding the spacer domain, nucleotide sequence encoding the transmembrane domain, nucleotide sequence encoding the intracellular signaling domain, encoding the self-cleaving peptide The nucleotide sequence of the sequence, the nucleotide sequence encoding the signal peptide-2, the nucleotide sequence encoding another biologically active molecule.
  • the eighth aspect of the present invention provides a vector, which contains the isolated nucleic acid molecule described in the second aspect and the sixth aspect, or the nucleic acid construct described in the seventh aspect.
  • the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors, viral vectors.
  • the vector is an expression vector.
  • the vector is an episomal vector.
  • the vector is a viral vector.
  • the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.
  • the vector is an episomal or non-integrating viral vector, such as an integration-deficient retrovirus or lentivirus.
  • a ninth aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect.
  • Vectors as described above can be introduced into host cells by various suitable means, such as calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vectors mediated transfection (such as liposomes) or viral vector-mediated transfection (such as lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological methods for transfer into host cells Means, such as transposon technology, CRISPR-Cas9 and other technologies.
  • the host cell comprises the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule, and the host cell expresses the chimeric antigen receptor of the invention.
  • the host cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct, the host cell expresses the chimeric antigen receptor of the invention and additional biologically active molecules .
  • the host cells are selected from mammalian (eg, human) immune cells.
  • the immune cells are derived from a patient or healthy donor.
  • the immune cells are selected from T lymphocytes, natural killer (NK) cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are derived from T lymphocytes or NK cells.
  • the tenth aspect of the present invention provides a method for preparing cells expressing the chimeric antigen receptor of the present invention, which includes: (1) providing a host cell; (2) converting the isolated nucleic acid molecule as described in the sixth aspect or containing the The vector of the nucleic acid molecule is introduced into the host cell to obtain a host cell capable of expressing the chimeric antigen receptor.
  • Also provided is a method for cells that co-express the chimeric antigen receptor of the present invention and other biologically active molecules which includes: (1) providing a host cell; (2) converting the nucleic acid construct described in the seventh aspect or containing the The vector of the nucleic acid construct is introduced into the host cell to obtain a host cell capable of co-expressing the chimeric antigen receptor and other biologically active molecules.
  • the host cells are selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages, and any combination thereof.
  • the immune cells are selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination of these cells.
  • the host cells are provided from a patient or a healthy donor and undergo pretreatment; the pretreatment includes sorting, activation and/or proliferation of immune cells;
  • the pretreating includes contacting the immune cells with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.
  • the nucleic acid molecule or vector in step (2), is introduced into the host cell via viral infection. In some embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host cell through non-viral vector transfection, such as through transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, Methods such as electroporation, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • non-viral vector transfection such as through transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, Methods such as electroporation, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • the method further includes: amplifying the host cells obtained in step (2).
  • the eleventh aspect of the present invention also provides a modified immune cell, which contains the expression of the second aspect of the present invention, The isolated nucleic acid molecule of the sixth aspect.
  • the modified immune cells express the chimeric antigen receptor described in the fifth aspect.
  • the twelfth aspect of the present invention also provides a modified immune cell, which contains the nucleic acid construct expressing the seventh aspect of the present invention.
  • the engineered immune cells express the chimeric antigen receptor of the fifth aspect and the additional biologically active molecule.
  • the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells, and any combination thereof.
  • the immune cells are obtained from a patient; alternatively, the immune cells are obtained from a healthy donor.
  • the immune cells are derived from T lymphocytes or NK cells.
  • the engineered immune cells have genes involved in immune rejection (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B, or HLA-C) and immune co-suppressive pathways or signaling molecules.
  • Transcription or expression of one or both target genes eg, PD-1, CTLA-4, or LAG-3 is inhibited.
  • the transcription or expression of the target gene is inhibited by a method selected from the group consisting of gene knockout (eg, CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
  • the present invention also provides a method for preparing modified immune cells, which includes: (1) providing immune cells from patients or healthy donors; (2) using the isolated nucleic acid molecules described in the sixth aspect, or the seventh aspect
  • the nucleic acid constructs, or vectors containing them, are introduced into the immune cells described in step (1) to obtain immune cells capable of expressing and optionally additional biologically active molecules.
  • the immune cells are pretreated, and the pretreatment includes sorting, activation, and/or proliferation of immune cells; more preferably, the pretreatment includes converting the immune cells into The cells are contacted with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating preconditioned immune cells.
  • the nucleic acid molecule or vector is introduced into the immune cell via viral infection in step (2).
  • the nucleic acid molecule or vector in step (2), is introduced into the immune cells by non-viral vector transfection, such as by calcium phosphate transfection, DEAE-dextran mediated transfection, Microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.
  • non-viral vector transfection such as by calcium phosphate transfection, DEAE-dextran mediated transfection, Microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.
  • step (2) is followed by a step of amplifying the immune cells obtained in step (2).
  • the present invention also provides an immune cell composition, the immune cell composition comprising any of the aforementioned
  • the immune cell composition comprising any of the aforementioned
  • modified immune cells and optionally unmodified and/or unsuccessfully modified immune cells.
  • These unmodified and/or unsuccessfully modified immune cells do not express the CAR of interest. Limited by the current technical level and some unknown reasons, not all immune cells can express the target CAR after modification.
  • immune cells that do not express CAR also have certain biological activities, so the immune cell composition can contain immune cells that express and do not express the CAR of interest, and the immune cell composition can still meet the needs of clinical application.
  • the engineered immune cells expressing the CAR of interest comprise approximately 10%-100%, preferably 40%-80%, of the total cell number of the immune cell composition.
  • the immune cell composition is cultured into an immune cell line, and thus, in another aspect, the invention also provides immune cell lines containing the immune cell composition.
  • the present invention provides a kit for preparing the modified immune cells described in any of the above aspects.
  • the kit includes the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a carrier containing them, and necessary solvents, such as sterile water, Physiological saline, or cell culture medium, such as LB culture medium, such as EliteCell primary T lymphocyte culture system (product number: PriMed-EliteCell-024), and optionally, instructions for use.
  • the present invention provides a pharmaceutical composition, which contains the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including the chimeric antigen receptor and another CAR construct co-expressing biologically active molecules), the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the The host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect, and a pharmaceutically acceptable carrier and/or excipients.
  • the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug with anti-tumor activity (e.g., anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti- CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX).
  • a drug with anti-tumor activity e.g., anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti- CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX.
  • the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the The modified exemption mentioned in the eleventh or twelfth aspect The immune cells, or the immune cell composition of the thirteenth aspect, and the additional pharmaceutically active agent may be administered simultaneously, separately, or sequentially.
  • the pharmaceutical composition of the present invention includes: the antigen-binding molecule described in the first aspect.
  • the pharmaceutical composition of the present invention includes: the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, or a vector containing them.
  • the pharmaceutical composition of the present invention includes: the modified immune cells described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect.
  • the modified immune cells described in the twelfth aspect, or the immune cell composition described in the thirteenth aspect can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, Gels, capsules, powders, granules, elixirs, tablets, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical compositions of the present invention should be sterile and stable under the conditions of production and storage.
  • One preferred dosage form is an injection.
  • Such injections may be sterile injectable solutions.
  • sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution such as 0.9% (w/v) NaCl
  • Glucose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • the modified immune cells described in the twelfth aspect, or the immune cell composition described in the thirteenth aspect can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, Intraocular, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection injection, intramuscular injection).
  • parenteral e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection injection, intramuscular injection.
  • route and/or mode of administration will vary depending on the intended purpose.
  • the antigen-binding molecule described in the first aspect of the invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) , the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect , the modified immune cells described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect are administered by intravenous injection or bolus injection.
  • the pharmaceutical composition of the present invention may include a "therapeutic effective amount” or a "preventive effective amount” of the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor (including chimeric antigen receptor) described in the fifth aspect. CAR construct co-expressed with another biologically active molecule), the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect , the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect.
  • “Prophylactically effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of disease.
  • a “therapeutically effective amount” means an amount sufficient to cure or at least partially prevent disease and its complications in a patient who is already suffering from the disease.
  • the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule), the second aspect or the sixth aspect The isolated nucleic acid molecule described in the seventh aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the eleventh aspect or the
  • the therapeutically effective amount of the modified immune cells described in the twelfth aspect, or the immune cell composition described in the thirteenth aspect may vary according to the following factors: the severity of the disease to be treated, the overall condition of the patient's own immune system status, general characteristics of the patient such as age, weight, and gender, the manner in which the drug is administered, and other treatments administered at the same time, etc.
  • the invention provides a method for preventing and/or treating a disease associated with expression of GPC3 in a subject (e.g., a human), said method comprising administering to a subject in need thereof An effective amount of the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule), the second aspect Or the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the eleventh aspect
  • the disease associated with expression of GPC3 is selected from proliferative diseases, such as tumors. In certain embodiments, the disease associated with expression of GPC3 is a non-tumor-related indication associated with expression of GPC3.
  • the tumor is a GPC3-positive tumor.
  • the tumor is selected from the group consisting of solid tumors (e.g., liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma, non-small cell One or a combination of lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma.
  • the tumor is selected from a hematological tumor (e.g., leukemia , lymphoma, etc.).
  • the method includes administering to the subject an effective amount of the antigen-binding molecule of the first aspect.
  • the method includes administering to the subject an effective amount of the modified immune cells described in the eleventh or twelfth aspect, or the immune cell combination described in the thirteenth aspect things.
  • the method includes the following steps: (1) providing the immune cells required by the subject (e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells); (2) introducing the isolated nucleic acid molecule described in the second or sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into step (1) of immune cells; (3) administering the immune cells obtained in step (2) to the subject for treatment.
  • the immune cells required by the subject e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells
  • the isolated nucleic acid molecule described in the second or sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into step (1) of immune cells
  • administering the immune cells obtained in step (2) to the subject for treatment.
  • the method administers immune cells expressing the CAR of interest to the subject by dose-fractionation, such as one, two, three or more divided administrations of partial doses, e.g., on the first day of treatment.
  • a first percentage of the total dose is administered on one day and a second percentage of the total dose is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day , e.g., administer a third percent of the total dose (e.g., remaining percentage).
  • 10% of the total dose of cells is administered on the first day of treatment, 30% of the total dose of cells is administered on the second day, and the remaining 60% of the total dose of cells is administered on the third day.
  • 50% of the total dose of cells is administered on the first day of treatment and on subsequent (e.g., second, third, fourth, fifth, sixth or seventh or later) treatment days. Apply 50% of the total dose to cells.
  • 1/3 of the total dose of cells is administered on the first day of treatment, and on subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later)
  • the total cell dose includes 1 ⁇ 10 7 to 10 ⁇ 10 8 CAR-positive immune cells of interest, for example, includes (1-5) ⁇ 10 7 to (5-10) ⁇ 10 8 CAR-positive immune cells of interest Immune Cells.
  • the physician may adjust the dosage or treatment regimen based on clinical circumstances such as the patient's status, tumor size and stage, or combination therapy agents.
  • the cells, the modified immune cells described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect are administered in combination with another agent .
  • the additional agents include (i) increasing cells comprising a CAR nucleic acid or CAR polypeptide (e.g., an immune cell expressing a CAR of the invention, a modified immune cell, or an immune cell composition of the invention) an agent that improves the efficacy of; (ii) improves a or Agents with multiple side effects; (iii) Additional pharmaceutically active agents with anti-tumor activity.
  • a CAR nucleic acid or CAR polypeptide e.g., an immune cell expressing a CAR of the invention, a modified immune cell, or an immune cell composition of the invention
  • reagents can be used in the administration of the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) described in the fifth aspect,
  • the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the The modified immune cells described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect are administered before, simultaneously or after.
  • the methods described above further include administering to the subject a second therapy, which may be any therapy known for use in tumors, such as surgery, chemotherapy, radiotherapy, immunotherapy, Gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof.
  • a second therapy which may be any therapy known for use in tumors, such as surgery, chemotherapy, radiotherapy, immunotherapy, Gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof.
  • the second therapy may be used separately or in combination with the methods described above; or, the second therapy may be used simultaneously or sequentially with the methods described above.
  • the subject can be a mammal, such as a human.
  • the invention provides the antigen-binding molecule described in the first aspect and the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) , the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect , the experience mentioned in the eleventh or twelfth aspect Use of modified immune cells, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect, in the preparation of medicaments for preventing and/or treating diseases related to the expression of GPC3 . All aspects such as dosage, dosage form, route of administration, indications, combination therapy, etc. in the aforementioned treatment methods can be applied to the use of the medicine.
  • the invention provides the antigen-binding molecule described in the first aspect and the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) , the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect , the modified immune cells described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect, for prevention and/or Treating diseases associated with GPC3 expression. All aspects such as dosage, dosage form, route of administration, indications, combination therapy, etc. in the aforementioned treatment methods can be applied to the use of the medicine.
  • CAR Chimeric Antigen Receptor CDR complementarity determining region in immunoglobulin variable region CDR-H1 complementarity determining region 1 in the immunoglobulin heavy chain variable region CDR-H2 complementarity determining region 2 in the immunoglobulin heavy chain variable region CDR-H3 Complementarity determining region 3 in the immunoglobulin heavy chain variable region CDR-L1 Complementarity determining region 1 in the immunoglobulin light chain variable region CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region FR antibody framework region: amino acid residues in the antibody variable region except CDR residues VH antibody heavy chain variable region VL antibody light chain variable region Kabat Immunoglobulin alignment and numbering system proposed by Elvin A.
  • IMGT International Immunogenetics Information System
  • Lefranc et al. Dev. Comparat. Immunol. 27:55-77, 2003.
  • Chothia Immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. Man (1989) Nature 342:878-883).
  • the definition of AbM AbM CDR comes from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272).
  • IL-2 Interleukin 2 IFN interferon PCR polymerase chain reaction FACS flow cytometry fluorescence sorting
  • antigen-binding molecule is an antibody molecule or antigen-binding fragment thereof.
  • the term “antibody” refers to an immunoglobulin molecule capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • Globulin molecules As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g. Fab, Fab', F(ab')2, Fv), single chain (e.g.
  • antibodies of the invention are not limited to any particular method of producing the antibodies.
  • Antibodies include antibodies of any type, such as IgG, IgA or IgM (or subclasses thereof), and the antibodies need not be of any particular type.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy chain constant regions corresponding to the different types of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • the subunit structures and three-dimensional configurations of different types of immunoglobulins are well known.
  • the heavy chain constant region consists of 4 domains (CH1, hinge region, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • VH and VL regions of antibodies can also be subdivided into highly denaturing regions called complementarity-determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • Each V H and V L consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • the variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDRs contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art.
  • the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof are preferably determined by the Kabat, Chothia, or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a fragment of a full-length antibody, that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and/ or compete with the full-length antibody for specific binding to the antigen, which is also referred to as the "antigen-binding portion.” See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes.
  • antigen-binding fragments of the antibody are generated by enzymatic or chemical cleavage of the intact antibody.
  • Non-limiting examples of antigen-binding fragments include camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab )' 3 fragments, Fd, Fv, scFv, di-scFv, (scFv) 2 , minibodies, diabodies, tribodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv”) and single structures Domain antibodies (sdAb, Nanobodies) and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136 middle.
  • the term “Fd” means an antibody fragment consisting of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • the term “F(ab') 2 fragment” means an antibody fragment consisting of two Fab fragments connected by a disulfide bridge on the hinge region Antibody fragment
  • the term “Fab'fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (from VH and CH1 domain).
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
  • Fc means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • a disulfide bond may also exist between VH and VL of scFv.
  • the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, scFv containing NH2 -VH-VH-COOH, NH2- VL-VL-COOH.
  • the scFv can form any engineering possible structure, single chain antibody (scFv), tandem antibody (tandem di-scFvs), bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond stabilized Fv protein, camel Ig , IgNAR, etc.
  • scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody. in this hair
  • scFv can form (scFv) 2 , which refers to two or more individual scFvs joining in parallel to form an antibody.
  • single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable Antibody fragments composed of regions) that retain the ability to specifically bind to the same antigen bound by the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003 ).
  • Single domain antibodies are also called nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
  • the expression “specific binding” or “specific targeting” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D the equilibrium dissociation constant
  • the term " KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473).
  • K D , kon and kdis values can be measured by any valid method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two Each peptide A certain position among them is occupied by lysine)
  • each molecule is identical at that position.
  • Percent identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared ⁇ 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions).
  • comparisons are made when two sequences are aligned to yield maximum identity.
  • alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the PAM120 weight residue table using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0).
  • the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm can be used using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector may include sequences that replicate directly and autonomously in the cell, or may include sequences sufficient to permit integration into the host cell DNA.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and viral vectors, etc.
  • Non-limiting examples of viral vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, vesicle viruses (such as SV40).
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "episomal vector” means that the vector is capable of replicating without integrating into the chromosomal DNA of the host and is not gradually lost by dividing host cells. It also means that the vector is extrachromosomally or episomally. copy.
  • viral vector is used broadly to refer to a nucleic acid molecule (eg, a transfer plasmid) that includes a virus-derived nucleic acid element that typically facilitates the transfer or integration of the nucleic acid molecule into the genome of a cell, or mediates the transfer of nucleic acid of virus particles.
  • viral particles will typically include various viral components and sometimes host cell components.
  • viral vector may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself.
  • Viral vectors and transfer plasmids contain structural and/or functional genetic elements derived primarily from viruses.
  • retroviral vector refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from retroviruses, or portions thereof.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from lentiviruses, or portions thereof (including LTRs).
  • the terms "lentiviral vector” and “lentiviral expression vector” may be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles. mentioned in this article and elements (such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.), it should be understood that the sequences of these elements exist in the form of RNA in the lentiviral particles of the invention and in the form of DNA in the DNA of the invention in the plasmid.
  • an "integration-deficient" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that is unable to integrate the viral genome into the genome of the host cell.
  • the integrase protein is mutated to specifically reduce its integrase activity.
  • Integration-deficient lentiviral vectors can be obtained by modifying the pol gene encoding the integrase protein to generate a mutant pol gene encoding an integration-deficient integrase.
  • integration-deficient viral vectors have been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes cells, NK cells, monocytes, macrophages or dendritic cells, etc.). Host cells can include single cells or populations of cells.
  • chimeric antigen receptor refers to a receptor that contains at least one extracellular antigen-binding domain, spacer domain, transmembrane domain, and cytoplasmic signaling domain (also referred to herein as "Intracellular signaling domain”) recombinant polypeptide construct that combines antibody-based specificity for an antigen of interest (e.g., GPC3) with an immune effector cell-activating intracellular domain to demonstrate resistance to expression of the antigen of interest (e.g., GPC3 ) cell-specific immune activity.
  • the expression “CAR-expressing immune effector cells” refers to immune effector cells that express CAR and have antigen specificity determined by the targeting domain of the CAR.
  • CARs for cancer treatment
  • Methods of making CARs are known in the art, see, e.g., Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368 :1509-1518, 2013; Han et al., J. Hematol. Oncol., 6:47, 2013; PCT patent publications WO2012/079000, WO2013/059593; and US patent publication 2012/0213783, all of which are incorporated by reference in their entirety Incorporated herein.
  • extracellular antigen-binding domain refers to a polypeptide capable of specifically binding to an antigen or receptor of interest. This domain will be able to interact with cell surface molecules. For example, the extracellular antigen-binding domain can be selected to recognize an antigen that is a cell surface marker on a target cell associated with a particular disease state.
  • intracellular signaling domain refers to the portion of a protein that conducts effector signaling functions and directs the cell to perform specialized functions. Therefore, the intracellular signaling domain has the ability to activate the expression of CAR The ability of an immune effector cell to perform at least one normal effector function.
  • the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • primary signaling domain refers to a portion of a protein capable of modulating primary activation of a TCR complex in a stimulatory manner or in an inhibitory manner.
  • Primary signaling domains that act in a stimulatory manner often contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • costimulatory signaling domain refers to the intracellular signaling domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes after binding to an antigen.
  • Non-limiting examples of costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278(ICOS), DAP10.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: sterile water, physiological saline, pH adjusters, surfactants , adjuvants, ionic strength enhancers, diluents, reagents to maintain osmotic pressure, reagents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solutions (eg, 0.01% polysorbate 20), pH buffer solutions (eg, phosphate buffer solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • glucose solution e.g., 5% glucose
  • surfactant-containing solutions eg, 0.01% polysorbate 20
  • pH buffer solutions eg, phosphate buffer solution
  • Ringer's solution e.g, Ringer's solution, and any combination thereof.
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom (eg, tumor) in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no further worsening) of the disease state, delaying or slowing the progression of the disease, ameliorating or alleviating the symptoms of the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
  • the term “subject” refers to a mammal, such as a primate mammal, such as a human.
  • the term “subject” is meant to include living organisms in which an immune response can be elicited.
  • the subject eg, a human
  • has a tumor eg, a GPC3-related tumor
  • the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect.
  • a disease-preventing (e.g., tumor) effective amount refers to an amount that is sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a tumor);
  • a disease-treating effective amount refers to an amount that is sufficient to cure or at least partially prevent an existing disease. The patient's disease and the amount of its complications. Determining such effective amounts is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
  • immune cell refers to a cell involved in an immune response, such as in promoting immune effector function.
  • immune 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 bone marrow-derived macrophages.
  • Immune cells of the invention may be self/autologous ("self") or non-self ("non-self", eg allogeneic, syngeneic or allogeneic).
  • autologous refers to cells from the same subject;
  • allogeneic refers to cells of the same species that are genetically different from the comparison cells;
  • isogenic means cells that are genetically different from the comparison cells.
  • allogeneic means cells from a different species than the comparison cells.
  • the cells of the invention are allogeneic.
  • T lymphocytes and/or NK cells.
  • T cell or “T lymphocyte” is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • the T cells may be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells.
  • T cells can be helper T cells (HTL; CD4 T cells) CD4 T cells, cytotoxic T cells (CTL; CD8 T cells), CD4CD8 T cells, CD4CD8 T cells, or any other subset of T cells.
  • T cells can include naive T cells and memory T cells.
  • immune cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils, and macrophages.
  • Immune cells also include progenitor cells of immune cells, wherein the progenitor cells can be induced to differentiate into immune cells in vivo or in vitro.
  • the immune cells include progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained within a population of CD34+ cells derived from cord blood, bone marrow, or circulating peripheral blood, which are administered in a subject Then differentiate into mature immune cells, or they can be induced to differentiate into mature immune cells in vitro.
  • HSCs hematopoietic stem cells
  • modified immune cell refers to an immune cell that expresses any of the CARs described herein, or has been introduced with any of the isolated nucleic acids or vectors described herein.
  • the CAR polypeptide can also be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods of introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell.
  • transient transformation methods can be used to transiently express the polynucleotide construct without the polynucleotide construct being integrated into the genome of the cell.
  • virus-mediated methods may be used.
  • Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (eg, retroviruses, adenoviruses), liposomes, and the like.
  • Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or microparticle bombardment.
  • the polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.
  • immune effector function refers to a function or response of an immune effector cell that enhances or promotes an immune attack on a target cell (eg, killing of the target cell, or inhibiting its growth or proliferation).
  • the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • the current therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient.
  • the main reason is that solid tumors have complex
  • the tumor microenvironment and tumor heterogeneity are high.
  • the present invention provides a GPC3-targeting CAR or an immune cell containing the CAR. By specifically targeting GPC3, it improves the killing of tumor antigen-expressing cells and reduces its off-target toxicity to a certain extent, thereby enhancing the tumor immunity of CAR-T cells. Killing effect.
  • Figure 1 shows the detection results of the killing activity of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T) on target cells.
  • Figure 2A shows the detection results of IFN- ⁇ secretion levels after activation of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T).
  • Figure 2B shows the detection results of IL-2 secretion levels after activation of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T).
  • Figure 3A shows the tumor volume change curve of B-NDG mice treated with PBS, blank T cells, CA3-T, CC8-T, and CH3-T.
  • Figure 3B shows the body weight change curve of B-NDG mice treated with PBS, blank T cells, CA3-T, CC8-T, and CH3-T.
  • sequence information involved in the present invention is provided as follows:
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was performed as described in FMAusubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the embodiments describe the invention by way of example and do not It is intended to limit the scope of the invention as claimed.
  • Biotinylated GPC3 and SV magnetic beads were used to screen the fully human phage library, and the screened products were tested for phage titration by plating. Mix the product of the first round of sifting with PBST and perform the second and third rounds of sifting according to the above steps.
  • a single colony was inoculated from the phage panning product titer plate into a 96-deep well plate, and monoclonal phage were detected by ELISA.
  • a total of 6 strains of fully human anti-GPC3 monoclonal antibodies CD4, CA3, CB5, CC8, CE9, and CH3 were screened.
  • the sequences of VH and VL were obtained, and CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 were obtained according to the Kabat, IMGT, Chothia, and AbM numbering systems. , CDR-L3 sequence (see Table 1 and Table 2 for specific sequences).
  • the candidate scFv sequence was constructed in the TGEX-KAL vector, and then transfected into expi293 cells for expression and purification of scFv-Fc protein.
  • SEC analysis experimental results show (Table 4) that the six candidate scfv sequence monomer peaks (main peak area) are all greater than 80%.
  • GPC3 scFv-Fc protein binding affinity a GPC3-expressing cell line (293T/GPC3+) was selected for cell binding assays.
  • Mouse IgG isotype antibody (Mouse IgG Isotype Control, from Thermo Fisher Sci.) was used as a negative control, and anti-GPC3 antibody GC33 (Ishiguro et al, 2008; for GC33 related sequences, see US20150259417A1) was used as a positive control.
  • Table 5 results of 6 candidate scFvs on GPC3 positive cells The affinity of 293T/GPC3+ was significantly better than that of the control group.
  • Example 2 Construction of lentiviral plasmid and virus packaging
  • a CAR lentiviral expression vector was further constructed.
  • the intracellular domain of CD137 (4-1BB) and the ITAM region of CD3 ⁇ are used as activation signals and fused with the above scFv.
  • the CD8 ⁇ signal peptide, CD8 hinge region, and CD8 transmembrane region are added to construct a chimeric antigen receptor expression Vector and constructed chimeric antigen receptor structure are shown in Table 6 below.
  • GE lymphocyte separation medium
  • cytokines and antibody complexes IL-2, 10ng/mL IL-7, 5ng/mL IL-15, 500ng/mL Anti at a final concentration of 300U/mL.
  • -CD3(OKT3), 2 ⁇ g/mL Anti-CD28 configuration continuously cultured for 48 hours.
  • CAR-T cells expressing the CARs (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T) described in Example 2 were obtained by the above method.
  • the nucleic acid sequence encoding the CAR is expressed under the drive of a promoter.
  • T cells transfected with lentivirus are labeled using GPC3 antigen and measured by flow cytometry to reflect the expression level of CAR on the surface of T cells.
  • the CAR positivity rate of the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS detection results are shown in Table 7 below. The results showed that the CAR positive rate of all CAR-T cells was greater than 10%, indicating that after lentivirus transfection of effector cells, CAR was successfully expressed and T cells expressing 6 types of GPC3-CAR chimeric antigen receptors were successfully constructed. (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T).
  • HEPG2-luc was placed in a 96-well plate in a 5% CO 2 37°C incubator for 30 minutes.
