[go: up one dir, main page]

WO2024114410A1 - 靶向gpc3的抗体及其用途 - Google Patents

靶向gpc3的抗体及其用途 Download PDF

Info

Publication number
WO2024114410A1
WO2024114410A1 PCT/CN2023/132296 CN2023132296W WO2024114410A1 WO 2024114410 A1 WO2024114410 A1 WO 2024114410A1 CN 2023132296 W CN2023132296 W CN 2023132296W WO 2024114410 A1 WO2024114410 A1 WO 2024114410A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
cdr
variant
antibody
Prior art date
Application number
PCT/CN2023/132296
Other languages
English (en)
French (fr)
Inventor
田海军
刘雪松
蔡珍珍
周阳
葛均友
卫立辛
Original Assignee
四川科伦博泰生物医药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 四川科伦博泰生物医药股份有限公司 filed Critical 四川科伦博泰生物医药股份有限公司
Priority to CN202380069639.2A priority Critical patent/CN119968398A/zh
Publication of WO2024114410A1 publication Critical patent/WO2024114410A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present application relates to the field of biomedicine, and more specifically, to antibodies or antigen-binding fragments thereof that specifically bind to GPC3.
  • the present invention also relates to the use of these antibodies or antigen-binding fragments thereof for preventing and/or treating diseases related to the expression of GPC3, such as liver cancer, melanoma, ovarian cancer and other cancers, and methods for preventing and/or treating GPC3-positive tumors such as liver cancer, melanoma, ovarian cancer and the like.
  • Glypican-3 (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS and SGBS1) is a member of the heparan sulfate proteoglycan family. It is anchored on the cell surface through glycosylated phosphatidylinositol and is one of the representative liver cancer markers in preclinical studies. 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-related death in my country, which seriously threatens the life and health of the Chinese people.
  • the metastasis and recurrence rate within 5 years after resection is as high as 60% to 70%, and the overall 5-year survival rate is low, only 7% to 10%.
  • GPC3-positive hepatocellular carcinoma HCC
  • melanoma ovarian clear cell carcinoma
  • the inventors have developed a fully human antibody that is low in immunogenicity and highly specific to GPC3 and is capable of specifically recognizing/binding to GPC3, which has the potential to prevent and/or treat diseases associated with the expression of GPC3, such as liver cancer, melanoma, ovarian cancer and other cancers, and has significant clinical value.
  • the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to GPC3, wherein the antibody or the antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):
  • the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering systems.
  • an antibody or antigen-binding fragment thereof of the invention comprises 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:
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with a sequence of SEQ ID NO: 54 or a variant thereof
  • CDR-H3 with a sequence of SEQ ID NO: 55 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (1a)-(1d) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • an antibody or antigen-binding fragment thereof of the invention comprises 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
  • CDR-H2 having a sequence of SEQ ID NO: 16 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 17 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with a sequence of SEQ ID NO: 31 or a variant thereof
  • CDR-H3 with a sequence of SEQ ID NO: 32 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with a sequence of SEQ ID NO: 60 or a variant thereof
  • CDR-H3 with a sequence of SEQ ID NO: 61 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (2a)-(2d) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions.
  • an antibody or antigen-binding fragment thereof of the invention comprises 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 having a sequence of SEQ ID NO: 21 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 11 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 21 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 26 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with a sequence of SEQ ID NO: 50 or a variant thereof
  • CDR-H3 with a sequence of SEQ ID NO: 40 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 65 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 55 or a variant thereof
  • VL light chain variable region
  • the variant described in any one of (3a)-(3d) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 23 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 11 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 37 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 26 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 52 or a variant thereof
  • CDR-H3 having a sequence of SEQ ID NO: 40 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (4a)-(4d) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises framework regions (FRs) from human immunoglobulins.
  • an antibody or antigen-binding fragment thereof of the invention comprises:
  • VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof;
  • VH comprising the sequence shown in SEQ ID NO:5 or a variant thereof and/or a 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 a VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof;
  • the 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 compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.
  • the antibodies or antigen-binding fragments thereof of the invention are murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.