  • CAR-T centrifuge and resuspend CAR-T cells, GPC3-CAR and untransfected CAR blank T cells (UTD) in 1640 medium with 10% FBS as effector cells, and then follow different E/T (effector cells/target cells) were added to a 96-well plate containing HEPG2-luc at 100 ⁇ L/well, and the final volume was added to 200 ⁇ L/well, and cultured in a 37°C incubator with 5% CO 2 for 18 to 24 hours. After the culture is completed, take the well plate out of the incubator, add 20ul of fluorescence detection reagent, and use a microplate reader to detect the fluorescence reading.
  • E/T effector cells/target cells
  • the killing activity test results of CAR-T are shown in Figure 1.
  • the 6 types of CAR-T cells (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) constructed in this application were detected in different It can effectively lyse tumor cells at any E/T ratio.
  • CA3-T, CC8-T, CH3-T and CE9-T are particularly effective.
  • the effector cell/target cell ratio is 10
  • the lysis rate of tumor cells is as high as 98%.
  • Collect HepG2-luc cells use culture medium to adjust the cell density to 1 ⁇ 10 5 cells/mL, inoculate target cells in a 96-well plate at 100 ⁇ L/well, and resuspend CAR-T cells, GPC3-CAR and Blank T cells that have not been transfected with CAR are used as effector cells, and then added to a 96-well plate containing target cells at an E/T (effector cell/target cell) ratio of 1:1, 100 ⁇ L/well, and the final volume is filled to 200 ⁇ L/ Wells were incubated overnight in a 37 °C incubator with 5% CO 2 .
  • CAR-T cells constructed in this application (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) can kill tumor cells to varying degrees and release IFN- ⁇ ( The detection results are shown in Figure 2A) or IL-2 (the detection results are shown in Figure 2B).
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 HepG2 tumor cells on the right side or right scapula. When the average tumor volume reached 100-150 mm 3 , they were randomly divided into 6 groups. Each mouse was given cyclophosphate intraperitoneally. amide 100 mg/kg, and 5 ⁇ 10 6 CAR-T cells and blank T cells not transfected with CAR were reinfused into the tail vein the next day. Tumor diameters were measured with vernier calipers and mice were weighed twice a week. The results of the killing ability of CAR-T cells on target cells are shown in Figure 3A, and the changes in mouse body weight are shown in Figure 3B.
  • the CAR-T cells (CA3-T, CC8-T, CH3-T) constructed in this application have good inhibitory effects on tumor cells. There were no animal deaths or significant weight loss in all treatment groups during the observation period, and no obvious drug toxic reactions were observed. The mice were well tolerated during the treatment period.

Abstract

A chimeric antigen receptor specifically binding to GPC3, a modified immune cell expressing the chimeric antigen receptor, and a method for preparing the modified immune cell. These chimeric antigens and immune cells are used for a method for preventing and/or treating diseases associated with GPC3 expression.

Description

特异性结合GPC3的嵌合抗原受体及其应用Chimeric antigen receptor that specifically binds GPC3 and its application 技术领域Technical field
本发明涉及生物医药领域，具体而言，本发明涉特异性结合GPC3的单链抗体及嵌合抗原受体(CAR)。本发明还涉及表达所述CAR的免疫细胞，编码这种CAR或共表达分子的核酸分子以及制备所述经改造的免疫细胞的方法。本发明还涉及这些CAR和免疫细胞用于预防和/或治疗肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌等GPC3阳性肿瘤的方法。The present invention relates to the field of biomedicine. Specifically, the present invention relates to single-chain antibodies and chimeric antigen receptors (CAR) that specifically bind to GPC3. The invention also relates to immune cells expressing the CAR, nucleic acid molecules encoding such CAR or co-expressed molecules and methods of preparing the engineered immune cells. The present invention also relates to methods of using these CARs and immune cells to prevent and/or treat GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma.
背景技术Background technique
磷脂酰肌醇蛋白聚糖3(Glypican-3，GPC3也称为DGSX，GTR2-2，MXR7，OCI-5，SDYS，SGB，SGBS和SGBS1)为硫酸乙酰肝素蛋白多糖家族一员，通过糖基化的磷脂酰肌醇锚定在细胞表面，是目前临床前研究中具有代表性的肝癌标志物之一。GPC3在许多人类恶性肿瘤细胞及血清中表达，包括肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌，在其他癌症和正常组织中表达很少。GPC3是HCC的潜在生物标志物，其与WNT形成复合体，激活下游信号通路，促进肝癌细胞的增殖，参与多个与肿瘤发生和发展密切相关的信号通路的调节。Glypican-3 (Glypican-3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS and SGBS1) is a member of the heparan sulfate proteoglycan family. Phosphatidylinositol is anchored on the cell surface and is one of the representative liver cancer markers in current preclinical research. GPC3 is expressed in many human malignant tumor cells and serum, including hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma, and is rarely expressed in other cancers and normal tissues. GPC3 is a potential biomarker for HCC. It forms a complex with WNT, activates downstream signaling pathways, promotes the proliferation of liver cancer cells, and participates in the regulation of multiple signaling pathways closely related to tumor occurrence and development.
原发性肝癌是我国第4位的常见恶性肿瘤和第3位的肿瘤致死原因，严重威胁我国人民的生命健康。我国每年肝癌新发病例约46.6万，约占全球每年新发病例的55％，我国每年约42.2万人因肝癌而死亡。大量肝细胞癌患者依然缺乏精准有效的临床治疗手段。肝癌患者诊断时多已处于进展期或晚期，仅30％的患者有手术切除机会，切除后5年内转移、复发率高达60％～70％，总体5年生存率低，仅7％～10％。嵌合抗原受体(CAR)-T细胞疗法被认为是最有前途的癌症治疗方法之一，成为了人类对抗癌症的新希望。它通过从病人体内采集的免疫细胞进行体外培养，在体外转导特定的外源基因，体外扩增后回输到病人体内，以非MHC限制性的方式达到治疗肿瘤的目的。CAR-T细胞疗法在血液恶行肿瘤治疗中取得了显著的疗效，对复发难治性B细胞白血病的完全缓解率超过90％。实体瘤约占所有恶行肿瘤的90％，其治疗药物需求量大。但是，CAR-T细胞治疗目前在实体瘤中的治疗效果仍不足，其主要原因是实体瘤有复杂的肿瘤微环境和肿瘤异质性高。Primary liver cancer is the fourth most common malignant tumor and the third leading cause of cancer death in my country, seriously threatening the lives and health of our people. There are about 466,000 new cases of liver cancer in my country every year, accounting for about 55% of the new cases in the world every year. About 422,000 people die from liver cancer in my country every year. A large number of patients with hepatocellular carcinoma still lack precise and effective clinical treatments. Most liver cancer patients are already in the advanced or late stage when diagnosed. Only 30% of patients have the opportunity for surgical resection. The metastasis and recurrence rate within 5 years after resection is as high as 60% to 70%. The overall 5-year survival rate is low, only 7% to 10%. . Chimeric antigen receptor (CAR)-T cell therapy is considered one of the most promising cancer treatments and has become a new hope for humans to fight cancer. It cultivates immune cells collected from patients in vitro, transduces specific exogenous genes in vitro, amplifies them in vitro and then infuses them back into the patient's body to achieve the purpose of treating tumors in a non-MHC-restricted manner. CAR-T cell therapy has achieved remarkable results in the treatment of hematological malignancies, with a complete remission rate of more than 90% for relapsed and refractory B-cell leukemia. Solid tumors account for approximately 90% of all malignant tumors, and their therapeutic drugs are in high demand. However, the current therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient. The main reason is that solid tumors have complex tumor microenvironments and high tumor heterogeneity.
本发明提供一种靶向GPC3的CAR或者共表达靶向GPC3的CAR的经改造的免疫 细胞，修饰后的免疫细胞可以用于GPC3阳性的肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌等的治疗。The present invention provides a GPC3-targeting CAR or a modified immune system that co-expresses a GPC3-targeting CAR. Cells and modified immune cells can be used in the treatment of GPC3-positive hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma.
发明内容Contents of the invention
在本申请中，发明人首先开发了免疫原性低的能够特异性识别/结合GPC3的全人源抗体。在此基础上，本发明进一步设计构建了靶向GPC3的CAR。本发明的CAR能够以非MHC限制的方式将免疫效应细胞特异性和反应性指向表达GPC3的细胞(例如肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌)从而使其被清除。因此，本发明的靶向GPC3的CAR具有用于预防和/或治疗例如肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌等GPC3阳性肿瘤的潜力，具有重大的临床价值。In this application, the inventor first developed a fully human antibody with low immunogenicity that can specifically recognize/bind GPC3. On this basis, the present invention further designed and constructed a CAR targeting GPC3. The CAR of the present invention can direct immune effector cells specifically and reactively to GPC3-expressing cells (such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma) in a non-MHC-restricted manner so that they can be eliminated. Therefore, the GPC3-targeting CAR of the present invention has the potential to be used to prevent and/or treat GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma, and has significant clinical value.
抗原结合分子antigen binding molecules
本发明第一方面提供了一种抗原结合分子，其包含能够与GPC3特异性结合的结合结构域；所述抗原结合分子包含如下的互补决定区(CDRs)：The first aspect of the present invention provides an antigen-binding molecule, which includes a binding domain capable of specifically binding to GPC3; the antigen-binding molecule includes the following complementarity determining regions (CDRs):
(a)SEQ ID NO:1所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:2所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(a) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and/or, the light chain variable region shown in SEQ ID NO: 2 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
或，or,
(b)SEQ ID NO:3所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:4所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(b) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:3; and/or, the light chain variable region shown in SEQ ID NO:4 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
或，or,
(c)SEQ ID NO:5所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:6所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(c) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:5; and/or, the variable light chain shown in SEQ ID NO:6 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
或，or,
(d)SEQ ID NO:7所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(d) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:7; and/or, the variable light chain shown in SEQ ID NO:8 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
或， or,
(e)SEQ ID NO:9所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:10所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(e) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:9; and/or, the variable light chain shown in SEQ ID NO:10 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
或，or,
(f)SEQ ID NO:11所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:12所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(f) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 11; and/or, the light chain variable region shown in SEQ ID NO: 12 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
或or
(g)下述重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3，和/或下述轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3，其中，所述重链可变区(VH)和/或轻链可变区(VL)与(a)至(f)任一所述的重链可变区和/或轻链可变区相比，至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。(g) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) described below, and/or CDR-L1, CDR-L1, CDR-H3 contained in the light chain variable region (VL) described below L2 and CDR-L3, wherein the heavy chain variable region (VH) and/or light chain variable region (VL) are consistent with the heavy chain variable region and/or any one of (a) to (f). Compared to the light chain variable region, at least one CDR contains a mutation, which is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids).
在某些实施方案中，所述的置换为保守置换。In certain embodiments, the substitutions are conservative substitutions.
在某些实施方案中，所述CDR根据Kabat、IMGT、Chothia或AbM编号系统定义。In certain embodiments, the CDRs are defined according to the Kabat, IMGT, Chothia or AbM numbering system.
在某些实施方案中，本发明的抗原结合分子包含重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：In certain embodiments, the antigen-binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:
(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：13或其变体的CDR-H1；序列为SEQ ID NO：14或其变体的CDR-H2；序列为SEQ ID NO：15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：16或其变体的CDR-L1；序列为SEQ ID NO：17或其变体的CDR-L2；序列为SEQ ID NO：18或其变体的CDR-L3；(1a) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 13 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 14 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 16 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 17 or its variant; CDR-L3 whose sequence is SEQ ID NO: 18 or its variant;
或，or,
(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：26或其变体的CDR-H1；序列为SEQ ID NO：27或其变体的CDR-H2；序列为SEQ ID NO：28或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：29或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：31或其变体的CDR-L3；(1b) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 29 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 31 or its variant;
或， or,
(1c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：39或其变体的CDR-H1；序列为SEQ ID NO：40或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：44或其变体的CDR-L3；(1c) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 39 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 40 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 42 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 43 or its variant; CDR-L3 whose sequence is SEQ ID NO: 44 or its variant;
或，or,
(1d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：26或其变体的CDR-H1；序列为SEQ ID NO：27或其变体的CDR-H2；序列为SEQ ID NO：52或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：53或其变体的CDR-L1；序列为SEQ ID NO：54或其变体的CDR-L2；序列为SEQ ID NO：55或其变体的CDR-L3；(1d) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 52 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 53 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 54 or its variants; CDR-L3 whose sequence is SEQ ID NO: 55 or its variants;
或，or,
(1e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：60或其变体的CDR-H1；序列为SEQ ID NO：61或其变体的CDR-H2；序列为SEQ ID NO：62或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：63或其变体的CDR-L1；序列为SEQ ID NO：64或其变体的CDR-L2；序列为SEQ ID NO：65或其变体的CDR-L3；(1e) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 60 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 61 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 63 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 64 or its variants; CDR-L3 whose sequence is SEQ ID NO: 65 or its variants;
或，or,
(1f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：26或其变体的CDR-H1；序列为SEQ ID NO：27或其变体的CDR-H2；序列为SEQ ID NO：73或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：74或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：75或其变体的CDR-L3；(1f) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 74 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 75 or its variant;
其中，(1a)-(1f)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Wherein, the variant described in any one of (1a)-(1f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition); preferably, the substitution is a conservative substitution.
在某些实施方案中，本发明的抗原结合分子包含重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：In certain embodiments, the antigen-binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:
(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：19或其变体的 CDR-H1；序列为SEQ ID NO：20或其变体的CDR-H2；序列为SEQ ID NO：21或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：22或其变体的CDR-L1；序列为SEQ ID NO：23或其变体的CDR-L2；序列为SEQ ID NO：18或其变体的CDR-L3；(2a) Heavy chain variable region (VH) containing the following 3 CDRs: the sequence is SEQ ID NO: 19 or a variant thereof CDR-H1; CDR-H2 whose sequence is SEQ ID NO: 20 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 21 or a variant thereof; and/or, a light chain variable comprising the following 3 CDRs Region (VL): CDR-L1 whose sequence is SEQ ID NO: 22 or its variant; CDR-L2 whose sequence is SEQ ID NO: 23 or its variant; CDR whose sequence is SEQ ID NO: 18 or its variant -L3;
或，or,
(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：32或其变体的CDR-H1；序列为SEQ ID NO：33或其变体的CDR-H2；序列为SEQ ID NO：34或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：35或其变体的CDR-L1；序列为SEQ ID NO：36或其变体的CDR-L2；序列为SEQ ID NO：31或其变体的CDR-L3；(2b) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 32 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 34 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 35 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 36 or its variant; CDR-L3 whose sequence is SEQ ID NO: 31 or its variant;
或，or,
(2c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：45或其变体的CDR-H1；序列为SEQ ID NO：46或其变体的CDR-H2；序列为SEQ ID NO：47或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：48或其变体的CDR-L1；序列为SEQ ID NO：49或其变体的CDR-L2；序列为SEQ ID NO：44或其变体的CDR-L3；(2c) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 45 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 46 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 47 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 48 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 49 or its variant; CDR-L3 whose sequence is SEQ ID NO: 44 or its variant;
或，or,
(2d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：32或其变体的CDR-H1；序列为SEQ ID NO：33或其变体的CDR-H2；序列为SEQ ID NO：56或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：57或其变体的CDR-L1；序列为SEQ ID NO：58或其变体的CDR-L2；序列为SEQ ID NO：59或其变体的CDR-L3；(2d) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 32 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 56 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 57 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 58 or its variant; CDR-L3 whose sequence is SEQ ID NO: 59 or its variant;
或，or,
(2e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：66或其变体的CDR-H1；序列为SEQ ID NO：67或其变体的CDR-H2；序列为SEQ ID NO：68或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：69或其变体的CDR-L1；序列为SEQ ID NO：70或其变体的CDR-L2；序列为SEQ ID NO：65或其变体的CDR-L3；(2e) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 66 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 67 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 68 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 69 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 70 or its variant; CDR-L3 whose sequence is SEQ ID NO: 65 or its variant;
或，or,
(2f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：32或其变体的 CDR-H1；序列为SEQ ID NO：33或其变体的CDR-H2；序列为SEQ ID NO：76或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：77或其变体的CDR-L1；序列为SEQ ID NO：36或其变体的CDR-L2；序列为SEQ ID NO：75或其变体的CDR-L3；(2f) A heavy chain variable region (VH) containing the following 3 CDRs: the sequence is SEQ ID NO: 32 or a variant thereof CDR-H1; CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 76 or a variant thereof; and/or, a light chain variable comprising the following 3 CDRs Region (VL): CDR-L1 whose sequence is SEQ ID NO: 77 or its variants; CDR-L2 whose sequence is SEQ ID NO: 36 or its variants; CDR whose sequence is SEQ ID NO: 75 or its variants -L3;
其中，(2a)-(2f)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Wherein, the variant described in any one of (2a)-(2f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition); preferably, the substitution is a conservative substitution.
在某些实施方案中，本发明的抗原结合分子包含重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：In certain embodiments, the antigen-binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:
(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：24或其变体的CDR-H1；序列为SEQ ID NO：25或其变体的CDR-H2；序列为SEQ ID NO：15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：16或其变体的CDR-L1；序列为SEQ ID NO：17或其变体的CDR-L2；序列为SEQ ID NO：18或其变体的CDR-L3；(3a) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 24 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 25 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 16 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 17 or its variant; CDR-L3 whose sequence is SEQ ID NO: 18 or its variant;
或，or,
(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：37或其变体的CDR-H1；序列为SEQ ID NO：38或其变体的CDR-H2；序列为SEQ ID NO：28或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：29或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：31或其变体的CDR-L3；(3b) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 37 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 29 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 31 or its variant;
或，or,
(3c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：50或其变体的CDR-H1；序列为SEQ ID NO：51或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：44或其变体的CDR-L3；(3c) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 50 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 51 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 42 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 43 or its variant; CDR-L3 whose sequence is SEQ ID NO: 44 or its variant;
或，or,
(3d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：37或其变体的CDR-H1；序列为SEQ ID NO：38或其变体的CDR-H2；序列为SEQ ID NO：52或其变 体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：53或其变体的CDR-L1；序列为SEQ ID NO：54或其变体的CDR-L2；序列为SEQ ID NO：55或其变体的CDR-L3；(3d) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 whose sequence is SEQ ID NO: 37 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof; The sequence is SEQ ID NO: 52 or a variant thereof CDR-H3 of the body; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 53 or its variant; whose sequence is SEQ ID NO: 54 or its variant CDR-L2 of the variant; CDR-L3 whose sequence is SEQ ID NO: 55 or a variant thereof;
或，or,
(3e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：71或其变体的CDR-H1；序列为SEQ ID NO：72或其变体的CDR-H2；序列为SEQ ID NO：62或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：63或其变体的CDR-L1；序列为SEQ ID NO：64或其变体的CDR-L2；序列为SEQ ID NO：65或其变体的CDR-L3；(3e) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 71 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 72 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 63 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 64 or its variants; CDR-L3 whose sequence is SEQ ID NO: 65 or its variants;
或，or,
(3f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：37或其变体的CDR-H1；序列为SEQ ID NO：38或其变体的CDR-H2；序列为SEQ ID NO：73或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：74或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：75或其变体的CDR-L3；(3f) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 37 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 74 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 75 or its variant;
其中，(3a)-(3f)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Wherein, the variant described in any one of (3a)-(3f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition); preferably, the substitution is a conservative substitution.
在某些实施方案中，本发明的抗体或其抗原结合片段包含重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：In certain embodiments, the antibodies of the invention or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:
(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：80或其变体的CDR-H1；序列为SEQ ID NO：81或其变体的CDR-H2；序列为SEQ ID NO：15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：16或其变体的CDR-L1；序列为SEQ ID NO：17或其变体的CDR-L2；序列为SEQ ID NO：18或其变体的CDR-L3；(4a) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 80 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 81 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 16 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 17 or its variant; CDR-L3 whose sequence is SEQ ID NO: 18 or its variant;
或，or,
(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：78或其变体的CDR-H1；序列为SEQ ID NO：79或其变体的CDR-H2；序列为SEQ ID NO：28或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：29或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO： 31或其变体的CDR-L3；(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 whose sequence is SEQ ID NO: 78 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 29 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or a variant thereof; whose sequence is SEQ ID NO: CDR-L3 of 31 or its variants;
或，or,
(4c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：96或其变体的CDR-H1；序列为SEQ ID NO：97或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：44或其变体的CDR-L3；(4c) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 96 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 97 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 42 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 43 or its variant; CDR-L3 whose sequence is SEQ ID NO: 44 or its variant;
或，or,
(4d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：78或其变体的CDR-H1；序列为SEQ ID NO：79或其变体的CDR-H2；序列为SEQ ID NO：52或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：53或其变体的CDR-L1；序列为SEQ ID NO：54或其变体的CDR-L2；序列为SEQ ID NO：55或其变体的CDR-L3；(4d) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 78 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 52 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 53 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 54 or its variants; CDR-L3 whose sequence is SEQ ID NO: 55 or its variants;
或，or,
(4e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：98或其变体的CDR-H1；序列为SEQ ID NO：99或其变体的CDR-H2；序列为SEQ ID NO：62或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：63或其变体的CDR-L1；序列为SEQ ID NO：64或其变体的CDR-L2；序列为SEQ ID NO：65或其变体的CDR-L3；(4e) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 98 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 99 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 63 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 64 or its variants; CDR-L3 whose sequence is SEQ ID NO: 65 or its variants;
或，or,
(4f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：78或其变体的CDR-H1；序列为SEQ ID NO：79或其变体的CDR-H2；序列为SEQ ID NO：73或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：74或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：75或其变体的CDR-L3；(4f) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 78 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 74 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 75 or its variant;
其中，(4a)-(4f)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。在某些实施方案中，所述的置换是保守置换。Wherein, the variant described in any one of (4a)-(4f) has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids) compared to the sequence from which it is derived. substitution, deletion or addition). In certain embodiments, the substitutions are conservative substitutions.
在某些实施方案中，本发明的抗原结合分子包含：In certain embodiments, the antigen-binding molecules of the invention comprise:
(a)包含如SEQ ID NO:1所示的序列或其变体的VH和/或包含如SEQ ID NO:2所 示的序列或其变体的VL；(a) A VH comprising a sequence as set forth in SEQ ID NO: 1 or a variant thereof and/or a VH comprising a sequence as set forth in SEQ ID NO: 2 VL of the sequence shown or its variant;
或，or,
(b)包含如SEQ ID NO:3所示的序列或其变体的VH和/或包含如SEQ ID NO:4所示的序列或其变体的VL；(b) VH comprising the sequence shown in SEQ ID NO:3 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:4 or a variant thereof;
或，or,
(c)包含如SEQ ID NO:5所示的序列或其变体的VH和/或包含如SEQ ID NO:6所示的序列或其变体的VL；(c) VH comprising the sequence shown in SEQ ID NO:5 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:6 or a variant thereof;
或or
(d)包含如SEQ ID NO:7所示的序列或其变体的VH和/或包含如SEQ ID NO:8所示的序列或其变体的VL；(d) VH comprising the sequence shown in SEQ ID NO:7 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof;
或，or,
(e)包含如SEQ ID NO:9所示的序列或其变体的VH和/或包含如SEQ ID NO:10所示的序列或其变体的VL；(e) VH comprising the sequence shown in SEQ ID NO:9 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:10 or a variant thereof;
或，or,
(f)包含如SEQ ID NO:11所示的序列或其变体的VH和/或包含如SEQ ID NO:12所示的序列或其变体的VL；(f) VH comprising the sequence shown in SEQ ID NO:11 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:12 or a variant thereof;
其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。wherein said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.
在某些实施方案中，上述任一实施方案中所述的抗原结合分子可以包含来自或源自人免疫球蛋白的恒定区。In certain embodiments, the antigen-binding molecule described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.
在某些实施方案中，所述抗原结合分子的重链包含来自或源自人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区。在某些实施方案中，所述抗原结合分子的重链包含野生型Fc区，或者包含突变的或化学修饰的Fc区，其与野生型Fc区相比具有改变的效应子功能(例如增强的ADCC活性)。In certain embodiments, the heavy chain of the antigen-binding molecule comprises a heavy chain constant region from or derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the heavy chain of the antigen-binding molecule comprises a wild-type Fc region, or a mutated or chemically modified Fc region that has altered effector function (e.g., enhanced ADCC activity).
在某些实施方案中，所述抗原结合分子的轻链包含来自或源自人免疫球蛋白(例如κ或λ)的轻链恒定区。 In certain embodiments, the light chain of the antigen-binding molecule comprises a light chain constant region from or derived from a human immunoglobulin (eg, kappa or lambda).
在某些实施方案中，上述任一实施方案中所述的抗原结合分子为鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。In certain embodiments, the antigen-binding molecule described in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
在某些实施方案中，所述抗原结合分子选自全长抗体、Fab片段、Fab'片段、F(ab)'2片段、F(ab)'3片段、单链抗体(例如scFv、di-scFv或(scFv)2)、微型抗体、二硫键稳定的Fv蛋白(dsFv)和单结构域抗体(sdAb，纳米抗体)。In certain embodiments, the antigen-binding molecule is selected from the group consisting of full-length antibodies, Fab fragments, Fab' fragments, F(ab)' 2 fragments, F(ab)' 3 fragments, single chain antibodies (e.g., scFv, di- scFv or (scFv) 2 ), minibodies, disulfide-stabilized Fv proteins (dsFv) and single domain antibodies (sdAb, Nanobodies).
在某些实施方案中，本发明的抗原结合分子的VH和VL通过一个或多个连接子连接。连接子通常是肽接头，例如柔性和/或可溶性肽接头，例如富含甘氨酸、丝氨酸和/或苏氨酸的肽接头。在一些实施方案中，连接子还包括带电荷的残基(如赖氨酸和/或谷氨酸)，其可以改善溶解性。在一些实施方案中，连接子还包括一个或多个脯氨酸。In certain embodiments, the VH and VL of the antigen-binding molecules of the invention are linked by one or more linkers. The linker is typically a peptide linker, for example a flexible and/or soluble peptide linker, for example a glycine, serine and/or threonine rich peptide linker. In some embodiments, the linker also includes charged residues (such as lysine and/or glutamic acid), which can improve solubility. In some embodiments, the linker further includes one or more prolines.
在某些实施方案中，所述连接子包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为1或2。在某些实施方案中，所述连接子具有SEQ ID NO：110的序列。In certain embodiments, the linker comprises one or several (eg, 1, 2, or 3) sequences represented by (GmS)n, where m is selected from an integer of 1-6 and n is selected from An integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2. In certain embodiments, the linker has the sequence of SEQ ID NO: 110.
在某些实施方案中，本发明的抗原结合分子是单链抗体，例如scFv、di-scFv或(scFv)2。In certain embodiments, the antigen-binding molecules of the invention are single chain antibodies, such as scFv, di-scFv, or (scFv) 2 .
在某些实施方案中，所述单链抗体从其N端至C端依次包括：In certain embodiments, the single-chain antibody sequentially includes from its N-terminus to its C-terminus:
(1)包含如SEQ ID NO:1所示的序列或其变体的VH-连接子-包含如SEQ ID NO:2所示的序列或其变体的VL；或，包含如SEQ ID NO:2所示的序列或其变体的VL-连接子-包含如SEQ ID NO:1所示的序列或其变体的VH；(1) A VH-linker comprising the sequence shown in SEQ ID NO: 1 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO: 2 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO: 2 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO: 1 or a variant thereof; VL-linker of the sequence shown in 2 or its variant - VH containing the sequence shown in SEQ ID NO:1 or its variant;
或or
(2)包含如SEQ ID NO:3所示的序列或其变体的VH-连接子-包含如SEQ ID NO:4所示的序列或其变体的VL；或，包含如SEQ ID NO:4所示的序列或其变体的VL-连接子-包含如SEQ ID NO:3所示的序列或其变体的VH；(2) A VH-linker comprising the sequence shown in SEQ ID NO:3 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:3 or a variant thereof; VL-linker of the sequence shown in 4 or a variant thereof - VH containing the sequence shown in SEQ ID NO:3 or a variant thereof;
或or
(3)包含如SEQ ID NO:5所示的序列或其变体的VH-连接子-包含如SEQ ID NO:6所示的序列或其变体的VL；或，包含如SEQ ID NO:6所示的序列或其变体的VL-连接子-包含如SEQ ID NO:5所示的序列或其变体的VH；(3) A VH-linker comprising the sequence shown in SEQ ID NO:5 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:6 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:6 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:5 or a variant thereof; The VL-linker of the sequence shown in 6 or its variant-comprising the VH of the sequence shown in SEQ ID NO:5 or its variant;
或or
(4)包含如SEQ ID NO:7所示的序列或其变体的VH-连接子-包含如SEQ ID NO:8所示的序列或其变体的VL；或，包含如SEQ ID NO:8所示的序列或其变体的VL-连接子- 包含如SEQ ID NO:7所示的序列或其变体的VH；(4) VH-linker comprising the sequence shown in SEQ ID NO:7 or a variant thereof - a VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof; or, comprising a VL such as SEQ ID NO: VL-linker of the sequence shown in 8 or its variant- A VH comprising the sequence shown in SEQ ID NO:7 or a variant thereof;
或or
(5)包含如SEQ ID NO:9所示的序列或其变体的VH-连接子-包含如SEQ ID NO:10所示的序列或其变体的VL；或，包含如SEQ ID NO:10所示的序列或其变体的VL-连接子-包含如SEQ ID NO:9所示的序列或其变体的VH；(5) A VH-linker comprising the sequence shown in SEQ ID NO:9 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:10 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:10 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO: The VL-linker of the sequence shown in 10 or its variant - the VH containing the sequence shown in SEQ ID NO:9 or its variant;
或or
(6)包含如SEQ ID NO:11所示的序列或其变体的VH-连接子-包含如SEQ ID NO:12所示的序列或其变体的VL；或，包含如SEQ ID NO:12所示的序列或其变体的VL-连接子-包含如SEQ ID NO:11所示的序列或其变体的VH；(6) A VH-linker comprising the sequence shown in SEQ ID NO:11 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:11 or a variant thereof; The VL-linker of the sequence shown in 12 or its variant-comprising the VH of the sequence shown in SEQ ID NO:11 or its variant;
其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；在某些实施方案中，所述的置换是保守置换。wherein said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, substitutions, deletions, or additions of 3, 4, or 5 amino acids); in certain embodiments, the substitutions are conservative substitutions.
在某些实施方案中，所述单链抗体包含SEQ ID NO:86、88、90、92、94、82任一项所示的序列或其变体，所述变体与SEQ ID NO:86、88、90、92、94或82相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列，或具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；在某些实施方案中，所述的置换是保守置换。In certain embodiments, the single-chain antibody comprises the sequence shown in any one of SEQ ID NO:86, 88, 90, 92, 94, 82 or a variant thereof, which variant is consistent with SEQ ID NO:86 , 88, 90, 92, 94 or 82 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least A sequence that is 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); in certain embodiments, the substitutions are conservative substitutions.
在某些实施方案中，本发明的抗原结合分子进一步包含来源于人免疫球蛋白的恒定区。在某些实施方案中，所述抗原结合分子的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗原结合分子的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。In certain embodiments, the antigen-binding molecules of the invention further comprise a constant region derived from a human immunoglobulin. In certain embodiments, the heavy chain of the antigen-binding molecule comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antigen-binding molecule comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4). The light chain constant region of an immunoglobulin (eg kappa or lambda).
在某些实施方案中，所述抗原结合分子的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，In certain embodiments, the heavy chain of the antigen-binding molecule comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof that has an or Substitution, deletion or addition of multiple amino acids (e.g., substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., 1, 2, 3, 4 or 5 substitution, deletion or addition of amino acids); and/or,
所述抗原结合分子的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变 体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。The light chain of the antigen-binding molecule includes the light chain constant region (CL) of a human immunoglobulin or a variant thereof, which variant The entity has one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived; For example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids).
在某些实施方案中，所述重链恒定区是IgG、IgM、IgE、IgD或IgA重链恒定区。在某些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。In certain embodiments, the heavy chain constant region is an IgG, IgM, IgE, IgD or IgA heavy chain constant region. In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
在某些实施方案中，所述轻链恒定区是κ或λ轻链恒定区。在某些优选的实施方案中，所述轻链恒定区是人κ轻链恒定区。In certain embodiments, the light chain constant region is a kappa or lambda light chain constant region. In certain preferred embodiments, the light chain constant region is a human kappa light chain constant region.
抗原结合分子的制备Preparation of Antigen Binding Molecules
本发明的抗原结合分子可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码该抗原结合分子的DNA分子，将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗原结合分子。The antigen-binding molecules of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology. For example, a DNA molecule encoding the antigen-binding molecule is obtained through chemical synthesis or PCR amplification, the obtained DNA molecule is inserted into an expression vector, and then the host cell is transfected. Then, the transfected host cells are cultured under specific conditions and express the antigen-binding molecule of the present invention.
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(Reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’)2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’)2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) . In addition, these antigen-binding fragments can also be produced directly from recombinant host cells (Reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000 )). For example, Fab’ fragments can be obtained directly from host cells; Fab’ fragments can be chemically coupled to form F(ab’)2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)). In addition, Fv, Fab or F(ab’)2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
因此，本发明第二方面提供了一种分离的核酸分子，其包含编码本发明的抗原结合分子的核苷酸序列。在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：Accordingly, a second aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antigen-binding molecule of the invention. In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO:100所示的核苷酸序列、(ii)与SEQ ID NO:100基本上相同的序列(例如，与SEQ ID NO:100相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列；和/或，The nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 100, (ii) a sequence substantially identical to SEQ ID NO: 100 (e.g., the same sequence as SEQ ID NO: 100) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 100, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:101所示的核苷酸序列、 (v)与SEQ ID NO:101基本上相同的序列(例如，与SEQ ID NO:101相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(vi)上述(iv)或(v)的简并序列。The nucleic acid molecule encoding the variable region of the antibody light chain includes: (iv) the nucleotide sequence shown in SEQ ID NO: 101, (v) A sequence that is substantially identical to SEQ ID NO:101 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO:101, or sequences with one or more nucleotide substitutions), or (vi) degenerate sequences of (iv) or (v) above.
在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO:102所示的核苷酸序列、(ii)与SEQ ID NO:102基本上相同的序列(例如，与SEQ ID NO:102相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列；和/或，The nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 102, (ii) a sequence substantially identical to SEQ ID NO: 102 (e.g., the same sequence as SEQ ID NO: 102). A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 102, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:103所示的核苷酸序列、(v)与SEQ ID NO:103基本上相同的序列(例如，与SEQ ID NO:103相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(vi)上述(iv)或(v)的简并序列。The nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 103, (v) a sequence substantially identical to SEQ ID NO: 103 (e.g., the same sequence as SEQ ID NO: 103) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 103, or a sequence having one or more nucleotide substitutions), or (vi) the above ( Degenerate sequence of iv) or (v).
在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO:104所示的核苷酸序列、(ii)与SEQ ID NO:104基本上相同的序列(例如，与SEQ ID NO:104相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列；和/或，The nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 104, (ii) a sequence substantially identical to SEQ ID NO: 104 (e.g., the same sequence as SEQ ID NO: 104). A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 104, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:105所示的核苷酸序列、(v)与SEQ ID NO:105基本上相同的序列(例如，与SEQ ID NO:105相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(vi)上述(iv)或(v)的简并序列。The nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 105, (v) a sequence substantially identical to SEQ ID NO: 105 (e.g., the same sequence as SEQ ID NO: 105) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 105, or a sequence having one or more nucleotide substitutions), or (vi) the above ( Degenerate sequence of iv) or (v).
在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO:106所示的核苷酸序列、(ii)与SEQ ID NO:106基本上相同的序列(例如，与SEQ ID NO:106相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列；和/或，The nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 106, (ii) a sequence that is substantially the same as SEQ ID NO: 106 (e.g., the same sequence as SEQ ID NO: 106). A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 106, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:107所示的核苷酸序列、 (v)与SEQ ID NO:107基本上相同的序列(例如，与SEQ ID NO:107相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(vi)上述(iv)或(v)的简并序列。The nucleic acid molecule encoding the variable region of the antibody light chain includes: (iv) the nucleotide sequence shown in SEQ ID NO: 107, (v) A sequence that is substantially identical to SEQ ID NO:107 (e.g., a sequence that has at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to SEQ ID NO:107, or sequences with one or more nucleotide substitutions), or (vi) degenerate sequences of (iv) or (v) above.
在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO:108所示的核苷酸序列、(ii)与SEQ ID NO:108基本上相同的序列(例如，与SEQ ID NO:108相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列；和/或，The nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO: 108, (ii) a sequence substantially identical to SEQ ID NO: 108 (e.g., the same sequence as SEQ ID NO: 108). A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity as compared to NO: 108, or a sequence having one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:109所示的核苷酸序列、(v)与SEQ ID NO:109基本上相同的序列(例如，与SEQ ID NO:109相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(vi)上述(iv)或(v)的简并序列。The nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO: 109, (v) a sequence substantially identical to SEQ ID NO: 109 (e.g., the same sequence as SEQ ID NO: 109) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO: 109, or a sequence having one or more nucleotide substitutions), or (vi) the above ( Degenerate sequence of iv) or (v).
在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO:84所示的核苷酸序列、(ii)与SEQ ID NO:84基本上相同的序列(例如，与SEQ ID NO:84相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列；和/或，The nucleic acid molecule encoding the antibody heavy chain variable region includes: (i) the nucleotide sequence shown in SEQ ID NO:84, (ii) a sequence substantially identical to SEQ ID NO:84 (e.g., the same sequence as SEQ ID NO:84). A sequence with at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO:84, or a sequence with one or more nucleotide substitutions), or (iii) the above ( A degenerate sequence of i) or (ii); and/or,
所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:85所示的核苷酸序列、(v)与SEQ ID NO:85基本上相同的序列(例如，与SEQ ID NO:85相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(vi)上述(iv)或(v)的简并序列。The nucleic acid molecule encoding the antibody light chain variable region includes: (iv) the nucleotide sequence shown in SEQ ID NO:85, (v) a sequence substantially identical to SEQ ID NO:85 (e.g., the same sequence as SEQ ID NO:85) A sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to NO:85, or a sequence having one or more nucleotide substitutions), or (vi) the above ( Degenerate sequence of iv) or (v).
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:87所示的核苷酸序列、(ii)与SEQ ID NO:87基本上相同的序列(例如，与SEQ ID NO:87相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO:87, (ii) a sequence substantially identical to SEQ ID NO:87 (e.g., a sequence similar to SEQ ID NO:87) A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO:87, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:89所示的核苷酸序列、(ii)与SEQ ID NO:89基本上相同的序列(例如，与SEQ ID NO:89相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的 序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO:89, (ii) a sequence substantially identical to SEQ ID NO:89 (e.g., a sequence similar to SEQ ID NO:89) Sequences having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO:89, or having one or more nucleotide substitutions sequence), or (iii) a degenerate sequence of (i) or (ii) above.
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:91所示的核苷酸序列、(ii)与SEQ ID NO:91基本上相同的序列(例如，与SEQ ID NO:91相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence set forth in SEQ ID NO: 91, (ii) a sequence substantially identical to SEQ ID NO: 91 (e.g., the same sequence as SEQ ID NO: 91 A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO: 91, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:93所示的核苷酸序列、(ii)与SEQ ID NO:93基本上相同的序列(例如，与SEQ ID NO:93相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO: 93, (ii) a sequence substantially identical to SEQ ID NO: 93 (e.g., a sequence similar to SEQ ID NO: 93) A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO: 93, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:95所示的核苷酸序列、(ii)与SEQ ID NO:95基本上相同的序列(例如，与SEQ ID NO:95相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO:95, (ii) a sequence substantially identical to SEQ ID NO:95 (e.g., a sequence similar to SEQ ID NO:95) A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO: 95, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:83所示的核苷酸序列、(ii)与SEQ ID NO:83基本上相同的序列(例如，与SEQ ID NO:83相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) a nucleotide sequence set forth in SEQ ID NO:83, (ii) a sequence substantially identical to SEQ ID NO:83 (e.g., a sequence similar to SEQ ID NO:83) A sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity as compared to ID NO:83, or a sequence having one or more nucleotide substitutions), or (iii) the above Degenerate sequence of (i) or (ii).
本发明第三方面提供了一种载体(例如克隆载体或表达载体)，其包含如上所述的分离的核酸分子。在某些实施方案中，本发明的载体是例如DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体或病毒载体；优选的，所述载体是表达载体；优选地，所述载体是游离型载体；优选地，所述载体是病毒载体；更优选地，所述病毒载体是慢病毒载体、腺病毒载体或逆转录病毒载体。A third aspect of the invention provides a vector (eg a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vector of the invention is, for example, a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector or a viral vector; preferably, the vector is an expression vector; preferably, the vector It is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.
本发明第四方面提供了一种宿主细胞，其包含本发明的分离的核酸分子或本发明的载体。宿主细胞可以是真核细胞(例如哺乳动物细胞、昆虫细胞、酵母细胞)或原核细胞(例如大肠杆菌)。合适的真核细胞包括但不限于NS0细胞、Vero细胞、Hela细胞、COS细胞、CHO细胞、ExpiCHO细胞、HEK293细胞、Expi293细胞、BHK细胞、和MDCKII细胞。适宜的昆虫细胞包括但不限于Sf9细胞。在某些实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DXB11、ExpiCHO、CHO DG44)。 A fourth aspect of the invention provides a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention. The host cell may be a eukaryotic cell (eg, mammalian cell, insect cell, yeast cell) or prokaryotic cell (eg, E. coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, host cells of the invention are mammalian cells, such as CHO (eg, CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).
在某些实施方案中，本发明的宿主细胞可以是嵌合抗原受体T细胞(CAR-T)。在此类实施方案中，所述宿主细胞所包含的分离的核酸分子可以包含编码嵌合抗原受体的核苷酸序列，所述编码嵌合抗原受体的核苷酸序列进一步包含编码本发明的抗原结合分子(例如ScFv)的核苷酸序列。在某些实施方案中，所述宿主细胞所包含的分离的核酸分子编码包含本发明抗原结合分子(例如scFv)的嵌合抗原受体。In certain embodiments, host cells of the invention may be chimeric antigen receptor T cells (CAR-T). In such embodiments, the isolated nucleic acid molecule comprised by the host cell may comprise a nucleotide sequence encoding a chimeric antigen receptor further comprising a nucleotide sequence encoding a chimeric antigen receptor of the invention. The nucleotide sequence of an antigen-binding molecule (e.g., ScFv). In certain embodiments, the host cell contains an isolated nucleic acid molecule encoding a chimeric antigen receptor comprising an antigen-binding molecule of the invention (eg, scFv).
在另一个方面，本发明还涉及制备本发明的抗原结合分子的方法，其包括，在允许蛋白表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗原结合分子。In another aspect, the present invention also relates to a method for preparing an antigen-binding molecule of the present invention, comprising culturing a host cell as described above under conditions that allow protein expression, and recovering said host cell culture from the cultured host cell culture. Antigen-binding molecules.
嵌合抗原受体chimeric antigen receptor
本发明涉及靶向GPC3的CAR，其特征包括非MHC限制的GPC3识别能力，其赋予表达该CAR的免疫细胞(例如，T细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞)不依赖于抗原加工及提呈而识别表达GPC3的细胞(例如肿瘤细胞)的能力。The present invention relates to a CAR targeting GPC3, the characteristics of which include non-MHC restricted GPC3 recognition ability, which confer an immune cell (for example, T cell, NK cell, monocyte, macrophage or dendritic cell) expressing the CAR The ability to recognize GPC3-expressing cells (such as tumor cells) independent of antigen processing and presentation.
因此，本发明第五方面提供了一种嵌合抗原受体，其包含抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域。Therefore, the fifth aspect of the present invention provides a chimeric antigen receptor comprising an antigen-binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain.
I.胞外抗原结合结构域I. Extracellular antigen binding domain
本发明的嵌合抗原受体中所包含的抗原结合结构域赋予所述CAR识别GPC3的能力。The antigen-binding domain contained in the chimeric antigen receptor of the present invention confers the ability of the CAR to recognize GPC3.
在某些实施方案中，所述抗原结合结构域包含第一方面所述的抗原结合分子。In certain embodiments, the antigen binding domain comprises the antigen binding molecule of the first aspect.
在某些实施方案中，所述抗原结合结构域包含所述抗原结合分子作为第一抗原结合结构域，并且进一步包含不结合GPC3的第二抗原结合结构域。在某些实施方案中，所述第二抗原结合结构域结合的抗原选自：PD-1、PD-L1、CTLA-、CD3、ASGPR1、CD19、MSLN、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2。In certain embodiments, the antigen binding domain comprises the antigen binding molecule as a first antigen binding domain and further comprises a second antigen binding domain that does not bind GPC3. In certain embodiments, the second antigen binding domain binds an antigen selected from: PD-1, PD-L1, CTLA-, CD3, ASGPR1, CD19, MSLN, PSMA, MUCl, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
在某些实施方案中，所述抗原结合结构域是单链抗体。In certain embodiments, the antigen binding domain is a single chain antibody.
在某些实施方案中，所述第一抗原结合结构域包含SEQ ID NO:86、88、90、92、94、82任一项所示的序列或其变体，所述变体与SEQ ID NO:86、88、90、92、94、82任一项相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列，或具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选 地，所述的置换是保守置换。In certain embodiments, the first antigen-binding domain comprises the sequence shown in any one of SEQ ID NO: 86, 88, 90, 92, 94, 82 or a variant thereof, which variant is identical to SEQ ID NO. NO: 86, 88, 90, 92, 94, 82 any one has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% , a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, or has one or several amino acid substitutions, deletions, or additions (e.g., 1 , substitution, deletion or addition of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids); preferred Specifically, the substitutions are conservative substitutions.
II.跨膜结构域II. Transmembrane domain
本发明的嵌合抗原受体所包含的跨膜结构域可以是本领域已知的任何蛋白结构，只要其能够在细胞膜(特别是真核细胞膜)中热力学稳定。适用于本发明的CAR的跨膜结构域可衍生自天然来源。在此类实施方案中，所述跨膜结构域可衍生自任何膜结合的或跨膜的蛋白质。或者，所述跨膜结构域可为合成的非天然存在的蛋白质区段，例如主要包含疏水残基例如亮氨酸和缬氨酸的蛋白质区段。The transmembrane domain contained in the chimeric antigen receptor of the present invention can be any protein structure known in the art, as long as it can be thermodynamically stable in the cell membrane (especially the eukaryotic cell membrane). The transmembrane domains of CARs suitable for use in the present invention can be derived from natural sources. In such embodiments, the transmembrane domain may be derived from any membrane-bound or transmembrane protein. Alternatively, the transmembrane domain may be a synthetic non-naturally occurring protein segment, such as a protein segment containing primarily hydrophobic residues such as leucine and valine.
在某些实施方案中，所述跨膜结构域是选自下列蛋白的跨膜区：T细胞受体的α、β或ζ链、CD28、CD45、CD3ε、CD3ζ、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD152、CD154和PD-1及其任意组合。在某些优选的实施方案中，所述跨膜结构域是选自下列蛋白的跨膜区：CD8、CD28、CD4、PD-1、CD152和CD154。在某些实施方案中，所述跨膜结构域包含序列如SEQ ID NO:111所示的CD8跨膜区。In certain embodiments, the transmembrane domain is a transmembrane region selected from the following proteins: alpha, beta or zeta chain of T cell receptor, CD28, CD45, CD3ε, CD3ζ, CD4, CD5, CD8, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1 and any combination thereof. In certain preferred embodiments, the transmembrane domain is a transmembrane region selected from the group consisting of CD8, CD28, CD4, PD-1, CD152 and CD154. In certain embodiments, the transmembrane domain comprises a CD8 transmembrane region with the sequence set forth in SEQ ID NO: 111.
III.间隔结构域III. Spacer domain
本发明的嵌合抗原受体所包含间隔结构域位于胞外抗原结合结构域与跨膜结构域之间。The chimeric antigen receptor of the present invention includes a spacer domain located between the extracellular antigen-binding domain and the transmembrane domain.
在某些实施方案中，所述间隔结构域包含免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区。在此类实施方案中，不受特定理论的约束，认为CH2和CH3使所述CAR的抗原结合结构域从表达CAR的细胞的细胞膜延伸出去，并且可更精确地模拟天然TCR的大小和结构域结构。In certain embodiments, the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (eg, IgG1 or IgG4). In such embodiments, without being bound by a particular theory, it is believed that CH2 and CH3 extend the antigen-binding domain of the CAR away from the membrane of the CAR-expressing cell and more accurately mimic the size and domain structure of the native TCR structure.
在某些实施方案中，所述间隔结构域包含铰链结构域。铰链结构域可以是通常在蛋白质的两个结构域之间发现的氨基酸区段，其可以允许蛋白质具有柔性并且允许一个或两个结构域相对于彼此的运动。因此，所述铰链结构域可以是任何氨基酸序列，只要其能够提供胞外抗原结合结构域的这种柔性以及其相对于跨膜结构域的这种运动性。In certain embodiments, the spacer domain comprises a hinge domain. A hinge domain may be a stretch of amino acids typically found between two domains of a protein that may allow flexibility of the protein and movement of one or both domains relative to each other. Therefore, the hinge domain may be any amino acid sequence that provides the flexibility of the extracellular antigen-binding domain and its mobility relative to the transmembrane domain.
在某些实施方案中，所述铰链结构域是天然存在的蛋白质的铰链区或其部分。在某些实施方案中，所述间隔结构域选自铰链结构域和/或免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区。在某些实施方案中，所述铰链结构域包含CD8、IgG4、PD-1、CD152或CD154的铰链区。在某些实施方案中，所述铰链结构域包含序列如SEQ ID NO:112所示的CD8铰链区。In certain embodiments, the hinge domain is the hinge region of a naturally occurring protein, or a portion thereof. In certain embodiments, the spacer domain is selected from the hinge domain and/or the CH2 and CH3 regions of an immunoglobulin (eg, IgG1 or IgG4). In certain embodiments, the hinge domain comprises the hinge region of CD8, IgG4, PD-1, CD152 or CD154. In certain embodiments, the hinge domain comprises a CD8 hinge region with the sequence set forth in SEQ ID NO: 112.
IV.信号肽 IV. Signal peptide
在某些实施方案中，本发明的CAR可进一步在其N端包含信号肽。通常，信号肽是将与其连接的序列靶向至所需位点的多肽序列。在某些实施方案中，所述信号肽可以将与其连接的CAR靶向至细胞的分泌途径，并允许该CAR进一步整合并锚定到脂质双分子层中。可用于CAR的信号肽是本领域技术人员已知的。在某些实施方案中，所述信号肽包含重链信号肽(例如IgG1的重链信号肽)、粒细胞-巨噬细胞集落刺激因子受体2(GM-CSFR2)信号肽、IL2信号肽、或CD8α信号肽。在某些优选的实施方案中，所述信号肽选自CD8α信号肽。在某些示例性实施方案中，所述信号肽包含SEQ ID NO:116所示的氨基酸序列。In certain embodiments, the CAR of the invention may further comprise a signal peptide at its N-terminus. Typically, a signal peptide is a polypeptide sequence that targets the sequence to which it is linked to a desired site. In certain embodiments, the signal peptide can target the CAR to which it is linked to the secretory pathway of the cell and allow further integration and anchoring of the CAR into the lipid bilayer. Signal peptides useful for CARs are known to those skilled in the art. In certain embodiments, the signal peptide comprises a heavy chain signal peptide (eg, a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or CD8α signal peptide. In certain preferred embodiments, the signal peptide is selected from the group consisting of CD8α signal peptides. In certain exemplary embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 116.
在某些实施方案中，本发明的CAR还可以与另外的生物活性分子共表达。所述另外的生物活性分子可以有其专有的信号肽，为与上一段的信号肽区别，此信号肽命名为信号肽-2。该信号肽-2引导另外的生物活性分子转运到细胞内特定的位点或细胞膜外。该述信号肽-2可与上一段所述的信号肽相同或不同。在某些实施方案中，该信号肽-2可与上一段所述的信号肽不同。在某些实施方案中，所述信号肽-2是IL2信号肽(例如，氨基酸序列如SEQ ID NO:129所示)。In certain embodiments, the CARs of the invention can also be co-expressed with additional biologically active molecules. The additional bioactive molecule may have its own proprietary signal peptide, which is named signal peptide-2 to distinguish it from the signal peptide in the previous paragraph. The signal peptide-2 guides the transport of additional bioactive molecules to specific sites within the cell or outside the cell membrane. The signal peptide-2 may be the same as or different from the signal peptide described in the previous paragraph. In certain embodiments, the signal peptide-2 may be different from the signal peptide described in the previous paragraph. In certain embodiments, the signal peptide-2 is an IL2 signal peptide (e.g., the amino acid sequence is set forth in SEQ ID NO: 129).
V.胞内信号传导结构域V. Intracellular signaling domain
本发明的CAR中所包含的胞内信号传导结构域参与将本发明的CAR与GPC3的结合所产生的信号传导进免疫效应细胞内部，激活表达CAR的免疫效应细胞的至少一种正常效应子功能，或增强表达CAR的免疫效应细胞的至少一种细胞因子的分泌(例如IL-2，IFN-γ)。The intracellular signaling domain contained in the CAR of the present invention is involved in transmitting the signal generated by the combination of the CAR of the present invention and GPC3 into the immune effector cells, activating at least one normal effector function of the immune effector cells expressing the CAR , or enhance the secretion of at least one cytokine (e.g., IL-2, IFN-γ) by CAR-expressing immune effector cells.
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域和/或共刺激信号传导结构域。In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain.
在某些实施方案中，所述初级信号传导结构域可以是包含免疫受体酪氨酸活化基序(ITAM)的任何胞内信号传导结构域。在某些实施方案中，所述初级信号传导结构域包含免疫受体酪氨酸活化基序(ITAM)。在某些实施方案中，所述初级信号传导结构域包含选自下列的蛋白的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CDS、CD22、CD79a、CD79b或CD66d。在某些实施方案中，所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域。In certain embodiments, the primary signaling domain can be any intracellular signaling domain comprising an immunoreceptor tyrosine activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of a protein selected from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the primary signaling domain comprises the intracellular signaling domain of CD3ζ.
在某些实施方案中，所述共刺激信号传导结构域可以是来自共刺激分子的胞内信号传导结构域。在某些实施方案中，所述共刺激信号传导结构域包含选自下列的蛋白的胞内信号传导结构域：CARD11、CD2、CD7、CD27、CD28、CD30、CD134(OX40)、 CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)或DAP10。In certain embodiments, the costimulatory signaling domain can be an intracellular signaling domain from a costimulatory molecule. In certain embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137(4-1BB), CD150(SLAMF1), CD270(HVEM), CD278(ICOS) or DAP10.
在某些实施方案中，所述共刺激信号传导结构域选自CD28的胞内信号传导结构域、或CD137(4-1BB)的胞内信号传导结构域、或二者片段的组合。In certain embodiments, the costimulatory signaling domain is selected from the intracellular signaling domain of CD28, or the intracellular signaling domain of CD137(4-1BB), or a combination of fragments thereof.