  • the antibody or antigen-binding fragment thereof of the present invention is selected from a full-length antibody, a single-chain antibody (e.g., scFv, di-scFv or (scFv) 2 ), Fab, Fab', Fab'-SH, F(ab') 2 , F(ab)' 3 fragment, Fv fragment, minibody, disulfide-linked Fv (dsFv), single domain antibody (sdAb, nanobody), diabody, bispecific antibody and multispecific antibody.
  • a single-chain antibody e.g., scFv, di-scFv or (scFv) 2
  • Fab Fab', Fab'-SH, F(ab') 2 , F(ab)' 3 fragment, Fv fragment, minibody, disulfide-linked Fv (dsFv), single domain antibody (sdAb, nanobody), diabody, bispecific antibody and multispecific antibody.
  • the VH and VL of the antibodies or antigen-binding fragments thereof of the present invention are connected by one or more linkers.
  • the linker is typically a peptide linker, such as a flexible and/or soluble peptide linker, such as a peptide linker rich in glycine, serine and/or threonine.
  • the linker also includes charged residues (such as lysine and/or glutamic acid), which can improve solubility.
  • the linker also includes one or more proline.
  • the linker comprises one or more (e.g., 1, 2, or 3) sequences as shown in ( GmS ) n , wherein 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 a sequence of SEQ ID NO: 76.
  • the antibodies or antigen-binding fragments thereof of the invention are single chain antibodies, such as scFv, di-scFv or (scFv) 2 .
  • the single-chain antibody comprises, from its N-terminus to its C-terminus:
  • VH-linker comprising the sequence shown in SEQ ID NO: 1 or a variant thereof-VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof;
  • VH-linker comprising the sequence shown in SEQ ID NO:3 or a variant thereof-VL comprising the sequence shown in SEQ ID NO:4 or a variant thereof;
  • VH-linker comprising the sequence shown in SEQ ID NO:5 or a variant thereof-VL comprising the sequence shown in SEQ ID NO:6 or a variant thereof;
  • VH-linker comprising the sequence shown in SEQ ID NO:7 or a variant thereof-VL comprising the sequence shown in SEQ ID NO:8 or a variant thereof;
  • VL-linker comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof-a VH comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof;
  • VL-linker comprising a sequence as shown in SEQ ID NO: 4 or a variant thereof-a VH comprising a sequence as shown in SEQ ID NO: 3 or a variant thereof;
  • VL-linker comprising a sequence as shown in SEQ ID NO:6 or a variant thereof-a VH comprising a sequence as shown in SEQ ID NO:5 or a variant thereof;
  • VL-linker comprising a sequence as shown in SEQ ID NO: 8 or a variant thereof-a VH comprising a sequence as shown in SEQ ID NO: 7 or a variant thereof;
  • the 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 compared to the sequence from which it is derived, or has one or more amino acid substitutions compared to the sequence from which it is derived, Deletion or addition (eg, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids). In certain embodiments, the substitution is a conservative substitution.
  • the single-chain antibody comprises an amino acid sequence selected from the following: (1) an amino acid sequence shown in any one of SEQ ID NOs: 68, 69, 70, and 71; (2) a sequence having 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 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence shown in any one of SEQ ID NOs: 68, 69, 70, and 71; or (3) a sequence having one or more 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) compared to the amino acid sequence shown in any one of SEQ ID NOs: 68, 69, 70, and 71.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the present invention further comprises a constant region derived from a human immunoglobulin.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., ⁇ or ⁇ ).
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the wild-type sequence from which it is derived; and/or,
  • CH heavy chain constant region
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, which 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; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived.
  • CL light chain constant region
  • 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 IgG1, 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 antibodies or antigen-binding fragments thereof of the present invention may be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein).
  • another molecule e.g., another polypeptide or protein.
  • the derivatization (e.g., labeling) of the antibody or antigen-binding fragment thereof will not adversely affect its binding to GPC3 (particularly human GPC3). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms.
  • the antibodies or antigen-binding fragments thereof of the present invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection agent, a pharmaceutical agent, and/or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or a polyhistidine tag).
  • another antibody e.g., to form a bispecific antibody
  • a detection agent e.g., to form a bispecific antibody
  • a pharmaceutical agent e.g., avidin or a polyhistidine tag
  • the present invention provides a conjugate, which comprises the antibody or antigen-binding fragment thereof of the present invention and a conjugated part.