在某些实施方案中，所述胞内信号传导结构域包含一个共刺激信号传导结构域。在某些实施方案中，所述胞内信号传导结构域包含两个或更多个共刺激信号传导结构域。在此类实施方案中，所述两个或更多个共刺激信号传导结构域可以是相同的，也可以是不同的。In certain embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains. In such embodiments, the two or more costimulatory signaling domains may be the same or different.
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域以及至少一个共刺激信号传导结构域。所述初级信号传导结构域以及至少一个共刺激信号传导结构域可以以任意顺序串联至跨膜结构域的羧基端。In certain embodiments, the intracellular signaling domain includes a primary signaling domain and at least one costimulatory signaling domain. The primary signaling domain and the at least one costimulatory signaling domain can be concatenated in any order to the carboxyl terminus of the transmembrane domain.
在某些实施方案中，所述胞内信号传导结构域可包含CD3ζ的胞内信号传导结构域和CD137(4-1BB)的胞内信号传导结构域。在某些示例性实施方案中，所述CD3ζ的胞内信号传导结构域包含SEQ ID NO:113所示的氨基酸序列。在某些示例性实施方案中，所述CD137(4-1BB)的胞内信号传导结构域包含SEQ ID NO:114所示的氨基酸序列。In certain embodiments, the intracellular signaling domain may comprise the intracellular signaling domain of CD3ζ and the intracellular signaling domain of CD137(4-1BB). In certain exemplary embodiments, the intracellular signaling domain of CD3ζ comprises the amino acid sequence set forth in SEQ ID NO: 113. In certain exemplary embodiments, the intracellular signaling domain of CD137(4-1BB) comprises the amino acid sequence set forth in SEQ ID NO: 114.
在某些示例性实施方案中，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO:115所示序列。In certain exemplary embodiments, the intracellular signaling domain of the chimeric antigen receptor has the sequence set forth in SEQ ID NO: 115.
VI.全长CARVI.Full length CAR
本发明提供了能够特异性地结合GPC3的嵌合抗原受体，所述嵌合抗原受体从其N端至C端依次包含抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域。在某些优选实施方案中，其中所述胞内信号传导结构域从N端到C端为共刺激信号传导结构域和初级信号传导结构域。The invention provides a chimeric antigen receptor that can specifically bind to GPC3. The chimeric antigen receptor sequentially includes an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal from its N-terminus to its C-terminus. conductive domain. In certain preferred embodiments, the intracellular signaling domain is a costimulatory signaling domain and a primary signaling domain from N-terminus to C-terminus.
在某些实施方案中，所述间隔结构域包含CD8(例如CD8α)的铰链区(例如，序列如SEQ ID NO:112所示的铰链区)。In certain embodiments, the spacer domain comprises a hinge region of CD8 (e.g., CD8α) (e.g., a hinge region with the sequence set forth in SEQ ID NO: 112).
在某些实施方案中，所述跨膜结构域包含CD8(例如CD8α)的跨膜区(例如，序列如SEQ ID NO:111所示的跨膜区)。In certain embodiments, the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8α) (e.g., a transmembrane region with the sequence set forth in SEQ ID NO: 111).
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域(例如，如SEQ ID NO:113所示序列)，所述共刺激信号传导结构域包含CD137(4-1BB)的胞内信号传导结构域(例如，如SEQ ID NO:114所示序列)；更优选地，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO:115所示序列。 In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises an intracellular signaling domain of CD3ζ (e.g., as Sequence shown in SEQ ID NO: 113), the costimulatory signaling domain includes the intracellular signaling domain of CD137 (4-1BB) (for example, sequence shown in SEQ ID NO: 114); More preferably, The intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 115.
在某些优选的实施方案中，所述嵌合抗原受体从其N端至C端依次包含所述信号肽、抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域(从N端到C端为共刺激信号传导结构域和初级信号传导结构域)。In certain preferred embodiments, the chimeric antigen receptor includes the signal peptide, antigen-binding domain, spacer domain, transmembrane domain, and intracellular signaling domain in order from its N-terminus to its C-terminus. (From N-terminus to C-terminus are the costimulatory signaling domain and the primary signaling domain).
在某些实施方案中，所述信号肽包含IgG1的重链信号肽或CD8α信号肽(例如，序列如SEQ ID NO:116所示的信号肽)。在某些示例性实施方案中，本发明的CAR包含SEQ ID NO:117、119、121、123、125、127任一项所示的序列或其变体，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换为保守置换。In certain embodiments, the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (e.g., a signal peptide with the sequence shown in SEQ ID NO: 116). In certain exemplary embodiments, the CAR of the present invention includes the sequence shown in any one of SEQ ID NO: 117, 119, 121, 123, 125, 127 or a variant thereof, which variant is the same as the sequence from which it is derived. Sequence comparison has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5) compared to the sequence from which it is derived substitution, deletion or addition of amino acids); preferably, the substitution is a conservative substitution.
VII.共表达的CAR和另外的生物活性分子VII. Co-expressed CAR and additional bioactive molecules
在一些情况下，本发明第五方面所述的CAR还可以与另外的生物活性分子共表达。自裂解肽能阻止翻译过程中氨基酸形成共价键并维持翻译继续进行，这样一来，翻译产物就被“自切割”，从而使本发明的嵌合抗原受体和另外的生物活性分子分离。因此，当本发明第五方面所述的CAR还可以与另外的生物活性分子共表达时，能够特异性地结合GPC3的嵌合抗原受体成为独立的具有胞外抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域的CAR，而另外的生物活性分子则能够分泌到细胞外或者表达成膜嵌合型的多肽或蛋白。随着表达CAR的免疫细胞在肿瘤微环境的扩增和富集，另外的生物活性分子富集在肿瘤微环境，与CAR协同发挥抗肿瘤效应。In some cases, the CAR described in the fifth aspect of the present invention can also be co-expressed with other biologically active molecules. The self-cleaving peptide can prevent amino acids from forming covalent bonds during translation and maintain translation to continue. In this way, the translation product is "self-cleaved", thereby separating the chimeric antigen receptor of the present invention from other biologically active molecules. Therefore, when the CAR described in the fifth aspect of the present invention can also be co-expressed with other biologically active molecules, the chimeric antigen receptor that can specifically bind to GPC3 becomes an independent extracellular antigen-binding domain and a spacer domain. , transmembrane domain and intracellular signaling domain of CAR, while other bioactive molecules can be secreted extracellularly or expressed into membrane chimeric polypeptides or proteins. As CAR-expressing immune cells expand and enrich in the tumor microenvironment, additional bioactive molecules are enriched in the tumor microenvironment and cooperate with CAR to exert anti-tumor effects.
在某些实施方案中，编码CAR的核酸序列通过自裂解肽的核酸序列与另外的生物活性分子的核酸序列连接。CAR可以在另外的生物活性分子的N端或C端。在某些示例的实施方案中，CAR在另外的生物活性分子的5’端。任何能够引起融合蛋白裂解成两个独立的蛋白的自裂解肽可以应用到本发明中。在某些示例的实施方案中，所述自裂解肽为P2A，优选地具有SEQ ID NO:130所示的序列，其核苷酸序列可以根据基因重组的需要进行优化。在此类实施方案中，包含CAR和另外的生物活性分子的融合蛋白具有如下的结构：In certain embodiments, a nucleic acid sequence encoding a CAR is linked to a nucleic acid sequence of another biologically active molecule through a nucleic acid sequence of a self-cleaving peptide. The CAR can be at the N-terminus or C-terminus of another biologically active molecule. In certain exemplary embodiments, the CAR is 5' to another biologically active molecule. Any self-cleaving peptide capable of causing the cleavage of the fusion protein into two independent proteins can be used in the present invention. In certain exemplary embodiments, the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 130, and its nucleotide sequence can be optimized according to the needs of genetic recombination. In such embodiments, a fusion protein comprising a CAR and an additional biologically active molecule has the following structure:
N’-信号肽--特异地结合GPC3的胞外抗原结合结构域--间隔结构域—跨膜结构域-胞内信号传导结构域-自裂解肽-信号肽-2--另外的生物活性分子-C’。其中信号肽-2与N末端信号肽相同或不同。 N'-Signal peptide--Extracellular antigen-binding domain that specifically binds GPC3--Spacer domain-Transmembrane domain-Intracellular signaling domain-Self-cleaving peptide-Signal peptide-2--Additional biological activities Molecule-C'. The signal peptide-2 is the same as or different from the N-terminal signal peptide.
在某些实施方案中，所述另外的生物活性分子N端的信号肽-2是IL2信号肽(例如，如SEQ ID NO:129所示)。In certain embodiments, the signal peptide-2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (e.g., as set forth in SEQ ID NO: 129).
嵌合抗原受体的制备Preparation of chimeric antigen receptors
生成嵌合抗原受体以及包含该嵌合抗原受体的免疫效应细胞(例如T细胞)的方法是本领域已知的，可包括用至少一种编码CAR的多核苷酸转染细胞，并在细胞中表达多核苷酸。例如，可将编码本发明的CAR的核酸分子包含于表达载体(例如，慢病毒载体)中，所述表达载体能够在宿主细胞例如T细胞中表达，以制造所述CAR。Methods of generating chimeric antigen receptors and immune effector cells (e.g., T cells) comprising the chimeric antigen receptors are known in the art and may include transfecting the cells with at least one polynucleotide encoding a CAR and in Expression of polynucleotides in cells. For example, a nucleic acid molecule encoding a CAR of the invention can be included in an expression vector (eg, a lentiviral vector) capable of expression in a host cell, such as a T cell, to produce the CAR.
因此，本发明第六方面提供了一种分离的核酸分子，其包含编码第五方面所述的嵌合抗原受体的核苷酸序列。Therefore, the sixth aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor according to the fifth aspect.
本领域技术人员理解，由于遗传密码的简并性，编码一种本发明的嵌合抗原受体的核苷酸序列可以具有多种不同的序列。因此，除非另有说明，否则“编码氨基酸序列的核苷酸序列”包括作为彼此的简并形式且编码相同氨基酸序列的所有核苷酸序列。Those skilled in the art understand that due to the degeneracy of the genetic code, the nucleotide sequence encoding a chimeric antigen receptor of the invention can have a variety of different sequences. Therefore, unless otherwise stated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.
在某些示例性实施方案中，所述编码第五方面所述的嵌合抗原受体的核苷酸序列选自：(1)SEQ ID NO:118、120、122、124、126、128任一项所示的序列或其简并变体；(2)与(1)所述的序列相比基本上相同的序列，例如，与(1)所述相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)所述的序列相比具有一个或更多个核苷酸取代的序列；并且所述序列基本保留了其所源自的核苷酸序列的至少一种生物学活性(例如，能够编码具有以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达GPC3的细胞的能力)。In certain exemplary embodiments, the nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect is selected from: (1) any of SEQ ID NO: 118, 120, 122, 124, 126, 128 The sequence shown in one item or a degenerate variant thereof; (2) A sequence that is substantially the same as the sequence described in (1), for example, at least 50%, at least 55% compared with the sequence described in (1) , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least A sequence that has 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or a sequence that has one or more nucleotide substitutions compared to the sequence described in (1); and The sequence substantially retains at least one biological activity of the nucleotide sequence from which it is derived (e.g., is capable of encoding a sequence having the ability to direct the specificity and reactivity of immune effector cells toward GPC3-expressing cells in a non-MHC-restricted manner). ability).
如上文第五方面中所述的，本发明的CAR还可以与另外的生物活性分子共表达，以协同发挥抗肿瘤作用。As described in the fifth aspect above, the CAR of the present invention can also be co-expressed with other bioactive molecules to synergistically exert anti-tumor effects.
因此，本发明第七方面还提供了一种核酸构建体，其包含编码第五方面所述的嵌合抗原受体的第一核酸序列，并且进一步包含编码另外的生物活性分子的第二核酸序列。Therefore, the seventh aspect of the present invention also provides a nucleic acid construct comprising a first nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect, and further comprising a second nucleic acid sequence encoding another biologically active molecule. .
在某些实施方案中，所述第二核酸序列所编码的另外的生物活性分子具有抗肿瘤活性。In certain embodiments, the second nucleic acid sequence encodes an additional biologically active molecule that has anti-tumor activity.
在某些实施方案中，所述第二核苷酸序列所编码的另外的生物活性分子在其N端进 一步包含信号肽-2。In certain embodiments, the second nucleotide sequence encodes an additional biologically active molecule at its N-terminus. One step contains signal peptide-2.
在某些实施方案中，所述第一核苷酸序列位于所述第二核苷酸序列的上游。In certain embodiments, the first nucleotide sequence is located upstream of the second nucleotide sequence.
在某些实施方案中，所述第一核酸序列和第二核酸序列通过编码自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)的核苷酸序列连接。在某些示例性实施方案中，所述编码自裂解肽的序列连接至所述第一核苷酸序列的3’端，并且连接至所述第二核苷酸序列的5’端。In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (eg, P2A, E2A, F2A, T2A, or any combination thereof). In certain exemplary embodiments, the sequence encoding a self-cleaving peptide is linked to the 3' end of the first nucleotide sequence and to the 5' end of the second nucleotide sequence.
在某些实施方案中，所述自裂解肽是P2A(例如，如SEQ ID NO:130所示)。In certain embodiments, the self-cleaving peptide is P2A (e.g., as set forth in SEQ ID NO: 130).
在某些示例性实施方案中，第七方面所述的核酸构建体从其5’端至3’端依次包含：编码所述信号肽的核苷酸序列、编码所述抗原结合结构域的核苷酸序列、编码所述间隔结构域的核苷酸序列、编码所述跨膜结构域的核苷酸序列、编码所述胞内信号传导结构域的核苷酸序列、编码所述自裂解肽序列的核苷酸序列、编码所述信号肽-2的核苷酸序列、编码另外的生物活性分子的核苷酸序列。In certain exemplary embodiments, the nucleic acid construct described in the seventh aspect sequentially includes from its 5' end to its 3' end: a nucleotide sequence encoding the signal peptide, a nucleic acid sequence encoding the antigen-binding domain Nucleotide sequence, nucleotide sequence encoding the spacer domain, nucleotide sequence encoding the transmembrane domain, nucleotide sequence encoding the intracellular signaling domain, encoding the self-cleaving peptide The nucleotide sequence of the sequence, the nucleotide sequence encoding the signal peptide-2, the nucleotide sequence encoding another biologically active molecule.
本发明第八方面提供了一种载体，其包含第二方面、第六方面所述的分离的核酸分子，或第七方面所述的核酸构建体。The eighth aspect of the present invention provides a vector, which contains the isolated nucleic acid molecule described in the second aspect and the sixth aspect, or the nucleic acid construct described in the seventh aspect.
在某些实施方案中，所述载体选自DNA载体，RNA载体，质粒，转座子载体，CRISPR/Cas9载体，病毒载体。In certain embodiments, the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors, viral vectors.
在某些实施方案中，所述载体是表达载体。In certain embodiments, the vector is an expression vector.
在某些实施方案中，所述载体是游离型载体。In certain embodiments, the vector is an episomal vector.
在某些实施方案中，所述载体是病毒载体。In certain embodiments, the vector is a viral vector.
在某些示例性实施方案中，所述病毒载体是慢病毒载体、腺病毒载体或逆转录病毒载体。In certain exemplary embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.
在某些实施方案中，所述载体是游离型或非整合病毒载体，例如整合缺陷型逆转录病毒或慢病毒。In certain embodiments, the vector is an episomal or non-integrating viral vector, such as an integration-deficient retrovirus or lentivirus.
本发明第九方面提供了一种宿主细胞，其包含如上第六方面所述的分离的核酸分子、第七方面所述的核酸构建体或第八方面所述的载体。可以通过各种合适的方式将如上所述的载体引入宿主细胞，例如磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、电穿孔、TALEN方法、ZFN方法、非病毒载体介导的转染(例如脂质体)或病毒载体介导的转染(如慢病毒感染，逆转录病毒感染，腺病毒感染)，以及其他用于转移入宿主细胞的物理、化学或生物学手段，如转座子技术，CRISPR-Cas9等技术。 A ninth aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect. Vectors as described above can be introduced into host cells by various suitable means, such as calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vectors mediated transfection (such as liposomes) or viral vector-mediated transfection (such as lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological methods for transfer into host cells Means, such as transposon technology, CRISPR-Cas9 and other technologies.
在某些实施方案中，所述宿主细胞包含第六方面所述的分离的核酸分子或包含所述核酸分子的载体，所述宿主细胞表达本发明的嵌合抗原受体。In certain embodiments, the host cell comprises the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule, and the host cell expresses the chimeric antigen receptor of the invention.
在某些实施方案中，所述宿主细胞包含第七方面所述的核酸构建体或包含所述核酸构建体的载体，所述宿主细胞表达本发明的嵌合抗原受体以及另外的生物活性分子。In certain embodiments, the host cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct, the host cell expresses the chimeric antigen receptor of the invention and additional biologically active molecules .
在某些实施方案中，所述宿主细胞选自哺乳动物(如人)的免疫细胞。在某些实施方案中，所述免疫细胞来源于患者或健康供体。在某些实施方案中，所述免疫细胞选自T淋巴细胞、自然杀伤(NK)细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合；优选地，所述免疫细胞来源于T淋巴细胞或NK细胞。In certain embodiments, the host cells are selected from mammalian (eg, human) immune cells. In certain embodiments, the immune cells are derived from a patient or healthy donor. In certain embodiments, the immune cells are selected from T lymphocytes, natural killer (NK) cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are derived from T lymphocytes or NK cells.
本发明第十方面提供了制备表达本发明的嵌合抗原受体的细胞的方法，其包括：(1)提供宿主细胞；(2)将如第六方面所述的分离的核酸分子或包含所述核酸分子的载体引入所述宿主细胞，以获得能够表达所述嵌合抗原受体的宿主细胞。还提供了共表达本发明的嵌合抗原受体以及另外的生物活性分子的细胞的方法，其包括：(1)提供宿主细胞；(2)将第七方面所述的核酸构建体或包含所述核酸构建体的载体引入所述宿主细胞，获得能够共表达所述嵌合抗原受体和另外的生物活性分子的宿主细胞。The tenth aspect of the present invention provides a method for preparing cells expressing the chimeric antigen receptor of the present invention, which includes: (1) providing a host cell; (2) converting the isolated nucleic acid molecule as described in the sixth aspect or containing the The vector of the nucleic acid molecule is introduced into the host cell to obtain a host cell capable of expressing the chimeric antigen receptor. Also provided is a method for cells that co-express the chimeric antigen receptor of the present invention and other biologically active molecules, which includes: (1) providing a host cell; (2) converting the nucleic acid construct described in the seventh aspect or containing the The vector of the nucleic acid construct is introduced into the host cell to obtain a host cell capable of co-expressing the chimeric antigen receptor and other biologically active molecules.
在某些实施方案中，所述宿主细胞选自免疫细胞，例如T淋巴细胞、NK细胞、单核细胞、树突状细胞、巨噬细胞及其任意组合。在某些实施方案中，所述免疫细胞选自T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及这些细胞的任意组合。In certain embodiments, the host cells are selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages, and any combination thereof. In certain embodiments, the immune cells are selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination of these cells.
在某些实施方案中，在步骤(1)中，所述宿主细胞提供自患者或者健康供体，并且经过预处理；所述预处理包括免疫细胞的分选、激活和/或增殖；在某些实施方案中，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞。In certain embodiments, in step (1), the host cells are provided from a patient or a healthy donor and undergo pretreatment; the pretreatment includes sorting, activation and/or proliferation of immune cells; In some embodiments, the pretreating includes contacting the immune cells with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.
在某些实施方案中，在步骤(2)中，将核酸分子或载体通过病毒感染引入宿主细胞。在某些实施方案中，在步骤(2)中将核酸分子或载体通过非病毒载体转染的方式引入宿主细胞，如通过转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法、电穿孔方法、磷酸钙转染、DEAE-葡聚糖介导的转染或显微注射等方法。In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host cell via viral infection. In some embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host cell through non-viral vector transfection, such as through transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, Methods such as electroporation, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
在某些实施方案中，在步骤(2)之后，所述方法还包括：扩增步骤(2)获得的宿主细胞。In certain embodiments, after step (2), the method further includes: amplifying the host cells obtained in step (2).
经改造的免疫细胞engineered immune cells
本发明第十一方面还提供了一种经改造的免疫细胞，其包含表达本发明第二方面、 第六方面所述的分离的核酸分子。所述经改造的免疫细胞表达第五方面所述的嵌合抗原受体。The eleventh aspect of the present invention also provides a modified immune cell, which contains the expression of the second aspect of the present invention, The isolated nucleic acid molecule of the sixth aspect. The modified immune cells express the chimeric antigen receptor described in the fifth aspect.
本发明第十二方面还提供了一种经改造的免疫细胞，其包含表达本发明第七方面所述的核酸构建体。所述经改造的免疫细胞表达第五方面所述的嵌合抗原受体以及所述另外的生物活性分子。The twelfth aspect of the present invention also provides a modified immune cell, which contains the nucleic acid construct expressing the seventh aspect of the present invention. The engineered immune cells express the chimeric antigen receptor of the fifth aspect and the additional biologically active molecule.
在某些实施方案中，所述免疫细胞来源于T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合。在某些实施方案中，所述免疫细胞得自患者；可选地，所述免疫细胞得自健康供体。在某些实施方案中，所述免疫细胞来源于T淋巴细胞或NK细胞。In certain embodiments, the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells, and any combination thereof. In certain embodiments, the immune cells are obtained from a patient; alternatively, the immune cells are obtained from a healthy donor. In certain embodiments, the immune cells are derived from T lymphocytes or NK cells.
在某些实施方案中，所述经改造的免疫细胞的免疫排斥有关的基因(例如，TRAC、TRBC、B2M、HLA-A、HLA-B或HLA-C)和免疫共抑制通路或信号分子的基因(例如，PD-1、CTLA-4或LAG-3)中的一种或两种靶基因的转录或表达被抑制。在某些实施方案中，所述靶基因的转录或表达被抑制采用的方法选自基因敲除(例如，CRISPR、CRISPR/Cas9)、同源重组、干扰RNA。In certain embodiments, the engineered immune cells have genes involved in immune rejection (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B, or HLA-C) and immune co-suppressive pathways or signaling molecules. Transcription or expression of one or both target genes (eg, PD-1, CTLA-4, or LAG-3) is inhibited. In certain embodiments, the transcription or expression of the target gene is inhibited by a method selected from the group consisting of gene knockout (eg, CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
本发明还提供了制备经改造的免疫细胞的方法，其包括：(1)提供来自患者或者健康供体的免疫细胞；(2)将第六方面所述的分离的核酸分子，或第七方面所述的核酸构建体、或包含它们的载体引入步骤(1)所述的免疫细胞，以获得能够表达以及任选的另外的生物活性分子的免疫细胞。The present invention also provides a method for preparing modified immune cells, which includes: (1) providing immune cells from patients or healthy donors; (2) using the isolated nucleic acid molecules described in the sixth aspect, or the seventh aspect The nucleic acid constructs, or vectors containing them, are introduced into the immune cells described in step (1) to obtain immune cells capable of expressing and optionally additional biologically active molecules.
在某些实施方案中，在步骤(1)中，所述免疫细胞经预处理，所述预处理包括免疫细胞的分选、激活和/或增殖；更优选地，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞。In certain embodiments, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation, and/or proliferation of immune cells; more preferably, the pretreatment includes converting the immune cells into The cells are contacted with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating preconditioned immune cells.
在某些实施方案中，在步骤(2)中将所述核酸分子或载体通过病毒感染引入免疫细胞。In certain embodiments, the nucleic acid molecule or vector is introduced into the immune cell via viral infection in step (2).
在某些实施方案中，在步骤(2)中将所述核酸分子或载体通过非病毒载体转染的方式引入免疫细胞，如通过磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法或电穿孔方法。In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cells by non-viral vector transfection, such as by calcium phosphate transfection, DEAE-dextran mediated transfection, Microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.
在某些实施方案中，在步骤(2)之后还包括扩增步骤(2)获得的免疫细胞的步骤。In some embodiments, step (2) is followed by a step of amplifying the immune cells obtained in step (2).
免疫细胞组合物immune cell composition
在第十三方面，本发明还提供了免疫细胞组合物，所述免疫细胞组合物包括前述任 一方面的经改造的免疫细胞，以及可选的未改造和/或未成功改造的免疫细胞，这些未改造和/或未成功改造的免疫细胞不表达目的的CAR。限制于当前的技术水平及一些未知的原因，并不是所有免疫细胞经过改造都能表达目的CAR。而且不表达CAR的免疫细胞也有一定的生物学活性，因此免疫细胞组合物可以含有表达和不表达目的CAR的免疫细胞，该免疫细胞组合物依然能够满足临床应用的需求。In a thirteenth aspect, the present invention also provides an immune cell composition, the immune cell composition comprising any of the aforementioned On the one hand, there are modified immune cells, and optionally unmodified and/or unsuccessfully modified immune cells. These unmodified and/or unsuccessfully modified immune cells do not express the CAR of interest. Limited by the current technical level and some unknown reasons, not all immune cells can express the target CAR after modification. Moreover, immune cells that do not express CAR also have certain biological activities, so the immune cell composition can contain immune cells that express and do not express the CAR of interest, and the immune cell composition can still meet the needs of clinical application.
在某些实施方案中，经改造的表达目的CAR的免疫细胞占免疫细胞组合物总细胞数的大约10％-100％，优选地40％-80％。In certain embodiments, the engineered immune cells expressing the CAR of interest comprise approximately 10%-100%, preferably 40%-80%, of the total cell number of the immune cell composition.
在某些实施方案中，免疫细胞组合物被培养成免疫细胞系，因此，另一方面，本发明还提供了含有免疫细胞组合物的免疫细胞系。In certain embodiments, the immune cell composition is cultured into an immune cell line, and thus, in another aspect, the invention also provides immune cell lines containing the immune cell composition.
在第十四方面，本发明提供了用于制备上述任一方面所述的经改造的免疫细胞的试剂盒。在某些实施方案中，所述试剂盒包括第六方面所述的分离的核酸分子，或第七方面所述的核酸构建体、或包含它们的载体，和必要的溶剂，如无菌水，生理盐水，或细胞培养液，如LB培养液，如EliteCell原代T淋巴细胞培养体系(产品编号：PriMed-EliteCell-024)，以及可选的，还包括使用说明书。In a fourteenth aspect, the present invention provides a kit for preparing the modified immune cells described in any of the above aspects. In certain embodiments, the kit includes the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a carrier containing them, and necessary solvents, such as sterile water, Physiological saline, or cell culture medium, such as LB culture medium, such as EliteCell primary T lymphocyte culture system (product number: PriMed-EliteCell-024), and optionally, instructions for use.
药物组合物pharmaceutical composition
在第十五方面，本发明提供了一种药物组合物，其含有本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物，以及药学上可接受的载体和/或赋形剂。In the fifteenth aspect, the present invention provides a pharmaceutical composition, which contains the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including the chimeric antigen receptor and another CAR construct co-expressing biologically active molecules), the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the The host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect, and a pharmaceutically acceptable carrier and/or excipients.
在某些实施方案中，所述药物组合物还包含另外的药学活性剂，例如具有抗肿瘤活性的药物(例如anti-PD1抗体、anti-PD-L1抗体、anti-CTLA-4抗体、anti-CD3抗体、anti-ASGPR1抗体、索拉菲尼或其衍生物、瑞格菲尼或其衍生物、培美曲塞、顺铂、紫杉醇、吉西他滨、卡培他滨或FOLFIRINOX)。In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug with anti-tumor activity (e.g., anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti- CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX).
在某些实施方案中，第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免 疫细胞、或第十三方面所述的免疫细胞组合物与所述另外的药学活性剂可以同时、分开或相继施用。In certain embodiments, the antigen-binding molecule described in the first aspect, the chimeric antigen receptor (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) described in the fifth aspect, The isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the The modified exemption mentioned in the eleventh or twelfth aspect The immune cells, or the immune cell composition of the thirteenth aspect, and the additional pharmaceutically active agent may be administered simultaneously, separately, or sequentially.
在某些实施方案中，本发明的药物组合物包含：第一方面所述的抗原结合分子。In certain embodiments, the pharmaceutical composition of the present invention includes: the antigen-binding molecule described in the first aspect.
在某些实施方案中，本发明的药物组合物包含：第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、或包含它们的载体。In certain embodiments, the pharmaceutical composition of the present invention includes: the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, or a vector containing them.
在某些实施方案中，本发明的药物组合物包含：第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物。In certain embodiments, the pharmaceutical composition of the present invention includes: the modified immune cells described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect.
本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。The antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule), the second aspect or the sixth aspect The isolated nucleic acid molecule described in the seventh aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the eleventh aspect or the The modified immune cells described in the twelfth aspect, or the immune cell composition described in the thirteenth aspect, can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, Gels, capsules, powders, granules, elixirs, tablets, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of production and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. Additionally, sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注 射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物通过静脉注射或推注给予。The antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule), the second aspect or the sixth aspect The isolated nucleic acid molecule described in the seventh aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the eleventh aspect or the The modified immune cells described in the twelfth aspect, or the immune cell composition described in the thirteenth aspect, can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, Intraocular, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection injection, intramuscular injection). The skilled artisan will understand that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the antigen-binding molecule described in the first aspect of the invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) , the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect , the modified immune cells described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect are administered by intravenous injection or bolus injection.
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物。“预防有效量”是指，足以预防，阻止，或延迟疾病的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。The pharmaceutical composition of the present invention may include a "therapeutic effective amount" or a "preventive effective amount" of the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor (including chimeric antigen receptor) described in the fifth aspect. CAR construct co-expressed with another biologically active molecule), the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect , the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect. "Prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the occurrence of disease. A "therapeutically effective amount" means an amount sufficient to cure or at least partially prevent disease and its complications in a patient who is already suffering from the disease. The antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule), the second aspect or the sixth aspect The isolated nucleic acid molecule described in the seventh aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the eleventh aspect or the The therapeutically effective amount of the modified immune cells described in the twelfth aspect, or the immune cell composition described in the thirteenth aspect, may vary according to the following factors: the severity of the disease to be treated, the overall condition of the patient's own immune system status, general characteristics of the patient such as age, weight, and gender, the manner in which the drug is administered, and other treatments administered at the same time, etc.
治疗方法及用途Treatment methods and uses
在另一方面，本发明提供了一种用于在受试者(例如人)中预防和/或治疗与GPC3的表达相关的疾病的方法，所述方法包括向有此需要的受试者施用有效量的本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物。In another aspect, the invention provides a method for preventing and/or treating a disease associated with expression of GPC3 in a subject (e.g., a human), said method comprising administering to a subject in need thereof An effective amount of the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule), the second aspect Or the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the eleventh aspect The modified immune cells described in the aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect.
在某些实施方案中，所述与GPC3的表达相关的疾病选自增生性疾病，例如肿瘤。 在某些实施方案中，所述与GPC3的表达相关的疾病是与GPC3的表达相关的非肿瘤相关的适应症。In certain embodiments, the disease associated with expression of GPC3 is selected from proliferative diseases, such as tumors. In certain embodiments, the disease associated with expression of GPC3 is a non-tumor-related indication associated with expression of GPC3.
在某些实施方案中，所述肿瘤是GPC3阳性肿瘤。在某些实施方案中，所述肿瘤选自实体瘤(例如肝癌、肝细胞癌、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、卵巢癌、卵巢透明细胞癌、黑色素瘤、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、直结肠癌、胃癌、神经胶质瘤中的一种或其组合。在某些实施方案中，所述肿瘤选自血液肿瘤(例如白血病、淋巴瘤等)。In certain embodiments, the tumor is a GPC3-positive tumor. In certain embodiments, the tumor is selected from the group consisting of solid tumors (e.g., liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma, non-small cell One or a combination of lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma. In certain embodiments, the tumor is selected from a hematological tumor (e.g., leukemia , lymphoma, etc.).
在某些实施方案中，所述方法包括向所述受试者施用有效量的第一方面所述的抗原结合分子。In certain embodiments, the method includes administering to the subject an effective amount of the antigen-binding molecule of the first aspect.
在某些实施方案中，所述方法包括向所述受试者施用有效量的第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物。In certain embodiments, the method includes administering to the subject an effective amount of the modified immune cells described in the eleventh or twelfth aspect, or the immune cell combination described in the thirteenth aspect things.
在某些实施方案中，所述方法包括以下步骤：(1)提供所述受试者所需的免疫细胞(例如T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突状细胞、或这些细胞的任意组合)；(2)将第二方面或第六方面所述的分离的核酸分子，或第七方面所述的核酸构建体、或包含它们的载体引入步骤(1)所述的免疫细胞；(3)将步骤(2)中获得的免疫细胞施用至所述受试者以进行治疗。In certain embodiments, the method includes the following steps: (1) providing the immune cells required by the subject (e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells); (2) introducing the isolated nucleic acid molecule described in the second or sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into step (1) of immune cells; (3) administering the immune cells obtained in step (2) to the subject for treatment.
在某些实施方案中，所述方法通过剂量分次，例如一次，两次，三次或更多次分开施用部分剂量，向所述受试者施用表达目的CAR的免疫细胞，例如在治疗的第一天施用总剂量的第一百分比，在随后的(例如第二，第三，第四，第五，第六或第七天或更晚)治疗日施用总剂量的第二百分比，例如在随后的(例如第三，第四，第五，第六，第七，第八，第九，第十天或更晚)治疗日施用总剂量的第三百分比(例如，剩余百分比)。In certain embodiments, the method administers immune cells expressing the CAR of interest to the subject by dose-fractionation, such as one, two, three or more divided administrations of partial doses, e.g., on the first day of treatment. A first percentage of the total dose is administered on one day and a second percentage of the total dose is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day , e.g., administer a third percent of the total dose (e.g., remaining percentage).
在某些实施方案中，在治疗的第一天施用总剂量的10％的细胞，在第二天施用总剂量的30％的细胞，并且在第三天施用总剂量的剩余60％的细胞。In certain embodiments, 10% of the total dose of cells is administered on the first day of treatment, 30% of the total dose of cells is administered on the second day, and the remaining 60% of the total dose of cells is administered on the third day.
在某些实施方案中，在治疗的第一天施用总剂量的50％的细胞，在随后的(例如第二，第三，第四，第五，第六或第七或更晚)治疗日施用总剂量的50％的细胞。在某些实施方案中，在治疗的第一天施用总剂量的1/3的细胞，在随后的(例如第二，第三，第四，第五，第六或第七天或更晚)治疗日施用总剂量的1/3的细胞，在随后的(例如第三，第四，第五，第六，第七，第八，第九，第十天或更晚)施用总剂量的1/3的细胞。In certain embodiments, 50% of the total dose of cells is administered on the first day of treatment and on subsequent (e.g., second, third, fourth, fifth, sixth or seventh or later) treatment days. Apply 50% of the total dose to cells. In certain embodiments, 1/3 of the total dose of cells is administered on the first day of treatment, and on subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) Administer 1/3 of the total dose of cells on the treatment day and 1/3 of the total dose on subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or later days) /3 cells.
在某些实施方案中，总细胞剂量包含1×107至10×108个目的CAR阳性免疫细胞，例如包含(1-5)×107至(5-10)×108个目的CAR阳性免疫细胞。 In certain embodiments, the total cell dose includes 1×10 7 to 10×10 8 CAR-positive immune cells of interest, for example, includes (1-5)×10 7 to (5-10)×10 8 CAR-positive immune cells of interest Immune Cells.
在某些实施方案中，医师可以根据病人的状态，肿瘤的大小和阶段，或联合治疗的药物等临床情况来调节剂量或治疗方案。In certain embodiments, the physician may adjust the dosage or treatment regimen based on clinical circumstances such as the patient's status, tumor size and stage, or combination therapy agents.
在某些实施方案中，将本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物与另外的试剂联合施用。在某些实施方案中，所述另外的试剂包括(i)增加包含CAR核酸或CAR多肽的细胞(例如表达本发明的CAR的免疫细胞，本发明的经改造的免疫细胞或免疫细胞组合物)的功效的作用剂；(ii)改善与施用包含CAR核酸或CAR多肽的细胞(例如表达本发明的CAR的免疫细胞，本发明的经改造的免疫细胞或免疫细胞组合物)相关的一种或多种副作用的作用剂；(iii)具有抗肿瘤活性的另外的药学活性剂。这些试剂可以在施用本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物之前、同时或之后施用。In certain embodiments, the antigen-binding molecule according to the first aspect of the invention, the chimeric antigen receptor according to the fifth aspect (including a CAR construct co-expressing the chimeric antigen receptor and another biologically active molecule) ), the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host described in the fourth or ninth aspect The cells, the modified immune cells described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect are administered in combination with another agent . In certain embodiments, the additional agents include (i) increasing cells comprising a CAR nucleic acid or CAR polypeptide (e.g., an immune cell expressing a CAR of the invention, a modified immune cell, or an immune cell composition of the invention) an agent that improves the efficacy of; (ii) improves a or Agents with multiple side effects; (iii) Additional pharmaceutically active agents with anti-tumor activity. These reagents can be used in the administration of the antigen-binding molecule described in the first aspect of the present invention, the chimeric antigen receptor (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) described in the fifth aspect, The isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the The modified immune cells described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect are administered before, simultaneously or after.
在某些实施方案中，以上所述方法还包括向所述受试者施用第二疗法，所述第二疗法可以是已知用于肿瘤的任何疗法，例如手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其任意组合。In certain embodiments, the methods described above further include administering to the subject a second therapy, which may be any therapy known for use in tumors, such as surgery, chemotherapy, radiotherapy, immunotherapy, Gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof.
在某些实施方案中，所述第二疗法可以与以上所述的方法分开或联合应用；或，所述第二疗法可以与以上所述的方法同时或相继应用。In certain embodiments, the second therapy may be used separately or in combination with the methods described above; or, the second therapy may be used simultaneously or sequentially with the methods described above.
在某些实施方案中，所述受试者可以为哺乳动物，例如人。In certain embodiments, the subject can be a mammal, such as a human.
在另一个方面，提供了本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经 改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物在制备用于预防和/或治疗与GPC3的表达相关的疾病的药物中的用途。前述治疗方法中的剂量，剂型，给药途径，适应症，联合治疗等各个方面都可以应用到所述药物的用途中。In another aspect, the invention provides the antigen-binding molecule described in the first aspect and the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) , the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect , the experience mentioned in the eleventh or twelfth aspect Use of modified immune cells, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect, in the preparation of medicaments for preventing and/or treating diseases related to the expression of GPC3 . All aspects such as dosage, dosage form, route of administration, indications, combination therapy, etc. in the aforementioned treatment methods can be applied to the use of the medicine.
在另一个方面，提供了本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物，用于预防和/或治疗与GPC3的表达相关的疾病。前述治疗方法中的剂量，剂型，给药途径，适应症，联合治疗等各个方面都可以应用到所述药物的用途中。In another aspect, the invention provides the antigen-binding molecule described in the first aspect and the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the chimeric antigen receptor is co-expressed with another biologically active molecule) , the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect , the modified immune cells described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect, for prevention and/or Treating diseases associated with GPC3 expression. All aspects such as dosage, dosage form, route of administration, indications, combination therapy, etc. in the aforementioned treatment methods can be applied to the use of the medicine.
缩略词
CAR        嵌合抗原受体
CDR        免疫球蛋白可变区中的互补决定区
CDR-H1     免疫球蛋白重链可变区中的互补决定区1
CDR-H2     免疫球蛋白重链可变区中的互补决定区2
CDR-H3     免疫球蛋白重链可变区中的互补决定区3
CDR-L1     免疫球蛋白轻链可变区中的互补决定区1
CDR-L2     免疫球蛋白轻链可变区中的互补决定区2
CDR-L3     免疫球蛋白轻链可变区中的互补决定区3
FR         抗体构架区：抗体可变区中除CDR残基以外的氨基酸残基
VH         抗体重链可变区
VL         抗体轻链可变区
Kabat      由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et 
al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
IMGT      基于由Lefranc等人发起的国际免疫遗传学信息系统(The international 
ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
Chothia   由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴
定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
AbM       AbM CDR定义方式来源于Martin的相关研究(Martin ACR,Cheetham 
JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268–9272)。
IL-2      白细胞介素2
IFN       干扰素
PCR       聚合酶链式反应
FACS      流式细胞荧光分选 abbreviation
CAR Chimeric Antigen Receptor
CDR complementarity determining region in immunoglobulin variable region
CDR-H1 complementarity determining region 1 in the immunoglobulin heavy chain variable region
CDR-H2 complementarity determining region 2 in the immunoglobulin heavy chain variable region
CDR-H3 Complementarity determining region 3 in the immunoglobulin heavy chain variable region
CDR-L1 Complementarity determining region 1 in the immunoglobulin light chain variable region
CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region
CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region
FR antibody framework region: amino acid residues in the antibody variable region except CDR residues
VH antibody heavy chain variable region
VL antibody light chain variable region
Kabat Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al.
al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
IMGT is based on the International Immunogenetics Information System (The International Immunogenetics Information System) initiated by Lefranc et al.
ImMunoGeneTics information system (IMGT)), please refer to Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
Chothia Immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. Man (1989) Nature 342:878-883).
The definition of AbM AbM CDR comes from Martin's related research (Martin ACR, Cheetham
JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272).
IL-2 Interleukin 2
IFN interferon
PCR polymerase chain reaction
FACS flow cytometry fluorescence sorting
术语定义Definition of Terms
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，本文中所用的分子生物学、微生物学、细胞生物学、生物化学、免疫学等操作步骤均为相应领域内广泛使用的常规步骤。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。In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the operating procedures used in this article such as molecular biology, microbiology, cell biology, biochemistry, and immunology are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.
如本文中所使用的，术语“抗原结合分子”是抗体分子或其抗原结合片段。As used herein, the term "antigen-binding molecule" is an antibody molecule or antigen-binding fragment thereof.
如本文中所使用的，术语“抗体”指能够通过位于免疫球蛋白分子可变区的至少一个抗原识别位点特异性结合靶(如碳水化合物、多核苷酸、脂质、多肽等)的免疫球蛋白分子。如本文所用，该术语不仅包括完整的多克隆或单克隆抗体，而且包括其片段(例如Fab、Fab'、F(ab')2、Fv)、单链(例如scFv，di-scFv，(scFv)2)和结构域抗体(包括例如鲨鱼和骆驼抗体)、以及包括抗体的融合蛋白、以及包括抗原识别位点的任何其它修饰构型的免疫球蛋白分子。本发明的抗体不受任何特定的产生抗体的方法限制。抗体包括任何类型的抗体，例如IgG、IgA或IgM(或其亚类)，并且抗体不需要属于任何特定的类型。取决于抗体重链恒定区的氨基酸序列，免疫球蛋白可以分配到不同的类型。有五种主要类型的免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中几种可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类型的免疫球蛋白的重链恒定区分别被称为α、δ、ε、γ和μ。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。不同类型的免疫球蛋白的亚基结构和三维构型是众所周知的。重链恒定区由4个结构域(CH1、hinge region、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Globulin molecules. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g. Fab, Fab', F(ab')2, Fv), single chain (e.g. scFv, di-scFv, (scFv ) 2 ) and domain antibodies (including, for example, shark and camel antibodies), as well as fusion proteins including antibodies, and any other modified configuration of immunoglobulin molecules including an antigen recognition site. The antibodies of the invention are not limited to any particular method of producing the antibodies. Antibodies include antibodies of any type, such as IgG, IgA or IgM (or subclasses thereof), and the antibodies need not be of any particular type. Depending on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulins can be assigned to different classes. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different types of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known. The heavy chain constant region consists of 4 domains (CH1, hinge region, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
抗体的VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDRs和4个FRs组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National  Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。The VH and VL regions of antibodies can also be subdivided into highly denaturing regions called complementarity-determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each V H and V L consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site. The assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDRs，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or IMGT numbering system (Lefranc et al. , Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).
在本发明中，抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof, are preferably determined by the Kabat, Chothia, or IMGT numbering systems.
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
如本文中所使用的，术语抗体的“抗原结合片段”是指抗体的片段的多肽，例如全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括骆驼Ig、Ig NAR、Fab片段、Fab'片段、F(ab)'2片段、F(ab)'3片段、Fd、Fv、scFv、di-scFv、(scFv)2、微型抗体、双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白(“dsFv”)和单结构域抗体(sdAb，纳米抗体)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a fragment of a full-length antibody, that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and/ or compete with the full-length antibody for specific binding to the antigen, which is also referred to as the "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. It can be obtained by recombinant DNA technology Or antigen-binding fragments of the antibody are generated by enzymatic or chemical cleavage of the intact antibody. Non-limiting examples of antigen-binding fragments include camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab )' 3 fragments, Fd, Fv, scFv, di-scFv, (scFv) 2 , minibodies, diabodies, tribodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv") and single structures Domain antibodies (sdAb, Nanobodies) and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136 middle.
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))； 术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989)); The term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab') 2 fragment" means an antibody fragment consisting of two Fab fragments connected by a disulfide bridge on the hinge region Antibody fragment; the term "Fab'fragment" means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (from VH and CH1 domain).
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDRs赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDRs)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。As used herein, the term "Fc" means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain. Antibody fragments. The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在某些实施方案中，VH和VL结构域可以以任何合适的排列彼此相对定位。例如，包含NH2-VH-VH-COOH、NH2-VL-VL-COOH的scFv。所述scFv可以形成任何工程上可能的结构，单链抗体(scFv)，串联抗体(tandem di-scFvs)，双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白，骆驼Ig、IgNAR等。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发 明的某些实施方案中，scFv可形成(scFv)2，其指的是两个或两个以上单个scFv并联而形成抗体。As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In certain embodiments, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, scFv containing NH2 -VH-VH-COOH, NH2- VL-VL-COOH. The scFv can form any engineering possible structure, single chain antibody (scFv), tandem antibody (tandem di-scFvs), bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond stabilized Fv protein, camel Ig , IgNAR, etc. In certain embodiments of the invention, scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody. in this hair In certain embodiments of the invention, scFv can form (scFv) 2 , which refers to two or more individual scFvs joining in parallel to form an antibody.
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力(Holt,L.等人,生物技术趋势(Trends in Biotechnology),21(11):484-490，2003)。单域抗体也称为纳米抗体(nanobody)。As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable Antibody fragments composed of regions) that retain the ability to specifically bind to the same antigen bound by the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003 ). Single domain antibodies are also called nanobodies.
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。Antigen-binding fragments of an antibody (e.g., the above-described antibody fragments) can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。As used herein, when the term "antibody" is mentioned, it includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
如本文中所使用的，表述“特异性结合”或“特异性针对”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。As used herein, the expression "specific binding" or "specific targeting" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction. In the present invention, the term " KD " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。The specific binding properties between two molecules can be determined using methods known in the art. One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187). The ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). K D , kon and kdis values can be measured by any valid method. In certain embodiments, dissociation constants can be measured in Biacore using surface plasmon resonance (SPR). Alternatively, bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一 个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two Each peptide A certain position among them is occupied by lysine), then each molecule is identical at that position. "Percent identity" between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions). Typically, comparisons are made when two sequences are aligned to yield maximum identity. Such alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the PAM120 weight residue table using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0). , a gap length penalty of 12 and a gap penalty of 4 to determine the percent identity between two amino acid sequences. Alternatively, the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm can be used using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA94:412-417(1997)，其通过引用并入本文)。 As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999) ; and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。The twenty conventional amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
如本文中所使用的，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。载体可以包括在细胞中直接自主复制的序列，或可以包括足以允许整合到宿主细胞DNA中的序列。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(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噬菌体及病毒载体等。病毒载体的非限制性实例包括，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，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。As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. The vector may include sequences that replicate directly and autonomously in the cell, or may include sequences sufficient to permit integration into the host cell DNA. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and viral vectors, etc. Non-limiting examples of viral vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, vesicle viruses (such as SV40). A vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.
如本文中所使用的，术语“游离型载体”中游离型是指载体能够复制而不整合到宿主的染色体DNA中并且不由分裂宿主细胞逐渐丧失，还意指所述载体在染色体外或游离地复制。As used herein, the term "episomal vector" means that the vector is capable of replicating without integrating into the chromosomal DNA of the host and is not gradually lost by dividing host cells. It also means that the vector is extrachromosomally or episomally. copy.
如本文中所使用的，术语“病毒载体”广泛用以指包括典型地促进核酸分子转移或整合到细胞的基因组中的病毒衍生的核酸元件的核酸分子(例如转移质粒)，或介导核酸转移的病毒颗粒。除了核酸之外，病毒颗粒典型地将包括各种病毒组分并且有时还包括宿主细胞组分。As used herein, the term "viral vector" is used broadly to refer to a nucleic acid molecule (eg, a transfer plasmid) that includes a virus-derived nucleic acid element that typically facilitates the transfer or integration of the nucleic acid molecule into the genome of a cell, or mediates the transfer of nucleic acid of virus particles. In addition to nucleic acid, viral particles will typically include various viral components and sometimes host cell components.
术语“病毒载体”可以指能够将核酸转移到细胞中的病毒或病毒颗粒，或指转移的核酸本身。病毒载体和转移质粒含有主要衍生自病毒的结构和/或功能遗传元件。The term "viral vector" may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements derived primarily from viruses.
如本文中所使用的，术语“逆转录病毒载体”是指含有主要衍生自逆转录病毒的结构和功能遗传元件或其部分的病毒载体或质粒。As used herein, the term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from retroviruses, or portions thereof.
如本文中所使用的，术语“慢病毒载体”是指含有主要衍生自慢病毒的结构和功能遗传元件或其部分(包括LTR)的病毒载体或质粒。在某些实施方案中，术语“慢病毒载体”、“慢病毒表达载体”可以用以指慢病毒转移质粒和/或感染性慢病毒颗粒。在本文提 及元件(例如克隆位点、启动子、调节元件、异源核酸等)时，应理解，这些元件的序列以RNA形式存在于本发明的慢病毒颗粒中并且以DNA形式存在于本发明的DNA质粒中。As used herein, the term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from lentiviruses, or portions thereof (including LTRs). In certain embodiments, the terms "lentiviral vector" and "lentiviral expression vector" may be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles. mentioned in this article and elements (such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.), it should be understood that the sequences of these elements exist in the form of RNA in the lentiviral particles of the invention and in the form of DNA in the DNA of the invention in the plasmid.
如本文中所使用的，“整合缺陷型”逆转录病毒或慢病毒是指具有不能将病毒基因组整合到宿主细胞的基因组中的整合酶的逆转录病毒或慢病毒。在某些实施方案中，整合酶蛋白突变以特异性降低其整合酶活性。整合缺陷型慢病毒载体可以通过修饰编码整合酶蛋白的pol基因，产生编码整合缺陷型整合酶的突变pol基因而获得。所述整合缺陷型病毒载体已经描述于专利申请WO 2006/010834中，所述专利申请以全文引用的方式并入本文中。As used herein, an "integration-deficient" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that is unable to integrate the viral genome into the genome of the host cell. In certain embodiments, the integrase protein is mutated to specifically reduce its integrase activity. Integration-deficient lentiviral vectors can be obtained by modifying the pol gene encoding the integrase protein to generate a mutant pol gene encoding an integration-deficient integrase. Such integration-deficient viral vectors have been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞，免疫细胞(如T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞等)。宿主细胞可以包括单个细胞或细胞群体。As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes cells, NK cells, monocytes, macrophages or dendritic cells, etc.). Host cells can include single cells or populations of cells.
如本文中所使用的，术语“嵌合抗原受体”或“CAR”是指包含至少一个细胞外抗原结合结构域，间隔结构域，跨膜结构域和细胞质信号传导结构域(本文也称为“胞内信号传导结构域”)的重组多肽构建体，其将针对目的抗原(例如GPC3)的基于抗体的特异性与免疫效应细胞活化胞内结构域组合以展现针对表达该目的抗原(例如GPC3)细胞的特异性免疫活性。在本发明中，表述“表达CAR的免疫效应细胞”是指表达CAR并且具有由该CAR的靶向结构域决定的抗原特异性的免疫效应细胞。制造CAR(例如，用于癌症治疗)的方法是本领域已知的，可参见例如，Park等人,Trends Biotechnol.,29:550-557,2011；Grupp等人,N Engl J Med.,368:1509-1518,2013；Han等人,J.Hematol.Oncol.,6:47,2013；PCT专利公开文本WO2012/079000、WO2013/059593；和美国专利公开文本2012/0213783，其全部通过引用整体并入本文。As used herein, the term "chimeric antigen receptor" or "CAR" refers to a receptor that contains at least one extracellular antigen-binding domain, spacer domain, transmembrane domain, and cytoplasmic signaling domain (also referred to herein as "Intracellular signaling domain") recombinant polypeptide construct that combines antibody-based specificity for an antigen of interest (e.g., GPC3) with an immune effector cell-activating intracellular domain to demonstrate resistance to expression of the antigen of interest (e.g., GPC3 ) cell-specific immune activity. In the present invention, the expression "CAR-expressing immune effector cells" refers to immune effector cells that express CAR and have antigen specificity determined by the targeting domain of the CAR. Methods of making CARs (e.g., for cancer treatment) are known in the art, see, e.g., Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368 :1509-1518, 2013; Han et al., J. Hematol. Oncol., 6:47, 2013; PCT patent publications WO2012/079000, WO2013/059593; and US patent publication 2012/0213783, all of which are incorporated by reference in their entirety Incorporated herein.
如本文中所使用的，术语“胞外抗原结合结构域”是指能够特异性结合目的抗原或受体的多肽。该结构域将能够与细胞表面分子相互作用。例如，可以选择胞外抗原结合结构域来识别作为与特定疾病状态相关的靶细胞细胞表面标志物的抗原。As used herein, the term "extracellular antigen-binding domain" refers to a polypeptide capable of specifically binding to an antigen or receptor of interest. This domain will be able to interact with cell surface molecules. For example, the extracellular antigen-binding domain can be selected to recognize an antigen that is a cell surface marker on a target cell associated with a particular disease state.
如本文中所使用的，术语“胞内信号传导结构域”是指传导效应信号功能信号并引导细胞进行专门的功能的蛋白质部分。因此，胞内信号传导结构域具有激活表达CAR 的免疫效应细胞的至少一种正常效应子功能的能力。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。As used herein, the term "intracellular signaling domain" refers to the portion of a protein that conducts effector signaling functions and directs the cell to perform specialized functions. Therefore, the intracellular signaling domain has the ability to activate the expression of CAR The ability of an immune effector cell to perform at least one normal effector function. For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
如本文中所使用的，术语“初级信号传导结构域”是指能够以刺激方式或以抑制方式调节TCR复合物的初级活化的蛋白质部分。以刺激方式作用的初级信号传导结构域通常含有已知为基于免疫受体酪氨酸的活化基序(ITAM)的信号传导基序。含有特别用于本发明中的初级信号传导结构域的ITAM的非限制性实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。As used herein, the term "primary signaling domain" refers to a portion of a protein capable of modulating primary activation of a TCR complex in a stimulatory manner or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner often contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.