  • the coupling moiety is selected from a detectable label.
  • the detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means.
  • labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acri
  • such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).
  • the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  • the detectable label as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention by linkers of different lengths to reduce potential steric hindrance.
  • the conjugated moiety is selected from a therapeutic agent.
  • the therapeutic agent is preferably an anti-tumor agent, such as a cytotoxic agent, a cytokine, a toxin or a radionuclide.
  • the conjugated moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, it can be a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a sugar group.
  • PEG polyethylene glycol
  • the present invention provides a multispecific antibody comprising the antibody of the present invention or an antigen-binding fragment thereof.
  • the multispecific antibody comprises an antibody of the present invention or an antigen-binding fragment thereof as a first antigen-binding domain, and further comprises at least one second antigen-binding domain directed against another target.
  • each antigen binding domain of the multispecific antibody retains its respective original binding specificity.
  • the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.
  • the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification.
  • the obtained DNA molecules are inserted into expression vectors and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibodies 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)). In addition, these antigen-binding fragments can also be directly produced by 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 directly obtained 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 culture medium of recombinant host cells.
  • Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.
  • the second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or the heavy chain variable region and/or light chain variable region thereof of the present invention.
  • the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO:89, (ii) a sequence substantially identical to SEQ ID NO:89 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:89), or (iii) a degenerate sequence of (i) or (ii) above; and/or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of SEQ ID NO:90, (v) a sequence substantially identical to SEQ ID NO:90 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:90), or (vi) a degenerate sequence of (
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:91, (ii) a sequence substantially identical to SEQ ID NO:91 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO:91, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above; and/or, the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence shown in SEQ ID NO:92, (v) a sequence substantially identical to SEQ ID NO:92 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO:92, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv)
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:93, (ii) a sequence substantially identical to SEQ ID NO:93 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:93), or (iii) a degenerate sequence of (i) or (ii) above; and/or, the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence shown in SEQ ID NO:94, (v) a sequence substantially identical to SEQ ID NO:94 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:94), or (vi) a degenerate
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:95, (ii) a sequence substantially identical to SEQ ID NO:95 (for example, a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO:95, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of the above (i) or (ii); and/or, the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence shown in SEQ ID NO:96, (v) a sequence substantially identical to SEQ ID NO:96 (for example, a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO:96, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of the above (iv) or (ii
  • the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (1) a nucleotide sequence as set forth in any one of SEQ ID NOs: 72, 73, 74, and 75; (2) a nucleotide sequence as set forth in any one of SEQ ID NOs: 72, 73, 74, and 75; Compared to the nucleotide sequence shown in any of the items, there is a sequence 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%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the third aspect of the present invention provides a kind of vector (such as cloning vector or expression vector), it comprises the nucleic acid molecule of separation as described above.
  • the vector of the present invention is such as DNA vector, RNA vector, plasmid, transposon vector, CRISPR/Cas9 vector or viral vector.
  • 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.
  • a fourth aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the present invention also relates to a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.
  • the present invention relates to a CAR targeting GPC3, which has the characteristics of non-MHC-restricted GPC3 recognition ability, which confers the ability of immune cells (e.g., T cells, NK cells, monocytes, macrophages or dendritic cells) expressing the CAR to recognize cells expressing GPC3 (e.g., tumor cells) independently of antigen processing and presentation.
  • immune cells e.g., T cells, NK cells, monocytes, macrophages or dendritic cells
  • the fifth aspect of the present invention provides a chimeric antigen receptor (CAR) capable of specifically binding to GPC3, which comprises an extracellular antigen binding domain (antigen binding domain), a spacer domain, a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • 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 an antigen binding domain
  • the antigen binding domain comprises an antibody or antigen binding fragment thereof that can specifically bind to GPC3 (e.g., human GPC3).
  • the antibody or antigen binding fragment thereof is selected from the antibody or antigen binding fragment thereof described in the first aspect.
  • the antibody or antigen-binding fragment thereof is a single chain antibody, such as a scFv, di-scFv, or (scFv) 2 .
  • the VH and VL of the antibody or antigen-binding fragment thereof are connected by a linker.