如本文中所使用的，术语“共刺激信号传导结构域”是指共刺激分子的胞内信号传导结构域。共刺激分子是除抗原受体或Fc受体以外的在结合到抗原后提供T淋巴细胞的高效活化和功能所需的第二信号的细胞表面分子。所述共刺激分子的非限制性实例包括CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)、DAP10。As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes after binding to an antigen. Non-limiting examples of costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278(ICOS), DAP10.
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：无菌水，生理盐水，pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些 示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。As used herein, the term "pharmaceutically acceptable carrier and/or excipient" means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: sterile water, physiological saline, pH adjusters, surfactants , adjuvants, ionic strength enhancers, diluents, reagents to maintain osmotic pressure, reagents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). in some In exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solutions (eg, 0.01% polysorbate 20), pH buffer solutions (eg, phosphate buffer solution), Ringer's solution, and any combination thereof.
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom (eg, tumor) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no further worsening) of the disease state, delaying or slowing the progression of the disease, ameliorating or alleviating the symptoms of the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging survival compared to expected survival if not receiving treatment.
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，术语“受试者”是指包括其中可以引出免疫应答的活生物体。在某些实施方式中，所述受试者(例如人)患有肿瘤(例如与GPC3相关的肿瘤)，或者，具有患有上述疾病的风险。As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the term "subject" is meant to include living organisms in which an immune response can be elicited. In certain embodiments, the subject (eg, a human) has a tumor (eg, a GPC3-related tumor), or is at risk of suffering from a disease described above.
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。As used herein, the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect. For example, a disease-preventing (e.g., tumor) effective amount refers to an amount that is sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a tumor); a disease-treating effective amount refers to an amount that is sufficient to cure or at least partially prevent an existing disease. The patient's disease and the amount of its complications. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
如本文中使用的，术语“免疫细胞”是指涉及免疫反应例如涉及促进免疫效应子功能的细胞。免疫细胞的实例包括T细胞(例如α/βT细胞和γ/δT细胞)、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞和骨髓来源巨噬细胞。As used herein, the term "immune cell" refers to a cell involved in an immune response, such as in promoting immune effector function. Examples of immune 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 bone marrow-derived macrophages.
本发明所述的免疫细胞可以是自身的/自体的(“自我”)或非自身的(“非自我”，例如同种异体的、同基因的或异基因的)。如本文中使用的，“自身的”是指来自同一受试者的细胞；“同种异体的”是指与比较细胞遗传不同的同一物种的细胞；“同基因的”是指与比较细胞遗传相同的来自不同受试者的细胞；“异基因的”是指与比较细胞来自不同物种的细胞。在优选实施例中，本发明的细胞是同种异体的。 Immune cells of the invention may be self/autologous ("self") or non-self ("non-self", eg allogeneic, syngeneic or allogeneic). As used herein, "autologous" refers to cells from the same subject; "allogeneic" refers to cells of the same species that are genetically different from the comparison cells; "isogenic" means cells that are genetically different from the comparison cells. Identical cells from different subjects; "allogeneic" means cells from a different species than the comparison cells. In preferred embodiments, the cells of the invention are allogeneic.
可用于本文所述的CAR的示例性免疫细胞包括T淋巴细胞和/或NK细胞。术语“T细胞”或“T淋巴细胞”是本领域公知的并且意图包括胸腺细胞、未成熟的T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化的T淋巴细胞。T细胞可以是T辅助(Th)细胞，例如T辅助1(Th1)或T辅助2(Th2)细胞。T细胞可以是辅助T细胞(HTL；CD4T细胞)CD4T细胞、细胞毒性T细胞(CTL；CD8T细胞)、CD4CD8T细胞、CD4CD8T细胞或任何其它T细胞子组。在某些实施方案中，T细胞可以包括原初T细胞和记忆T细胞。Exemplary immune cells useful in the CARs described herein include T lymphocytes and/or NK cells. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cells may be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 T cells) CD4 T cells, cytotoxic T cells (CTL; CD8 T cells), CD4CD8 T cells, CD4CD8 T cells, or any other subset of T cells. In certain embodiments, T cells can include naive T cells and memory T cells.
本领域技术人员将理解，其它细胞也可以用作具有如本文所述的CAR的免疫细胞。具体来说，免疫细胞还包括NK细胞、单核细胞、巨噬细胞或树突状细胞、NKT细胞、嗜中性白细胞和巨噬细胞。免疫细胞还包括免疫细胞的祖细胞，其中所述祖细胞可以在体内或体外经诱导以分化成免疫细胞。因此，在某些实施方案中，免疫细胞包括免疫细胞的祖细胞，例如含于衍生自脐血、骨髓或流动周边血液的CD34+细胞群体内的造血干细胞(HSC)，其在受试者中投与后分化成成熟免疫细胞，或其可以在体外经诱导以分化成成熟免疫细胞。Those skilled in the art will understand that other cells may also be used as immune cells with a CAR as described herein. Specifically, immune cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils, and macrophages. Immune cells also include progenitor cells of immune cells, wherein the progenitor cells can be induced to differentiate into immune cells in vivo or in vitro. Thus, in certain embodiments, the immune cells include progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained within a population of CD34+ cells derived from cord blood, bone marrow, or circulating peripheral blood, which are administered in a subject Then differentiate into mature immune cells, or they can be induced to differentiate into mature immune cells in vitro.
如本文中使用的，术语“经改造的免疫细胞”是指，表达本文所述的任何一种CAR，或导入了本文所述的任何一种分离的核酸或载体的免疫细胞。可以用多种方法将编码CAR多肽的多核苷酸引入细胞后，也可以在细胞中原位合成CAR多肽。或者，可以在细胞外生产CAR多肽，然后将其引入细胞。将多核苷酸构建体引入细胞的方法是本领域已知的。在一些实施方案中，可以使用稳定的转化方法将多核苷酸构建体整合到细胞的基因组中。在其他实施方案中，瞬时转化方法可用于瞬时表达多核苷酸构建体，并且多核苷酸构建体未整合到细胞的基因组中。在其它实施方案中，可以使用病毒介导的方法。多核苷酸可以通过任何合适的方法引入细胞，例如重组病毒载体(例如逆转录病毒、腺病毒)，脂质体等。瞬时转化方法包括，例如但不限于显微注射、电穿孔或微粒轰击。多核苷酸可以包括在载体中，例如质粒载体或病毒载体。As used herein, the term "modified immune cell" refers to an immune cell that expresses any of the CARs described herein, or has been introduced with any of the isolated nucleic acids or vectors described herein. After the polynucleotide encoding the CAR polypeptide is introduced into the cell using various methods, the CAR polypeptide can also be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods of introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct without the polynucleotide construct being integrated into the genome of the cell. In other embodiments, virus-mediated methods may be used. Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (eg, retroviruses, adenoviruses), liposomes, and the like. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or microparticle bombardment. The polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.
如本文中使用的，术语“免疫效应子功能”是指免疫效应细胞的增强或促进对靶细胞的免疫攻击(例如对靶细胞的杀伤，或者抑制其生长或增殖)的功能或反应。例如，T细胞的效应子功能可以是溶细胞活性或辅助活性，包括细胞因子的分泌。As used herein, the term "immune effector function" refers to a function or response of an immune effector cell that enhances or promotes an immune attack on a target cell (eg, killing of the target cell, or inhibiting its growth or proliferation). For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
发明的有益效果Beneficial effects of the invention
CAR-T细胞治疗目前在实体瘤中的治疗效果仍不足，其主要原因是实体瘤有复杂的 肿瘤微环境和肿瘤异质性高。本发明提供一种靶向GPC3的CAR或包含所述CAR的免疫细胞，通过特异性靶向GPC3提高对于表达肿瘤抗原细胞的杀伤并在一定程度上降低其脱靶毒性，增强CAR-T细胞的肿瘤杀伤效果。The current therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient. The main reason is that solid tumors have complex The tumor microenvironment and tumor heterogeneity are high. The present invention provides a GPC3-targeting CAR or an immune cell containing the CAR. By specifically targeting GPC3, it improves the killing of tumor antigen-expressing cells and reduces its off-target toxicity to a certain extent, thereby enhancing the tumor immunity of CAR-T cells. Killing effect.
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. The various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments.
附图说明Description of the drawings
图1显示了CAR-T细胞(空白T，CD4-T、CA3-T、CB5-T、CC8-T、CE9-T和CH3-T)对靶细胞的杀伤活性检测结果。Figure 1 shows the detection results of the killing activity of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T) on target cells.
图2A显示了CAR-T细胞(空白T，CD4-T、CA3-T、CB5-T、CC8-T、CE9-T和CH3-T)激活后IFN-γ分泌水平检测结果。图2B显示了CAR-T细胞(空白T，CD4-T、CA3-T、CB5-T、CC8-T、CE9-T和CH3-T)激活后IL-2分泌水平检测结果。Figure 2A shows the detection results of IFN-γ secretion levels after activation of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T). Figure 2B shows the detection results of IL-2 secretion levels after activation of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T).
图3A显示了经PBS，空白T细胞，CA3-T，CC8-T，CH3-T治疗的B-NDG小鼠的肿瘤体积变化曲线。图3B显示了经PBS，空白T细胞，CA3-T，CC8-T，CH3-T治疗的B-NDG小鼠的体重变化曲线。Figure 3A shows the tumor volume change curve of B-NDG mice treated with PBS, blank T cells, CA3-T, CC8-T, and CH3-T. Figure 3B shows the body weight change curve of B-NDG mice treated with PBS, blank T cells, CA3-T, CC8-T, and CH3-T.
序列信息sequence information
本发明涉及的序列信息提供如下：The sequence information involved in the present invention is provided as follows:
表1

















Table 1

















具体实施方式Detailed ways
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。The invention will now be described with reference to the following examples which are intended to illustrate but not to limit the invention.
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行。本领域技术人员知晓，实施例以举例方式描述本发明，且不 意欲限制本发明所要求保护的范围。Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was performed as described in FMAusubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the embodiments describe the invention by way of example and do not It is intended to limit the scope of the invention as claimed.
实施例1：结合人类GPC3的特异性单链抗体(scFv)的制备Example 1: Preparation of specific single chain antibodies (scFv) binding to human GPC3
1)噬菌体库筛选GPC3 scFv1) Phage library screening for GPC3 scFv
采用生物素化GPC3与SV磁珠对全人源噬菌体文库进行筛选，筛选产物通过铺板检测噬菌体滴定。取第一轮淘筛产物与PBST混合，按上述步骤进行第2轮、第3轮淘筛。Biotinylated GPC3 and SV magnetic beads were used to screen the fully human phage library, and the screened products were tested for phage titration by plating. Mix the product of the first round of sifting with PBST and perform the second and third rounds of sifting according to the above steps.
2)ELISA检测单克隆噬菌体2) ELISA detection of monoclonal phage
从噬菌体淘筛产物滴定板中接种单个菌落到96深孔板中，ELISA检测单克隆噬菌体。A single colony was inoculated from the phage panning product titer plate into a 96-deep well plate, and monoclonal phage were detected by ELISA.
共筛选得到6株全人源抗GPC3单克隆抗体CD4，CA3，CB5，CC8，CE9，CH3。对上述单克隆抗体进行测序和分析后，获得VH、VL的序列，并根据Kabat、IMGT和、Chothia和AbM编号系统获得CDR-H1，CDR-H2，CDR-H3，CDR-L1，CDR-L2，CDR-L3的序列(具体序列参见表1和表2)。A total of 6 strains of fully human anti-GPC3 monoclonal antibodies CD4, CA3, CB5, CC8, CE9, and CH3 were screened. After sequencing and analyzing the above monoclonal antibodies, the sequences of VH and VL were obtained, and CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 were obtained according to the Kabat, IMGT, Chothia, and AbM numbering systems. , CDR-L3 sequence (see Table 1 and Table 2 for specific sequences).
表2.抗GPC3单克隆抗体的序列

Table 2. Sequences of anti-GPC3 monoclonal antibodies

3)scFv-Fc的构建与抗体聚体分析3) Construction of scFv-Fc and analysis of antibody aggregates
将上述全人源抗体的VH和VL通过linker(SEQ ID NO:110)连接，获得scFv，各scFv的序列信息如下表所示。Connect the VH and VL of the above fully human antibodies through a linker (SEQ ID NO: 110) to obtain scFv. The sequence information of each scFv is shown in the table below.
表3：scFv的结构
Table 3: Structure of scFv
将候选scfv序列构建在TGEX-KAL载体中，然后转染expi293细胞进行表达和纯化scFv-Fc蛋白。SEC分析实验结果表明(表4)，6个候选scfv序列单体峰(main peak area)均大于80％。The candidate scFv sequence was constructed in the TGEX-KAL vector, and then transfected into expi293 cells for expression and purification of scFv-Fc protein. SEC analysis experimental results show (Table 4) that the six candidate scfv sequence monomer peaks (main peak area) are all greater than 80%.
表4：scFv-Fc蛋白SEC数据
Table 4: scFv-Fc protein SEC data
4)GPC3细胞结合试验4) GPC3 cell binding assay
为鉴定GPC3 scFv-Fc蛋白结合亲和力，选择表达GPC3的细胞系(293T/GPC3+)用于细胞结合测定。以鼠IgG同型抗体(Mouse IgG Isotype Control，来自Thermo Fisher Sci.)作为阴性对照，抗GPC3抗体GC33(Ishiguro et al,2008；GC33相关序列参见US20150259417A1)作为阳性对照。表5结果为6个候选scFvs对GPC3阳性细胞 293T/GPC3+的亲和力明显优于对照组。To characterize GPC3 scFv-Fc protein binding affinity, a GPC3-expressing cell line (293T/GPC3+) was selected for cell binding assays. Mouse IgG isotype antibody (Mouse IgG Isotype Control, from Thermo Fisher Sci.) was used as a negative control, and anti-GPC3 antibody GC33 (Ishiguro et al, 2008; for GC33 related sequences, see US20150259417A1) was used as a positive control. Table 5 results of 6 candidate scFvs on GPC3 positive cells The affinity of 293T/GPC3+ was significantly better than that of the control group.
表5.GPC3细胞结合试验结果
Table 5. GPC3 cell binding test results
实施例2：慢病毒质粒的构建及病毒包装Example 2: Construction of lentiviral plasmid and virus packaging
1)慢病毒质粒的构建：1) Construction of lentiviral plasmid:
基于上述实施例中的scFv序列，进一步构建CAR慢病毒表达载体。以CD137(4-1BB)的胞内结构域和CD3ζ的ITAM区作为激活信号，与上述scFv进行融合，同时加上CD8α信号肽，CD8铰链区，CD8跨膜区，构建嵌合抗原受体表达载体，构建的嵌合抗原受体结构如下表6所示。Based on the scFv sequence in the above example, a CAR lentiviral expression vector was further constructed. The intracellular domain of CD137 (4-1BB) and the ITAM region of CD3ζ are used as activation signals and fused with the above scFv. At the same time, the CD8α signal peptide, CD8 hinge region, and CD8 transmembrane region are added to construct a chimeric antigen receptor expression Vector and constructed chimeric antigen receptor structure are shown in Table 6 below.
表6.嵌合抗原受体的结构
Table 6. Structure of chimeric antigen receptor
2)病毒包装：2) Virus packaging:
将以上构建的CAR慢病毒质粒与转染试剂混合液逐滴加入到293T(来源：ATCC)细胞中，轻轻晃动培养皿，充分混匀。将培养皿置于37℃、5％CO2培养箱；培养6～8小时后，将含有转染试剂的培养基去掉，更换为新鲜的完全培养基。连续培养48小时后，收集培养皿中含有病毒的培养基上清，用0.45μm的滤膜过滤，转至离心管中，配平后，20000×g 4℃离心2小时。离心结束后，在生物安全柜中，小心将离心管中的液体吸去，加入500μL PBS缓冲液将沉淀重悬，将病毒置于-80℃保存。Add the mixture of CAR lentiviral plasmid and transfection reagent constructed above into 293T (source: ATCC) cells drop by drop, shake the culture dish gently, and mix thoroughly. Place the culture dish in a 37°C, 5% CO2 incubator; after 6 to 8 hours of incubation, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium. After continuous culture for 48 hours, collect the culture supernatant containing the virus in the culture dish, filter it with a 0.45 μm filter, transfer it to a centrifuge tube, balance it, and centrifuge it at 20,000 × g at 4°C for 2 hours. After centrifugation, carefully remove the liquid from the centrifuge tube in a biological safety cabinet, add 500 μL PBS buffer to resuspend the pellet, and store the virus at -80°C.
实验例3：CAR-T细胞制备Experimental Example 3: CAR-T cell preparation
1)原代T细胞分离：1) Primary T cell isolation:
(1)采用淋巴细胞分离液(GE)分离得到人的PBMC细胞，置于37℃，5％ CO2的培养箱中培养，加入100μL/mL的CD3抗体和CD28抗体，充分混匀后，室温孵育15分钟。(1) Use lymphocyte separation medium (GE) to isolate human PBMC cells, place them at 37°C, 5% Incubate in a CO 2 incubator, add 100 μL/mL CD3 antibody and CD28 antibody, mix thoroughly, and incubate at room temperature for 15 minutes.
(2)取出磁珠，用移液枪上下吹打至少5次，充分混匀。(2) Take out the magnetic beads, pipet up and down at least 5 times with a pipette, and mix thoroughly.
(3)吸取50μL磁珠/mL至上述样品中，充分混匀后，室温孵育10分钟。(3) Add 50 μL magnetic beads/mL to the above sample, mix thoroughly, and incubate at room temperature for 10 minutes.
(4)添加完全培养基至管内总体积为2.5mL，将管子(开盖)插入磁极中，室温静置5分钟。(4) Add complete culture medium to a total volume of 2.5 mL in the tube, insert the tube (open the cap) into the magnetic pole, and let stand at room temperature for 5 minutes.
(5)孵育完毕后，管子继续留在磁极中，轻轻倒置，将管内的细胞倒出。(5) After the incubation is completed, keep the tube in the magnetic pole, gently invert it, and pour out the cells in the tube.
(6)将细胞重悬于X-vivo 15培养基中，并添加10％FBS，300U/mL IL-2,5ng/mL IL-15和10ng/mL IL-7。(6) Resuspend the cells in X-vivo 15 medium and add 10% FBS, 300U/mL IL-2, 5ng/mL IL-15 and 10ng/mL IL-7.
2)T细胞的激活：2) Activation of T cells:
调整细胞密度至1×106细胞/mL，加入细胞因子及抗体复合物(按终浓度为300U/mL的IL-2、10ng/mL IL-7、5ng/mL IL-15、500ng/mL Anti-CD3(OKT3)、2μg/mL Anti-CD28配置)，连续培养48小时。Adjust the cell density to 1×10 6 cells/mL, and add cytokines and antibody complexes (IL-2, 10ng/mL IL-7, 5ng/mL IL-15, 500ng/mL Anti at a final concentration of 300U/mL). -CD3(OKT3), 2μg/mL Anti-CD28 configuration), continuously cultured for 48 hours.
3)病毒感染：3) Viral infection:
(1)按照MOI＝5，计算所需要的病毒量。计算公式如下：所需病毒量(mL)＝(MOI*细胞数量)/病毒滴度(1) Calculate the required amount of virus according to MOI=5. The calculation formula is as follows: required amount of virus (mL) = (MOI*number of cells)/virus titer
(2)将病毒迅速复温到37℃。在六孔板中加入上述计算所得的病毒量，添加终浓度为5μg/mL的DEAE，充分混匀后，离心。(2) Rapidly rewarm the virus to 37°C. Add the amount of virus calculated above to the six-well plate, add DEAE with a final concentration of 5 μg/mL, mix thoroughly, and centrifuge.
(3)离心结束后，将六孔板置于37℃5％CO2的培养箱中，继续培养备用。(3) After centrifugation, place the six-well plate in an incubator at 37°C and 5% CO2 , and continue culturing for later use.
(4)250×g离心10分钟，去掉含有病毒的培养基上清，用新鲜培养基重悬细胞沉淀，将细胞转移至新的六孔板中，继续培养3-6天备用。(4) Centrifuge at 250×g for 10 minutes, remove the virus-containing culture medium supernatant, resuspend the cell pellet in fresh culture medium, transfer the cells to a new six-well plate, and continue to culture for 3-6 days before use.
通过上述方法分别获得表达实施例2中所述的CAR(CD4-T、CA3-T、CB5-T、CC8-T、CE9-T和CH3-T)的CAR-T细胞。CAR-T cells expressing the CARs (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T and CH3-T) described in Example 2 were obtained by the above method.
实验例4：CAR-T细胞的阳性率检测Experimental Example 4: Detection of Positive Rate of CAR-T Cells
编码CAR的核酸序列在启动子的驱动下表达，使用GPC3抗原对慢病毒转染的T细胞进行标记并通过流式进行测定，反映CAR在T细胞表面的表达水平。通过如上方法检测实施例3获得的CAR-T细胞的CAR阳性率进行检测，FACS检测结果如下表7所示。结果显示，所有CAR-T细胞的CAR阳性率均大于10％，表明慢病毒转染效应细胞后，成功表达了CAR，成功构建了表达了6种GPC3-CAR嵌合抗原受体T细胞 (CD4-T，CA3-T，CB5-T，CC8-T，CE9-T，CH3-T)。The nucleic acid sequence encoding the CAR is expressed under the drive of a promoter. T cells transfected with lentivirus are labeled using GPC3 antigen and measured by flow cytometry to reflect the expression level of CAR on the surface of T cells. The CAR positivity rate of the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS detection results are shown in Table 7 below. The results showed that the CAR positive rate of all CAR-T cells was greater than 10%, indicating that after lentivirus transfection of effector cells, CAR was successfully expressed and T cells expressing 6 types of GPC3-CAR chimeric antigen receptors were successfully constructed. (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T).
表7：CAR的阳性率检测结果
Table 7: CAR positive rate test results
实验例5：CAR-T对HepG2靶细胞的杀伤活性评价Experimental Example 5: Evaluation of the killing activity of CAR-T on HepG2 target cells
使用0.25％胰酶消化HEPG2-luc细胞，含10％FBS的1640培养基终止消化，离心后，重悬细胞，调整细胞密度至1×105个/mL，按照100μL/孔的量接种靶细胞HEPG2-luc于96孔板中，5％CO2 37℃培养箱静置30min。收集CAR-T，离心收集并用10％FBS的1640培养基重悬CAR-T细胞，GPC3-CAR以及未转染CAR的空白T细胞(UTD)作为效应细胞，然后按照不同的E/T(效应细胞/靶细胞)比例加入到含有HEPG2-luc的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO2 37℃培养箱中培养18～24h。培养结束后，将孔板从培养箱中取出，加入20ul荧光检测试剂，使用酶标仪检测荧光读值。Use 0.25% trypsin to digest HEPG2-luc cells, stop digestion with 1640 medium containing 10% FBS, centrifuge, resuspend the cells, adjust the cell density to 1×10 5 cells/mL, and inoculate target cells at 100 μL/well. HEPG2-luc was placed in a 96-well plate in a 5% CO 2 37°C incubator for 30 minutes. Collect CAR-T, centrifuge and resuspend CAR-T cells, GPC3-CAR and untransfected CAR blank T cells (UTD) in 1640 medium with 10% FBS as effector cells, and then follow different E/T (effector cells/target cells) were added to a 96-well plate containing HEPG2-luc at 100 μL/well, and the final volume was added to 200 μL/well, and cultured in a 37°C incubator with 5% CO 2 for 18 to 24 hours. After the culture is completed, take the well plate out of the incubator, add 20ul of fluorescence detection reagent, and use a microplate reader to detect the fluorescence reading.
CAR-T的杀伤活性检测结果如图1，本申请构建的6种CAR-T细胞(CD4-T，CA3-T，CB5-T，CC8-T，CE9-T，CH3-T)在不同的E/T比例下都能有效裂解肿瘤细胞。其中，CA3-T，CC8-T，CH3-T和CE9-T的效果尤为突出，在效应细胞/靶细胞比例为10时，对肿瘤细胞的裂解率高达98％。The killing activity test results of CAR-T are shown in Figure 1. The 6 types of CAR-T cells (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) constructed in this application were detected in different It can effectively lyse tumor cells at any E/T ratio. Among them, CA3-T, CC8-T, CH3-T and CE9-T are particularly effective. When the effector cell/target cell ratio is 10, the lysis rate of tumor cells is as high as 98%.
实验例6：CAR-T与HepG2靶细胞共孵育时的细胞因子释放Experimental Example 6: Cytokine release when CAR-T and HepG2 target cells are co-incubated
收集HepG2-luc细胞，使用培养基调整细胞密度至1×105个/mL，按照100μL/孔的量接种靶细胞于96孔板中，并用培养基重悬CAR-T细胞，GPC3-CAR以及未转染CAR的空白T细胞作为效应细胞，然后按照1:1的E/T(效应细胞/靶细胞)比例加入到含有靶细胞的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO2 37℃培养箱中培养过夜。培养结束后，将孔板从培养箱中取出，离心，取上清，使用ELISA试剂盒(IL2、IFN-γ)检测细胞因子释放。本申请构建的6种CAR-T细胞(CD4-T，CA3-T，CB5-T，CC8-T，CE9-T，CH3-T)能够在不同程度上杀伤肿瘤细胞，并释放IFN-γ(检测结果如图2A所示)或IL-2(检测结果如图2B所示)。 Collect HepG2-luc cells, use culture medium to adjust the cell density to 1×10 5 cells/mL, inoculate target cells in a 96-well plate at 100 μL/well, and resuspend CAR-T cells, GPC3-CAR and Blank T cells that have not been transfected with CAR are used as effector cells, and then added to a 96-well plate containing target cells at an E/T (effector cell/target cell) ratio of 1:1, 100 μL/well, and the final volume is filled to 200 μL/ Wells were incubated overnight in a 37 °C incubator with 5% CO 2 . After the culture, remove the well plate from the incubator, centrifuge, take the supernatant, and use an ELISA kit (IL2, IFN-γ) to detect cytokine release. The six types of CAR-T cells constructed in this application (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) can kill tumor cells to varying degrees and release IFN-γ ( The detection results are shown in Figure 2A) or IL-2 (the detection results are shown in Figure 2B).
实验例7：体内模型评估CAR-T细胞对靶细胞的杀伤能力Experimental Example 7: In vivo model to evaluate the killing ability of CAR-T cells against target cells
36只B-NDG小鼠右侧或右侧肩胛处皮下接种5×106个HepG2肿瘤细胞，待肿瘤平均体积达到100～150mm3时，随机分为6组，每只小鼠腹腔给予环磷酰胺100mg/kg，次日尾静脉回输5×106个CAR-T以及未转染CAR的空白T细胞。每周两次用游标卡尺测量肿瘤直径和称量小鼠体重。CAR-T细胞对靶细胞的杀伤能力结果如图3A，小鼠体重变化情况如图3B。与阴性对照组相比，本申请构建的CAR-T细胞(CA3-T，CC8-T，CH3-T)对肿瘤细胞有良好的抑制作用。所有治疗组在观察期内均无动物死亡及显著动物体重降低，未见明显的药物毒性反应，治疗期间小鼠耐受性良好。36 B-NDG mice were subcutaneously inoculated with 5 × 10 6 HepG2 tumor cells on the right side or right scapula. When the average tumor volume reached 100-150 mm 3 , they were randomly divided into 6 groups. Each mouse was given cyclophosphate intraperitoneally. amide 100 mg/kg, and 5 × 10 6 CAR-T cells and blank T cells not transfected with CAR were reinfused into the tail vein the next day. Tumor diameters were measured with vernier calipers and mice were weighed twice a week. The results of the killing ability of CAR-T cells on target cells are shown in Figure 3A, and the changes in mouse body weight are shown in Figure 3B. Compared with the negative control group, the CAR-T cells (CA3-T, CC8-T, CH3-T) constructed in this application have good inhibitory effects on tumor cells. There were no animal deaths or significant weight loss in all treatment groups during the observation period, and no obvious drug toxic reactions were observed. The mice were well tolerated during the treatment period.
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。 Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all teachings that have been published, and these changes are within the protection scope of the present invention. . The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims (31)

  1. 嵌合抗原受体，其包含抗原结合结构域，所述抗原结合结构域包含如下的互补决定区(CDRs)：Chimeric antigen receptor, which includes an antigen-binding domain that includes the following complementarity-determining regions (CDRs):
    (a)SEQ ID NO:1所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:2所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(a) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and/or, the light chain variable region shown in SEQ ID NO: 2 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
    或，or,
    (b)SEQ ID NO:3所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:4所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(b) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:3; and/or, the light chain variable region shown in SEQ ID NO:4 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
    或，or,
    (c)SEQ ID NO:5所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:6所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(c) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:5; and/or, the variable light chain shown in SEQ ID NO:6 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
    或，or,
    (d)SEQ ID NO:7所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(d) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:7; and/or, the variable light chain shown in SEQ ID NO:8 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
    或，or,
    (e)SEQ ID NO:9所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:10所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(e) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:9; and/or, the variable light chain shown in SEQ ID NO:10 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
    或，or,
    (f)SEQ ID NO:11所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:12所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(f) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 11; and/or, the light chain variable region shown in SEQ ID NO: 12 CDR-L1, CDR-L2 and CDR-L3 contained in the region (VL);
    或or
    (g)下述重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3，和/或下述轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3，其中，所述重链可变区 (VH)和/或轻链可变区(VL)与(a)至(f)任一所述的重链可变区和/或轻链可变区相比，至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换为保守置换；(g) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) described below, and/or CDR-L1, CDR-L1, CDR-H3 contained in the light chain variable region (VL) described below L2 and CDR-L3, wherein the heavy chain variable region (VH) and/or light chain variable region (VL) compared with the heavy chain variable region and/or light chain variable region described in any one of (a) to (f), at least one CDR contains a mutation, so The mutation is the substitution, deletion or addition of one or several amino acids (for example, the substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution;
    优选地，所述CDR根据Kabat、IMGT、Chothia或AbM编号系统定义。Preferably, the CDRs are defined according to the Kabat, IMGT, Chothia or AbM numbering system.
  2. 权利要求1所述的嵌合抗原受体，其中，所述抗原结合结构域包含：The chimeric antigen receptor of claim 1, wherein the antigen-binding domain comprises:
    (1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：(1) The following heavy chain variable region (VH) and/or light chain variable region (VL), where CDRs are defined according to the Kabat numbering system:
    (1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：13或其变体的CDR-H1；序列为SEQ ID NO：14或其变体的CDR-H2；序列为SEQ ID NO：15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：16或其变体的CDR-L1；序列为SEQ ID NO：17或其变体的CDR-L2；序列为SEQ ID NO：18或其变体的CDR-L3；(1a) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 13 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 14 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 16 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 17 or its variant; CDR-L3 whose sequence is SEQ ID NO: 18 or its variant;
    或，or,
    (1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：26或其变体的CDR-H1；序列为SEQ ID NO：27或其变体的CDR-H2；序列为SEQ ID NO：28或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：29或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：31或其变体的CDR-L3；(1b) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 29 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 31 or its variant;
    或，or,
    (1c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：39或其变体的CDR-H1；序列为SEQ ID NO：40或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：44或其变体的CDR-L3；(1c) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 39 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 40 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 42 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 43 or its variant; CDR-L3 whose sequence is SEQ ID NO: 44 or its variant;
    或，or,
    (1d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：26或其变体的CDR-H1；序列为SEQ ID NO：27或其变体的CDR-H2；序列为SEQ ID NO：52或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：53或其变体的CDR-L1；序列为SEQ ID NO：54或其变体的CDR-L2；序列为SEQ ID NO： 55或其变体的CDR-L3；(1d) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 52 or a variant thereof; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 53 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 54 or a variant thereof; whose sequence is SEQ ID NO: 55 or its variant CDR-L3;
    或，or,
    (1e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：60或其变体的CDR-H1；序列为SEQ ID NO：61或其变体的CDR-H2；序列为SEQ ID NO：62或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：63或其变体的CDR-L1；序列为SEQ ID NO：64或其变体的CDR-L2；序列为SEQ ID NO：65或其变体的CDR-L3；(1e) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 60 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 61 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 63 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 64 or its variants; CDR-L3 whose sequence is SEQ ID NO: 65 or its variants;
    或，or,
    (1f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：26或其变体的CDR-H1；序列为SEQ ID NO：27或其变体的CDR-H2；序列为SEQ ID NO：73或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：74或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：75或其变体的CDR-L3；(1f) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 74 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 75 or its variant;
    或，or,
    (2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(2) The following heavy chain variable region (VH) and/or light chain variable region (VL), where CDRs are defined according to the IMGT numbering system:
    (2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：19或其变体的CDR-H1；序列为SEQ ID NO：20或其变体的CDR-H2；序列为SEQ ID NO：21或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：22或其变体的CDR-L1；序列为SEQ ID NO：23或其变体的CDR-L2；序列为SEQ ID NO：18或其变体的CDR-L3；(2a) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 19 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 20 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 21 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 22 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 23 or its variant; CDR-L3 whose sequence is SEQ ID NO: 18 or its variant;
    或，or,
    (2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：32或其变体的CDR-H1；序列为SEQ ID NO：33或其变体的CDR-H2；序列为SEQ ID NO：34或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：35或其变体的CDR-L1；序列为SEQ ID NO：36或其变体的CDR-L2；序列为SEQ ID NO：31或其变体的CDR-L3；(2b) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 32 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 34 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 35 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 36 or its variant; CDR-L3 whose sequence is SEQ ID NO: 31 or its variant;
    或，or,
    (2c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：45或其变体的CDR-H1；序列为SEQ ID NO：46或其变体的CDR-H2；序列为SEQ ID NO：47或其变 体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：48或其变体的CDR-L1；序列为SEQ ID NO：49或其变体的CDR-L2；序列为SEQ ID NO：44或其变体的CDR-L3；(2c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 whose sequence is SEQ ID NO: 45 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 46 or a variant thereof; The sequence is SEQ ID NO: 47 or a variant thereof CDR-H3 of the body; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 48 or a variant thereof; and/or a CDR-L1 whose sequence is SEQ ID NO: 49 or its variant. CDR-L2 of the variant; CDR-L3 whose sequence is SEQ ID NO: 44 or a variant thereof;
    或，or,
    (2d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：32或其变体的CDR-H1；序列为SEQ ID NO：33或其变体的CDR-H2；序列为SEQ ID NO：56或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：57或其变体的CDR-L1；序列为SEQ ID NO：58或其变体的CDR-L2；序列为SEQ ID NO：59或其变体的CDR-L3；(2d) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 32 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 56 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 57 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 58 or its variant; CDR-L3 whose sequence is SEQ ID NO: 59 or its variant;
    或，or,
    (2e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：66或其变体的CDR-H1；序列为SEQ ID NO：67或其变体的CDR-H2；序列为SEQ ID NO：68或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：69或其变体的CDR-L1；序列为SEQ ID NO：70或其变体的CDR-L2；序列为SEQ ID NO：65或其变体的CDR-L3；(2e) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 66 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 67 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 68 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 69 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 70 or its variant; CDR-L3 whose sequence is SEQ ID NO: 65 or its variant;
    或，or,
    (2f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：32或其变体的CDR-H1；序列为SEQ ID NO：33或其变体的CDR-H2；序列为SEQ ID NO：76或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：77或其变体的CDR-L1；序列为SEQ ID NO：36或其变体的CDR-L2；序列为SEQ ID NO：75或其变体的CDR-L3；(2f) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 32 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 33 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 76 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 77 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 36 or its variant; CDR-L3 whose sequence is SEQ ID NO: 75 or its variant;
    或，or,
    (3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(3) The following heavy chain variable region (VH) and/or light chain variable region (VL), where CDRs are defined according to the Chothia numbering system:
    (3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：24或其变体的CDR-H1；序列为SEQ ID NO：25或其变体的CDR-H2；序列为SEQ ID NO：15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：16或其变体的CDR-L1；序列为SEQ ID NO：17或其变体的CDR-L2；序列为SEQ ID NO：18或其变体的CDR-L3；(3a) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 24 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 25 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 16 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 17 or its variant; CDR-L3 whose sequence is SEQ ID NO: 18 or its variant;
    或， or,
    (3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：37或其变体的CDR-H1；序列为SEQ ID NO：38或其变体的CDR-H2；序列为SEQ ID NO：28或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：29或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：31或其变体的CDR-L3；(3b) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 37 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 29 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 31 or its variant;
    或，or,
    (3c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：50或其变体的CDR-H1；序列为SEQ ID NO：51或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：44或其变体的CDR-L3；(3c) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 50 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 51 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 42 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 43 or its variant; CDR-L3 whose sequence is SEQ ID NO: 44 or its variant;
    或，or,
    (3d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：37或其变体的CDR-H1；序列为SEQ ID NO：38或其变体的CDR-H2；序列为SEQ ID NO：52或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：53或其变体的CDR-L1；序列为SEQ ID NO：54或其变体的CDR-L2；序列为SEQ ID NO：55或其变体的CDR-L3；(3d) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 37 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 52 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 53 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 54 or its variants; CDR-L3 whose sequence is SEQ ID NO: 55 or its variants;
    或，or,
    (3e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：71或其变体的CDR-H1；序列为SEQ ID NO：72或其变体的CDR-H2；序列为SEQ ID NO：62或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：63或其变体的CDR-L1；序列为SEQ ID NO：64或其变体的CDR-L2；序列为SEQ ID NO：65或其变体的CDR-L3；(3e) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 71 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 72 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 63 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 64 or its variants; CDR-L3 whose sequence is SEQ ID NO: 65 or its variants;
    或，or,
    (3f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：37或其变体的CDR-H1；序列为SEQ ID NO：38或其变体的CDR-H2；序列为SEQ ID NO：73或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：74或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：75或其变体的CDR-L3；(3f) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 37 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 38 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 74 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 75 or its variant;
    或， or,
    (4)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：(4) The following heavy chain variable region (VH) and/or light chain variable region (VL), where CDRs are defined according to the AbM numbering system:
    (4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：80或其变体的CDR-H1；序列为SEQ ID NO：81或其变体的CDR-H2；序列为SEQ ID NO：15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：16或其变体的CDR-L1；序列为SEQ ID NO：17或其变体的CDR-L2；序列为SEQ ID NO：18或其变体的CDR-L3；(4a) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 80 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 81 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 15 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 16 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 17 or its variant; CDR-L3 whose sequence is SEQ ID NO: 18 or its variant;
    或，or,
    (4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：78或其变体的CDR-H1；序列为SEQ ID NO：79或其变体的CDR-H2；序列为SEQ ID NO：28或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：29或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：31或其变体的CDR-L3；(4b) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 78 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 28 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 29 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 31 or its variant;
    或，or,
    (4c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：96或其变体的CDR-H1；序列为SEQ ID NO：97或其变体的CDR-H2；序列为SEQ ID NO：41或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：42或其变体的CDR-L1；序列为SEQ ID NO：43或其变体的CDR-L2；序列为SEQ ID NO：44或其变体的CDR-L3；(4c) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 96 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 97 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 41 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 42 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 43 or its variant; CDR-L3 whose sequence is SEQ ID NO: 44 or its variant;
    或，or,
    (4d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：78或其变体的CDR-H1；序列为SEQ ID NO：79或其变体的CDR-H2；序列为SEQ ID NO：52或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：53或其变体的CDR-L1；序列为SEQ ID NO：54或其变体的CDR-L2；序列为SEQ ID NO：55或其变体的CDR-L3；(4d) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 78 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 52 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 53 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 54 or its variants; CDR-L3 whose sequence is SEQ ID NO: 55 or its variants;
    或，or,
    (4e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：98或其变体的CDR-H1；序列为SEQ ID NO：99或其变体的CDR-H2；序列为SEQ ID NO：62或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：63或其变体的CDR-L1；序列为SEQ ID NO：64或其变体的CDR-L2；序列为SEQ ID NO： 65或其变体的CDR-L3；(4e) A heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 98 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 99 or a variant thereof; CDR-H3 whose sequence is SEQ ID NO: 62 or a variant thereof; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 whose sequence is SEQ ID NO: 63 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 64 or a variant thereof; whose sequence is SEQ ID NO: CDR-L3 of 65 or its variants;
    或，or,
    (4f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：78或其变体的CDR-H1；序列为SEQ ID NO：79或其变体的CDR-H2；序列为SEQ ID NO：73或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：74或其变体的CDR-L1；序列为SEQ ID NO：30或其变体的CDR-L2；序列为SEQ ID NO：75或其变体的CDR-L3；(4f) A heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 whose sequence is SEQ ID NO: 78 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 79 or a variant thereof; CDR-H3 with the sequence SEQ ID NO: 73 or a variant thereof; and/or, a light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with the sequence SEQ ID NO: 74 or a variant thereof ; CDR-L2 whose sequence is SEQ ID NO: 30 or its variant; CDR-L3 whose sequence is SEQ ID NO: 75 or its variant;
    其中，(1a)-(1f)、(2a)-(2f)、(3a)-(3f)、(4a)-(4f)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Among them, the variant described in any one of (1a)-(1f), (2a)-(2f), (3a)-(3f), (4a)-(4f) is compared with the sequence from which it is derived. There is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution.
  3. 权利要求1或2所述的嵌合抗原受体，其中，所述抗原结合结构域包含：The chimeric antigen receptor of claim 1 or 2, wherein the antigen-binding domain comprises:
    (a)包含如SEQ ID NO:1所示的序列或其变体的VH和/或包含如SEQ ID NO:2所示的序列或其变体的VL；(a) VH comprising the sequence shown in SEQ ID NO:1 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:2 or a variant thereof;
    或，or,
    (b)包含如SEQ ID NO:3所示的序列或其变体的VH和/或包含如SEQ ID NO:4所示的序列或其变体的VL；(b) VH comprising the sequence shown in SEQ ID NO:3 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:4 or a variant thereof;
    或，or,
    (c)包含如SEQ ID NO:5所示的序列或其变体的VH和/或包含如SEQ ID NO:6所示的序列或其变体的VL；(c) VH comprising the sequence shown in SEQ ID NO:5 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:6 or a variant thereof;
    或or
    (d)包含如SEQ ID NO:7所示的序列或其变体的VH和/或包含如SEQ ID NO:8所示的序列或其变体的VL；(d) VH comprising the sequence shown in SEQ ID NO:7 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof;
    或，or,
    (e)包含如SEQ ID NO:9所示的序列或其变体的VH和/或包含如SEQ ID NO:10所示的序列或其变体的VL；(e) VH comprising the sequence shown in SEQ ID NO:9 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:10 or a variant thereof;
    或，or,
    (f)包含如SEQ ID NO:11所示的序列或其变体的VH和/或包含如SEQ ID NO:12所示的序列或其变体的VL；(f) VH comprising the sequence shown in SEQ ID NO:11 or a variant thereof and/or VL comprising the sequence shown in SEQ ID NO:12 or a variant thereof;
    其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少 91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。wherein said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived Compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); preferably, the substitution is a conservative substitution.
  4. 权利要求1-3任一项所述的嵌合抗原受体，其中，所述抗原结合结构域选自全长抗体、Fab片段、Fab'片段、Fab’-SH、F(ab)'2片段、F(ab)'3片段、Fv片段、单链抗体(例如scFv、di-scFv或(scFv)2)、微型抗体、二硫键稳定的Fv蛋白(dsFv)、单结构域抗体(sdAb，纳米抗体)、双抗体(diabody)、双特异性抗体和多特异性抗体。The chimeric antigen receptor according to any one of claims 1 to 3, wherein the antigen-binding domain is selected from the group consisting of full-length antibodies, Fab fragments, Fab' fragments, Fab'-SH, and F(ab)' 2 fragments , F(ab)' 3 fragment, Fv fragment, single chain antibody (such as scFv, di-scFv or (scFv) 2 ), minibody, disulfide bond stabilized Fv protein (dsFv), single domain antibody (sdAb, Nanobodies), diabodies, bispecific antibodies and multispecific antibodies.
  5. 权利要求1-4任一项所述的嵌合抗原受体，其中，所述抗原结合结构域是单链抗体，例如scFv、di-scFv或(scFv)2；The chimeric antigen receptor of any one of claims 1-4, wherein the antigen-binding domain is a single-chain antibody, such as scFv, di-scFv or (scFv) 2 ;
    优选地，所述单链抗体从其N端至C端依次包括：Preferably, the single-chain antibody includes in sequence from its N-terminus to its C-terminus:
    (1)包含如SEQ ID NO:1所示的序列或其变体的VH-连接子-包含如SEQ ID NO:2所示的序列或其变体的VL；或，包含如SEQ ID NO:2所示的序列或其变体的VL-连接子-包含如SEQ ID NO:1所示的序列或其变体的VH；(1) A VH-linker comprising the sequence shown in SEQ ID NO: 1 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO: 2 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO: 2 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO: 1 or a variant thereof; VL-linker of the sequence shown in 2 or its variant - VH containing the sequence shown in SEQ ID NO:1 or its variant;
    或or
    (2)包含如SEQ ID NO:3所示的序列或其变体的VH-连接子-包含如SEQ ID NO:4所示的序列或其变体的VL；或，包含如SEQ ID NO:4所示的序列或其变体的VL-连接子-包含如SEQ ID NO:3所示的序列或其变体的VH；(2) A VH-linker comprising the sequence shown in SEQ ID NO:3 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:4 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:3 or a variant thereof; VL-linker of the sequence shown in 4 or a variant thereof - VH containing the sequence shown in SEQ ID NO:3 or a variant thereof;
    或or
    (3)包含如SEQ ID NO:5所示的序列或其变体的VH-连接子-包含如SEQ ID NO:6所示的序列或其变体的VL；或，包含如SEQ ID NO:6所示的序列或其变体的VL-连接子-包含如SEQ ID NO:5所示的序列或其变体的VH；(3) A VH-linker comprising the sequence shown in SEQ ID NO:5 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:6 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:6 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:5 or a variant thereof; The VL-linker of the sequence shown in 6 or its variant-comprising the VH of the sequence shown in SEQ ID NO:5 or its variant;
    或or
    (4)包含如SEQ ID NO:7所示的序列或其变体的VH-连接子-包含如SEQ ID NO:8所示的序列或其变体的VL；或，包含如SEQ ID NO:8所示的序列或其变体的VL-连接子-包含如SEQ ID NO:7所示的序列或其变体的VH；(4) A VH-linker comprising the sequence shown in SEQ ID NO:7 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:8 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:8 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:7 or a variant thereof; VL-linker of the sequence shown in 8 or a variant thereof - VH containing the sequence shown in SEQ ID NO:7 or a variant thereof;
    或or
    (5)包含如SEQ ID NO:9所示的序列或其变体的VH-连接子-包含如SEQ ID NO:10 所示的序列或其变体的VL；或，包含如SEQ ID NO:10所示的序列或其变体的VL-连接子-包含如SEQ ID NO:9所示的序列或其变体的VH；(5) VH-linker comprising the sequence shown in SEQ ID NO:9 or a variant thereof - comprising SEQ ID NO:10 VL of the sequence shown or a variant thereof; or, a VL-linker comprising the sequence shown in SEQ ID NO: 10 or a variant thereof - a VL-linker comprising the sequence shown in SEQ ID NO: 9 or a variant thereof VH;
    或or
    (6)包含如SEQ ID NO:11所示的序列或其变体的VH-连接子-包含如SEQ ID NO:12所示的序列或其变体的VL；或，包含如SEQ ID NO:12所示的序列或其变体的VL-连接子-包含如SEQ ID NO:11所示的序列或其变体的VH；(6) A VH-linker comprising the sequence shown in SEQ ID NO:11 or a variant thereof - a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:12 or a variant thereof; or, a VL comprising a sequence shown in SEQ ID NO:11 or a variant thereof; The VL-linker of the sequence shown in 12 or its variant-comprising the VH of the sequence shown in SEQ ID NO:11 or its variant;
    其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；wherein said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% sequence identity, or having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, Substitution, deletion or addition of 3, 4 or 5 amino acids); preferably, the substitution is a conservative substitution;
    优选地，所述连接子为多肽；优选地，所述连接子包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为1或2；更优选地，所述连接子具有SEQ ID NO：110的序列。Preferably, the linker is a polypeptide; preferably, the linker includes one or several (eg 1, 2 or 3) sequences as shown in (GmS)n, where m is selected from 1-6 The integer, n is selected from the integer of 1-6; Preferably, m is 3, 4, or 5; Preferably, n is 1 or 2; More preferably, the linker has the sequence of SEQ ID NO: 110.
  6. 权利要求1-5任一项所述的嵌合抗原受体，其中，所述抗原结合结构域是单链抗体，所述单链抗体包含SEQ ID NO:86、88、90、92、94、82任一项所示的序列或其变体，所述变体与SEQ ID NO:86、88、90、92、94或82相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。The chimeric antigen receptor of any one of claims 1-5, wherein the antigen-binding domain is a single-chain antibody, and the single-chain antibody includes SEQ ID NOs: 86, 88, 90, 92, 94, The sequence shown in any one of 82 or a variant thereof, the variant has at least 70%, at least 75%, at least 80%, at least 85% compared to SEQ ID NO: 86, 88, 90, 92, 94 or 82 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, Or have one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or addition); preferably, the substitution is a conservative substitution.
  7. 权利要求1-6任一项所述的嵌合抗原受体，其进一步包含跨膜结构域，所述跨膜结构域选自下列蛋白的跨膜区：T细胞受体的α、β或ζ链、CD28、CD45、CD3ε、CD3ζ、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD152、CD154和PD-1；The chimeric antigen receptor according to any one of claims 1 to 6, further comprising a transmembrane domain selected from the transmembrane region of the following proteins: α, β or ζ of the T cell receptor chain, CD28, CD45, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1;
    优选地，所述跨膜结构域选自下列蛋白的跨膜区：CD8、CD28、CD4、PD-1、CD152和CD154； Preferably, the transmembrane domain is selected from the transmembrane regions of the following proteins: CD8, CD28, CD4, PD-1, CD152 and CD154;
    优选地，所述跨膜结构域包含SEQ ID NO:111所示的CD8跨膜区。Preferably, the transmembrane domain includes the CD8 transmembrane region shown in SEQ ID NO: 111.
  8. 权利要求1-7任一项所述的嵌合抗原受体，其进一步包含间隔结构域，所述间隔结构域位于抗原结合结构域与跨膜结构域之间，所述间隔结构域选自铰链结构域和/或免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区；The chimeric antigen receptor according to any one of claims 1 to 7, further comprising a spacer domain located between the antigen-binding domain and the transmembrane domain, the spacer domain being selected from the group consisting of hinges Domains and/or CH2 and CH3 regions of immunoglobulins (e.g., IgG1 or IgG4);
    优选地，所述铰链结构域包含CD8、IgG4、PD-1、CD152或CD154的铰链区；更优选地，所述铰链结构域包含SEQ ID NO:112所示的CD8铰链区。Preferably, the hinge domain comprises the hinge region of CD8, IgG4, PD-1, CD152 or CD154; more preferably, the hinge domain comprises the CD8 hinge region shown in SEQ ID NO:112.
  9. 权利要求1-8任一项所述的嵌合抗原受体，其进一步包含胞内信号传导结构域，所述胞内信号传导结构域包含初级信号传导结构域和/或共刺激信号传导结构域；The chimeric antigen receptor of any one of claims 1 to 8, further comprising an intracellular signaling domain comprising a primary signaling domain and/or a costimulatory signaling domain ;
    优选地，所述胞内信号传导结构域从N端到C端依次包含共刺激信号传导结构域和初级信号传导结构域；Preferably, the intracellular signaling domain includes a costimulatory signaling domain and a primary signaling domain in sequence from the N-terminus to the C-terminus;
    优选地，所述胞内信号传导结构域包含初级信号传导结构域以及至少一个共刺激信号传导结构域；Preferably, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain;
    优选地，所述初级信号传导结构域包含免疫受体酪氨酸活化基序(ITAM)；Preferably, the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM);
    优选地，所述初级信号传导结构域包含选自以下蛋白的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CDS、CD22、CD79a、CD79b或CD66d；更优选地，所述初级信号传导结构域包含SEQ ID NO:113所示的CD3ζ胞内信号传导结构域；Preferably, the primary signaling domain comprises an intracellular signaling domain selected from the following proteins: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b or CD66d; more preferably, the The primary signaling domain includes the CD3ζ intracellular signaling domain shown in SEQ ID NO: 113;
    优选地，所述共刺激信号传导结构域包含选自下列蛋白的胞内信号传导结构域：CARD11、CD2、CD7、CD27、CD28、CD30、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)或DAP10；Preferably, the costimulatory signaling domain comprises an intracellular signaling domain selected from the following proteins: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 ( SLAMF1), CD270(HVEM), CD278(ICOS) or DAP10;
    优选地，所述共刺激信号传导结构域选自CD28的胞内信号传导结构域或CD137(4-1BB)的胞内信号传导结构域或二者片段的组合；更优选地，所述共刺激信号传导结构域包含SEQ ID NO:114所示的CD137(4-1BB)胞内信号传导结构域；Preferably, the costimulatory signaling domain is selected from the intracellular signaling domain of CD28 or the intracellular signaling domain of CD137 (4-1BB) or a combination of fragments of both; more preferably, the costimulation The signaling domain includes the CD137(4-1BB) intracellular signaling domain shown in SEQ ID NO:114;
    更优选地，所述胞内信号传导结构域序列包含SEQ ID NO:115所示的序列。More preferably, the intracellular signaling domain sequence includes the sequence shown in SEQ ID NO: 115.
  10. 权利要求1-9任一项所述的嵌合抗原受体，其中，所述嵌合抗原受体进一步在其N端包含信号肽；The chimeric antigen receptor according to any one of claims 1 to 9, wherein the chimeric antigen receptor further comprises a signal peptide at its N-terminus;
    优选地，所述信号肽包含重链信号肽(例如IgG1的重链信号肽)、粒细胞-巨噬细胞集落刺激因子受体2(GM-CSFR2)信号肽、IL2信号肽、或CD8α信号肽；更优选地，所 述信号肽包含SEQ ID NO:116所示的序列。Preferably, the signal peptide comprises a heavy chain signal peptide (eg, heavy chain signal peptide of IgG1), granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, IL2 signal peptide, or CD8α signal peptide ; More preferably, the The signal peptide includes the sequence shown in SEQ ID NO:116.
  11. 权利要求1-10任一项所述的嵌合抗原受体，其中，所述嵌合抗原受体从其N端至C端依次包含信号肽、抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域；The chimeric antigen receptor according to any one of claims 1 to 10, wherein the chimeric antigen receptor includes a signal peptide, an antigen-binding domain, a spacer domain, and a transmembrane structure in sequence from its N-terminus to its C-terminus. domain, intracellular signaling domain;
    优选地，所述信号肽包含IgG1的重链信号肽或CD8α信号肽(例如，序列如SEQ ID NO:116所示的信号肽)；Preferably, the signal peptide includes the heavy chain signal peptide of IgG1 or the CD8α signal peptide (for example, the signal peptide with the sequence shown in SEQ ID NO: 116);
    优选地，所述抗原结合结构域如权利要求1-6任一项中所定义(例如，包含SEQ ID NO:86、88、90、92、94、82任一项所示的序列)；Preferably, the antigen-binding domain is as defined in any one of claims 1-6 (for example, comprising the sequence shown in any one of SEQ ID NO: 86, 88, 90, 92, 94, 82);
    优选地，所述间隔结构域包含CD8(例如CD8α)的铰链区(例如，序列如SEQ ID NO:112所示的铰链区)；Preferably, the spacer domain comprises a hinge region of CD8 (e.g., CD8α) (e.g., a hinge region with a sequence as shown in SEQ ID NO: 112);
    优选地，所述跨膜结构域包含CD8(例如CD8α)的跨膜区(例如，序列如SEQ ID NO:111所示的跨膜区)；Preferably, the transmembrane domain includes the transmembrane region of CD8 (e.g., CD8α) (e.g., the transmembrane region whose sequence is shown in SEQ ID NO: 111);
    优选地，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域(例如，如SEQ ID NO:113所示序列)，所述共刺激信号传导结构域包含CD137(4-1BB)的胞内信号传导结构域(例如，如SEQ ID NO:114所示序列)；更优选地，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO:115所示序列；Preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., as in SEQ ID NO: Sequence shown in SEQ ID NO: 113), the costimulatory signaling domain includes the intracellular signaling domain of CD137 (4-1BB) (for example, the sequence shown in SEQ ID NO: 114); more preferably, the chimeric The intracellular signaling domain of the antigen receptor has the sequence shown in SEQ ID NO: 115;
    优选地，所述嵌合抗原受体包含SEQ ID NO:117、119、121、123、125、127任一项所示的序列或其变体，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换为保守置换。Preferably, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NO: 117, 119, 121, 123, 125, 127 or a variant thereof, which variant is compared with the sequence from which it is derived. With at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acids) compared to the sequence from which it is derived substitution, deletion or addition); preferably, the substitution is a conservative substitution.
  12. 权利要求1-11任一项所述的嵌合抗原受体，其中，所述抗原结合结构域包含权利要求1-6任一项中所定义的抗原结合结构域作为第一抗原结合结构域，并且进一步包含不结合GPC3的第二抗原结合结构域；更优选地，所述第二抗原结合结构域结合的抗原选自：PD-1、PD-L1、CTLA-4、CD3、ASGPR1、CD19、MSLN、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2； The chimeric antigen receptor of any one of claims 1-11, wherein the antigen-binding domain includes the antigen-binding domain defined in any one of claims 1-6 as the first antigen-binding domain, And further includes a second antigen-binding domain that does not bind GPC3; more preferably, the antigen bound by the second antigen-binding domain is selected from: PD-1, PD-L1, CTLA-4, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2;
    优选地，所述抗原结合结构域是单链抗体，例如scFv、di-scFv或(scFv)2；Preferably, the antigen-binding domain is a single chain antibody, such as scFv, di-scFv or (scFv) 2 ;
    优选地，所述抗原结合结构域中的VH和VL通过连接子连接；优选地，所述连接子包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为1或2；更优选地，所述连接子具有SEQ ID NO：110的序列；Preferably, VH and VL in the antigen-binding domain are connected through a linker; preferably, the linker includes one or several (for example, 1, 2 or 3) as represented by (G m S) n The sequence shown, wherein m is selected from an integer of 1-6, n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, the The linker has the sequence of SEQ ID NO: 110;
    优选地，所述第一抗原结合结构域和第二抗原结合结构域通过自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)连接；Preferably, the first antigen-binding domain and the second antigen-binding domain are connected by a self-cleaving peptide (such as P2A, E2A, F2A, T2A or any combination thereof);
    优选地，所述自裂解肽是P2A；例如，所述自裂解肽的氨基酸序列如SEQ ID NO:130所示；Preferably, the self-cleaving peptide is P2A; for example, the amino acid sequence of the self-cleaving peptide is as shown in SEQ ID NO: 130;
    优选地，所述第二抗原结合结构域在其N端进一步包含信号肽；优选地，所述信号肽不同于所述GPC3特异性嵌合抗原受体所包含的信号肽；优选地，所述第二抗原结合结构域N端的信号肽是IL2信号肽(例如，氨基酸序列如SEQ ID NO:129所示)。Preferably, the second antigen-binding domain further comprises a signal peptide at its N-terminus; preferably, the signal peptide is different from the signal peptide comprised by the GPC3-specific chimeric antigen receptor; preferably, the signal peptide The signal peptide at the N-terminus of the second antigen-binding domain is the IL2 signal peptide (for example, the amino acid sequence is shown in SEQ ID NO: 129).
  13. 分离的核酸分子，其包含编码权利要求1-12任一项所述的嵌合抗原受体的核苷酸序列。An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-12.
  14. 权利要求13所述的分离的核酸分子，其包含选自下列的核苷酸序列：(1)SEQ ID NO:118、120、122、124、126、128任一项所示的序列或其简并变体；(2)与(1)中任一项所示序列相比基本上相同的序列(例如，与(1)中任一项所示的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)中任一项所示的序列相比具有一个或更多个核苷酸取代的序列)。The isolated nucleic acid molecule of claim 13, which comprises a nucleotide sequence selected from the following: (1) the sequence shown in any one of SEQ ID NO: 118, 120, 122, 124, 126, 128 or its abbreviation and variants; (2) A sequence that is substantially identical to the sequence shown in any one of (1) (for example, at least 50%, at least 55% compared to the sequence shown in any one of (1) , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least A sequence that has 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or has one or more nucleotide substitutions compared to the sequence shown in any of (1) the sequence of).
  15. 核酸构建体，其包含编码GPC3特异性嵌合抗原受体的核酸序列；其中，所述GPC3特异性嵌合抗原受体包含靶向GPC3的抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域，所述靶向GPC3的抗原结合结构域包含权利要求1-6任一项中定义的抗原结合结构域。A nucleic acid construct comprising a nucleic acid sequence encoding a GPC3-specific chimeric antigen receptor; wherein the GPC3-specific chimeric antigen receptor includes an antigen-binding domain, a spacer domain, a transmembrane domain and a GPC3-targeting Intracellular signaling domain, the GPC3-targeting antigen-binding domain comprises the antigen-binding domain defined in any one of claims 1-6.
  16. 权利要求15所述的核酸构建体，其具备以下特征中的一项或多项： The nucleic acid construct of claim 15, which possesses one or more of the following characteristics:
    (1)所述跨膜结构域如权利要求7中定义；(1) The transmembrane domain is as defined in claim 7;
    (2)所述间隔结构域如权利要求8中定义；(2) The spacer domain is as defined in claim 8;
    (3)所述胞内信号传导结构域如权利要求9中定义；(3) The intracellular signaling domain is as defined in claim 9;
    (4)所述核酸序列所编码的GPC3特异性嵌合抗原受体进一步在其N端包含信号肽，所述信号肽如权利要求10中定义。(4) The GPC3-specific chimeric antigen receptor encoded by the nucleic acid sequence further contains a signal peptide at its N-terminus, and the signal peptide is as defined in claim 10.
  17. 权利要求15-16任一项所述的核酸构建体，其中，所述核酸构建体从其5’端至3’端依次包含：编码所述信号肽的核苷酸序列、编码所述靶向GPC3的抗原结合结构域的核苷酸序列、编码所述间隔结构域的核苷酸序列、编码所述跨膜结构域的核苷酸序列、编码所述胞内信号传导结构域的核苷酸序列；The nucleic acid construct of any one of claims 15-16, wherein the nucleic acid construct sequentially includes from its 5' end to its 3' end: a nucleotide sequence encoding the signal peptide, a nucleotide sequence encoding the targeting Nucleotide sequence of the antigen-binding domain of GPC3, nucleotide sequence encoding the spacer domain, nucleotide sequence encoding the transmembrane domain, and nucleotide sequence encoding the intracellular signaling domain sequence;
    优选地，所述信号肽包含IgG1的重链信号肽或CD8α信号肽(例如，序列如SEQ ID NO:116所示的信号肽)；Preferably, the signal peptide includes the heavy chain signal peptide of IgG1 or the CD8α signal peptide (for example, the signal peptide whose sequence is shown in SEQ ID NO: 116);
    优选地，所述靶向GPC3的抗原结合结构域选自权利要求1-6任一项中所定义的抗原结合结构域(例如，包含SEQ ID NO:86、88、90、92、94、82任一项所示的序列)；Preferably, the antigen-binding domain targeting GPC3 is selected from the antigen-binding domain defined in any one of claims 1-6 (e.g., comprising SEQ ID NO: 86, 88, 90, 92, 94, 82 any sequence shown);
    优选地，所述间隔结构域包含CD8(例如CD8α)的铰链区(例如，序列如SEQ ID NO:112所示的铰链区)；Preferably, the spacer domain comprises a hinge region of CD8 (e.g., CD8α) (e.g., a hinge region with a sequence as shown in SEQ ID NO: 112);
    优选地，所述跨膜结构域包含CD8(例如CD8α)的跨膜区(例如，序列如SEQ ID NO:111所示的跨膜区)；Preferably, the transmembrane domain includes the transmembrane region of CD8 (e.g., CD8α) (e.g., the transmembrane region whose sequence is shown in SEQ ID NO: 111);
    优选地，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域(例如，如SEQ ID NO:113所示序列)，所述共刺激信号传导结构域包含CD137(4-1BB)的胞内信号传导结构域(例如，如SEQ ID NO:114所示序列)；更优选地，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO:115所示序列。Preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., as in SEQ ID NO: Sequence shown in SEQ ID NO: 113), the costimulatory signaling domain includes the intracellular signaling domain of CD137 (4-1BB) (for example, the sequence shown in SEQ ID NO: 114); more preferably, the chimeric The intracellular signaling domain of the antigen receptor has the sequence shown in SEQ ID NO: 115.
  18. 权利要求15-17任一项所述的核酸构建体，其中，所述核酸构建体包含选自下列的核苷酸序列：(1)SEQ ID NO:118、120、122、124、126、128任一项所示的序列或其简并变体；(2)与(1)中任一项所示序列相比基本上相同的序列(例如，与(1)中任一项所示的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或， 与(1)中任一项所示的序列相比具有一个或更多个核苷酸取代的序列)。The nucleic acid construct of any one of claims 15-17, wherein the nucleic acid construct comprises a nucleotide sequence selected from the following: (1) SEQ ID NO: 118, 120, 122, 124, 126, 128 The sequence shown in any one of the above or a degenerate variant thereof; (2) A sequence that is substantially the same as the sequence shown in any one of (1) (for example, the sequence shown in any one of (1)) Compared to having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% , a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical, or, A sequence having one or more nucleotide substitutions compared to the sequence shown in any of (1)).
  19. 权利要求15-18任一项所述的核酸构建体，其包含：(1)编码GPC3特异性嵌合抗原受体的第一核酸序列；和The nucleic acid construct of any one of claims 15-18, comprising: (1) a first nucleic acid sequence encoding a GPC3-specific chimeric antigen receptor; and
    (2)编码另外的生物活性分子的第二核酸序列；(2) A second nucleic acid sequence encoding an additional biologically active molecule;
    优选地，所述GPC3特异性嵌合抗原受体包含靶向GPC3的抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域，所述靶向GPC3的抗原结合结构域包含权利要求1-7任一项中定义的第一抗原结合结构域；Preferably, the GPC3-specific chimeric antigen receptor includes an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain targeting GPC3, and the antigen-binding domain targeting GPC3 includes The first antigen binding domain defined in any one of claims 1-7;
    优选地，所述第二核酸序列所编码的另外的生物活性分子选自下列组分的一种或一种以上：免疫检查点抑制剂(例如，抗PD-1、PD-L1、CTLA-4或LAG3抗体或其抗原结合片段、细胞因子(例如，IL-15、IL-7、IL-12、IL-18或IL-21)、T细胞表面抗原(例如，CD3等)或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18或mIL-21)；优选地，所述第一核酸序列和第二核酸序列通过编码自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)的核苷酸序列连接；Preferably, the additional biologically active molecule encoded by the second nucleic acid sequence is selected from one or more of the following components: immune checkpoint inhibitors (e.g., anti-PD-1, PD-L1, CTLA-4 or LAG3 antibody or antigen-binding fragment thereof, cytokine (e.g., IL-15, IL-7, IL-12, IL-18 or IL-21), T cell surface antigen (e.g., CD3, etc.) or membrane chimeric type polypeptide (e.g., mIL-15, mIL-7, mIL-12, mIL-18 or mIL-21); preferably, the first nucleic acid sequence and the second nucleic acid sequence encode a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof) nucleotide sequence connection;
    优选地，所述自裂解肽是P2A；例如，所述自裂解肽的氨基酸序列如SEQ ID NO:130所示；Preferably, the self-cleaving peptide is P2A; for example, the amino acid sequence of the self-cleaving peptide is as shown in SEQ ID NO: 130;
    优选地，所述第二核苷酸序列所编码的另外的生物活性分子在其N端进一步包含信号肽；优选地，所述信号肽不同于所述第一核酸序列所编码的GPC3特异性嵌合抗原受体所包含的信号肽；优选地，所述另外的生物活性分子N端的信号肽是IL2信号肽(例如，所述IL2信号肽的氨基酸序列如SEQ ID NO:129所示)。Preferably, the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide at its N-terminus; preferably, the signal peptide is different from the GPC3-specific chimeric gene encoded by the first nucleic acid sequence. The signal peptide included in the combined antigen receptor; preferably, the signal peptide at the N-terminus of the additional biologically active molecule is the IL2 signal peptide (for example, the amino acid sequence of the IL2 signal peptide is shown in SEQ ID NO: 129).
  20. 载体，其包含权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体；A vector comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15-19;
    优选地，所述载体选自DNA载体，RNA载体，质粒，转座子载体，CRISPR/Cas9载体，或病毒载体；Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, or a viral vector;
    优选的，所述载体是表达载体；Preferably, the vector is an expression vector;
    优选地，所述载体是游离型载体；Preferably, the carrier is a free carrier;
    优选地，所述载体是病毒载体；更优选地，所述病毒载体是慢病毒载体，腺病毒载体或逆转录病毒载体。 Preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.
  21. 宿主细胞，其包含权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体，或权利要求20所述的载体。A host cell comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15-19, or the vector of claim 20.
  22. 制备表达嵌合抗原受体细胞的方法，其包括：(1)提供宿主细胞；(2)将权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体、或包含它们的载体引入步骤(1)所述的宿主细胞，获得能够共表达所述嵌合抗原受体以及任选的另外的生物活性分子的宿主细胞；A method for preparing cells expressing chimeric antigen receptors, comprising: (1) providing a host cell; (2) converting the isolated nucleic acid molecule described in claim 13 or 14, or the nucleic acid molecule described in any one of claims 15-19 Nucleic acid constructs or vectors containing them are introduced into the host cell described in step (1) to obtain a host cell capable of co-expressing the chimeric antigen receptor and optionally additional biologically active molecules;
    优选地，所述宿主细胞选自免疫细胞，例如T淋巴细胞、NK细胞、单核细胞、树突状细胞、巨噬细胞及其任意组合；Preferably, the host cells are selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages and any combination thereof;
    优选地，所述免疫细胞选自T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及这些细胞的任意组合；Preferably, the immune cells are selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination of these cells;
    优选地，在步骤(1)中，所述宿主细胞提供自患者或者健康供体，并且经过预处理；所述预处理包括免疫细胞的分选、激活和/或增殖；优选地，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞；Preferably, in step (1), the host cells are provided from patients or healthy donors and undergo pretreatment; the pretreatment includes sorting, activation and/or proliferation of immune cells; preferably, the pretreatment Treating includes contacting the immune cells with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cells and inducing proliferation thereof, thereby generating pretreated immune cells;
    优选地，在步骤(2)中，将核酸分子或载体通过病毒感染引入宿主细胞；Preferably, in step (2), the nucleic acid molecule or vector is introduced into the host cell through viral infection;
    优选地，在步骤(2)中将核酸分子或载体通过非病毒载体转染的方式引入宿主细胞，如通过转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法、电穿孔方法、磷酸钙转染、DEAE-葡聚糖介导的转染或显微注射等方法；Preferably, in step (2), the nucleic acid molecule or vector is introduced into the host cell through non-viral vector transfection, such as through a transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, electroporation method, Methods such as calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection;
    优选地，在步骤(2)之后，所述方法还包括：扩增步骤(2)获得的宿主细胞。Preferably, after step (2), the method further includes: amplifying the host cells obtained in step (2).
  23. 经改造的免疫细胞，其包含权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体，或包含它们的载体；A modified immune cell comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15-19, or a vector containing them;
    优选地，所述经改造的免疫细胞表达权利要求1-12任一项所述的嵌合抗原受体。Preferably, the engineered immune cells express the chimeric antigen receptor according to any one of claims 1-12.
  24. 权利要求23所述的经改造的免疫细胞，其中，所述免疫细胞来源于T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合；优选地，所述免疫细胞得自患者；可选地，所述免疫细胞得自健康供体；优选地，所述免疫细胞来源于T淋巴细胞或NK细胞。 The modified immune cell of claim 23, wherein the immune cell is derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cell The cells are obtained from the patient; optionally, the immune cells are obtained from a healthy donor; preferably, the immune cells are derived from T lymphocytes or NK cells.
  25. 权利要求23或24所述的经改造的免疫细胞，其中，所述免疫细胞还表达不特异性针对GPC3的CAR；优选地，所述不特异性针对GPC3的CAR具有针对选自下列靶点的特异性：PD-1、PD-L1、CTLA-4、CD3、ASGPR1、CD19、MSLN、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2。The modified immune cell of claim 23 or 24, wherein the immune cell also expresses a CAR that is not specific for GPC3; preferably, the CAR that is not specific for GPC3 has a target selected from the following Specificity: PD-1, PD-L1, CTLA-4, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
  26. 权利要求23-25任一项所述的经改造的免疫细胞，其中，所述经改造的免疫细胞的免疫排斥有关的基因(例如，TRAC、TRBC、B2M、HLA-A、HLA-B或HLA-C)和免疫共抑制通路或信号分子的基因(例如，PD-1、CTLA-4或LAG-3)中的一种或两种靶基因的转录或表达被抑制；优选地，所述靶基因的转录或表达被抑制采用的方法选自基因敲除(例如，CRISPR、CRISPR/Cas9)、同源重组、干扰RNA。The modified immune cell of any one of claims 23-25, wherein the modified immune cell has a gene related to immune rejection (for example, TRAC, TRBC, B2M, HLA-A, HLA-B or HLA -C) and the transcription or expression of one or both target genes of immune co-suppression pathways or signaling molecules (for example, PD-1, CTLA-4 or LAG-3) are inhibited; preferably, the target genes The transcription or expression of the gene is inhibited by a method selected from gene knockout (eg, CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
  27. 制备经改造的免疫细胞的方法，其包括：(1)提供来自患者或者健康供体的免疫细胞；(2)将权利要求13或14所述的分离的核酸分子或包含其的载体引入步骤(1)所述的免疫细胞，以获得能够表达嵌合抗原受体的免疫细胞；或者，将权利要求15-19任一项所述的核酸构建体或包含其的载体引入步骤(1)所述的免疫细胞，以获得能够共表达嵌合抗原受体和另外的生物活性分子的免疫细胞；A method for preparing modified immune cells, which includes: (1) providing immune cells from patients or healthy donors; (2) introducing the isolated nucleic acid molecule of claim 13 or 14 or a vector containing it into the step ( 1) the immune cells described in order to obtain immune cells capable of expressing chimeric antigen receptors; or, introducing the nucleic acid construct or the vector comprising the same according to any one of claims 15-19 into step (1) immune cells to obtain immune cells capable of co-expressing chimeric antigen receptors and additional bioactive molecules;
    优选地，在步骤(1)中，所述免疫细胞经预处理，所述预处理包括免疫细胞的分选、激活和/或增殖；更优选地，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞；Preferably, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of immune cells; more preferably, the pretreatment includes combining the immune cells with anti-CD3 Contacting the antibody with an anti-CD28 antibody thereby stimulating the immune cells and inducing their proliferation, thereby generating preconditioned immune cells;
    优选地，在步骤(2)中将所述核酸分子或载体通过病毒感染引入免疫细胞；Preferably, in step (2), the nucleic acid molecule or vector is introduced into immune cells through viral infection;
    优选地，在步骤(2)中将所述核酸分子或载体通过非病毒载体转染的方式引入免疫细胞，如通过磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法或电穿孔方法；Preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune cells through non-viral vector transfection, such as through calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, Transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method;
    优选地，在步骤(2)之后还包括扩增步骤(2)获得的免疫细胞的步骤。Preferably, the step of amplifying the immune cells obtained in step (2) is further included after step (2).
  28. 免疫细胞组合物，包括权利要求23-26任一项所述的经改造的免疫细胞；可选地，所述组合物还包括未改造和/或未成功改造的免疫细胞；优选地，所述经改造的免疫细胞数占所述免疫细胞组合物细胞总数的10％-100％，更优选40％-80％。Immune cell composition, including the modified immune cells of any one of claims 23-26; optionally, the composition also includes unmodified and/or unsuccessfully modified immune cells; preferably, the The number of modified immune cells accounts for 10%-100% of the total number of cells in the immune cell composition, more preferably 40%-80%.
  29. 试剂盒，所述试剂盒包括权利要求1-12任一项所述的嵌合抗原受体，或权利要求 15-19任一项所述的核酸构建体；A test kit comprising the chimeric antigen receptor according to any one of claims 1 to 12, or the claim The nucleic acid construct described in any one of 15-19;
    优选地，所述试剂盒包括权利要求13或14所述的分离的核酸分子或包含所述分离的核酸分子的载体；所述试剂盒用于制备权利要求1-12任一项所述的嵌合抗原受体；Preferably, the kit includes the isolated nucleic acid molecule of claim 13 or 14 or a vector containing the isolated nucleic acid molecule; the kit is used to prepare the embedded nucleic acid molecule of any one of claims 1-12. Synthetic antigen receptor;
    优选地，所述试剂盒包括权利要求13或14所述的分离的核酸分子或包含所述分离的核酸分子的载体；所述试剂盒用于制备权利要求23-26所述的经改造的免疫细胞或包含所述经改造的免疫细胞的免疫细胞组合物；Preferably, the kit includes the isolated nucleic acid molecule of claim 13 or 14 or a vector comprising the isolated nucleic acid molecule; the kit is used to prepare the modified immune cells of claims 23-26. Cells or immune cell compositions comprising said engineered immune cells;
    优选地，所述试剂盒包括权利要求15-19任一项所述的核酸构建体或包含所述核酸构建体的载体；所述试剂盒用于制备权利要求23-26所述的经改造的免疫细胞或包含所述经改造的免疫细胞的免疫细胞组合物。Preferably, the kit includes the nucleic acid construct of any one of claims 15-19 or a vector containing the nucleic acid construct; the kit is used to prepare the modified nucleic acid construct of claims 23-26. Immune cells or immune cell compositions comprising said engineered immune cells.
  30. 药物组合物，其含有权利要求1-12任一项所述的嵌合抗原受体，或权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体，或权利要求20所述的载体，或权利要求21所述的宿主细胞，或权利要求23-26任一项所述的经改造的免疫细胞，或权利要求28所述的免疫细胞组合物，以及药学上可接受的载体和/或赋形剂；Pharmaceutical composition, which contains the chimeric antigen receptor described in any one of claims 1-12, or the isolated nucleic acid molecule described in claims 13 or 14, or the nucleic acid described in any one of claims 15-19 Construct, or the vector of claim 20, or the host cell of claim 21, or the modified immune cell of any one of claims 23-26, or the immune cell combination of claim 28 substances, and pharmaceutically acceptable carriers and/or excipients;
    优选地，所述药物组合物还包含另外的药学活性剂，例如具有抗肿瘤活性的药物；优选地，所述另外的药学活性剂包括anti-PD1抗体、anti-PD-L1抗体、anti-CTLA-4抗体、anti-CD3抗体、anti-ASGPR1抗体、索拉菲尼或其衍生物、瑞格菲尼或其衍生物、培美曲塞、顺铂、紫杉醇、吉西他滨、卡培他滨或FOLFIRINOX；Preferably, the pharmaceutical composition also contains additional pharmaceutically active agents, such as drugs with anti-tumor activity; preferably, the additional pharmaceutically active agents include anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA -4 antibody, anti-CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX ;
    可选地，所述药物组合物包含的嵌合抗原受体、或分离的核酸分子、或载体、或宿主细胞、或核酸构建体、或经改造的免疫细胞、或免疫细胞组合物，可与所述另外的药学活性剂同时、分开或相继施用。Alternatively, the chimeric antigen receptor, or isolated nucleic acid molecule, or vector, or host cell, or nucleic acid construct, or modified immune cell, or immune cell composition contained in the pharmaceutical composition can be combined with The additional pharmaceutically active agents are administered simultaneously, separately or sequentially.
  31. 权利要求1-12任一项所述的嵌合抗原受体，或权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体，或权利要求20所述的载体，或权利要求21所述的宿主细胞，或权利要求23-26任一项所述的经改造的免疫细胞，或权利要求28所述的免疫细胞组合物，或权利要求30所述的药物组合物，在制备用于预防和/或治疗与GPC3的表达相关的疾病的药物中的用途；The chimeric antigen receptor according to any one of claims 1-12, or the isolated nucleic acid molecule according to any one of claims 13 or 14, or the nucleic acid construct according to any one of claims 15-19, or the claim The vector of claim 20, or the host cell of claim 21, or the modified immune cell of any one of claims 23-26, or the immune cell composition of claim 28, or claim 30 The use of the pharmaceutical composition in preparing drugs for preventing and/or treating diseases related to the expression of GPC3;
    优选地，所述与GPC3的表达相关的疾病选自增生性疾病，例如肿瘤，或是与GPC3的表达相关的非肿瘤相关的适应症； Preferably, the disease associated with the expression of GPC3 is selected from proliferative diseases, such as tumors, or is a non-tumor-related indication associated with the expression of GPC3;
    优选地，所述肿瘤是GPC3阳性肿瘤；Preferably, the tumor is a GPC3 positive tumor;
    优选地，所述肿瘤选自实体瘤；优选地，所述实体瘤选自肝癌、肝细胞癌、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、卵巢癌、卵巢透明细胞癌、黑色素瘤、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、直结肠癌、胃癌、神经胶质瘤中的一种或其组合；Preferably, the tumor is selected from solid tumors; preferably, the solid tumors are selected from liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma , one or a combination of non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, and glioma;
    优选地，所述肿瘤选自血液肿瘤；优选地，所述血液肿瘤选自白血病、淋巴瘤。 Preferably, the tumor is selected from hematological tumors; preferably, the hematological tumors are selected from leukemia and lymphoma.
PCT/CN2023/100146 2022-06-24 2023-06-14 Chimeric antigen receptor specifically binding to gpc3 and use thereof WO2023246578A1 (en)

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