  • the linker comprises one or more (e.g., 1, 2, or 3) sequences as shown in ( GmS ) n , wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 1 or 2. In certain embodiments, the linker has a sequence of SEQ ID NO: 76.
  • the transmembrane domain included 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 a cell membrane (particularly a eukaryotic cell membrane).
  • the transmembrane domain suitable for the CAR of the present invention can be derived from a natural source.
  • the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain can be a synthetic non-naturally occurring protein segment, such as a protein segment mainly comprising hydrophobic residues such as leucine and valine.
  • the transmembrane domain is selected from the transmembrane region of the following proteins: ⁇ , ⁇ or ⁇ 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 selected from the transmembrane region of the following proteins: CD8, CD4, PD-1, CD152 and CD154.
  • the transmembrane domain comprises the transmembrane region of CD8.
  • the transmembrane domain comprises the CD8 transmembrane region as shown in SEQ ID NO: 77.
  • the chimeric antigen receptor of the present invention comprises 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 (e.g., IgG1 or IgG4).
  • an immunoglobulin e.g., IgG1 or IgG4
  • CH2 and CH3 extend the antigen binding domain of the CAR from the cell membrane of the cell expressing the CAR, and can more accurately mimic the size and domain structure of a natural TCR.
  • the spacer domain comprises a hinge domain.
  • a hinge domain can be an amino acid segment usually found between two domains of a protein, which can allow the protein to be flexible and allow one or two domains to move relative to each other. Therefore, the hinge domain can be any amino acid sequence as long as it can provide this flexibility of the extracellular antigen binding domain and its mobility relative to the transmembrane domain.
  • the hinge domain is a hinge region or a portion thereof of a naturally occurring protein.
  • the hinge domain comprises a hinge region or a portion thereof of CD8, such as a fragment of at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge region of CD8.
  • the hinge domain comprises a hinge region of CD8, IgG4, PD-1, CD152, or CD154.
  • the spacer domain comprises the amino acid sequence shown in SEQ ID NO:78.
  • the CAR of the present invention may further include a signal peptide at its N-terminus.
  • a signal peptide is a polypeptide sequence that targets the sequence connected thereto to the desired site.
  • the signal peptide can target the CAR connected thereto to the secretory pathway of the cell and allow the CAR to be further integrated and anchored into the lipid bilayer.
  • Signal peptides that can be used for CAR are known to those skilled in the art.
  • the signal peptide comprises a heavy chain signal peptide (e.g., a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or a CD8 ⁇ signal peptide.
  • the signal peptide is selected from a CD8 ⁇ signal peptide.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:82.
  • the CAR of the present invention is co-expressed with another bioactive molecule.
  • the other 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.
  • Signal peptide-2 guides the transport of other bioactive molecules to a specific site in the cell or outside the cell membrane.
  • the signal peptide-2 may be the same 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 shown in SEQ ID NO:84).
  • the intracellular signaling domain contained in the CAR of the present invention participates in the signal transduction generated by the binding of the CAR of the present invention to GPC3 into the interior of the immune effector cell, activates at least one normal effector function of the immune effector cell expressing CAR, or enhances the secretion of at least one cytokine (e.g., IL-2, IFN- ⁇ ) of the immune effector cell expressing CAR.
  • cytokine e.g., IL-2, IFN- ⁇
  • the intracellular signaling domain comprises a primary signaling domain and/or a co-stimulatory signaling domain.
  • the primary signaling domain may be any intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain selected from the following proteins: CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b or CD66d. In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of CD3 ⁇ .
  • the costimulatory signaling domain may be an intracellular signaling domain from a costimulatory molecule.
  • the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10.
  • the co-stimulatory 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.
  • the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains. In such embodiments, the two or more co-stimulatory signaling domains may be the same or different.
  • the intracellular signaling domain comprises a primary signaling domain and at least one co-stimulatory signaling domain.
  • the primary signaling domain and at least one co-stimulatory signaling domain can be connected in series to the carboxyl terminus of the transmembrane domain in any order.
  • the intracellular signaling domain may include the intracellular signaling domain of CD3 ⁇ and the intracellular signaling domain of CD137.
  • the intracellular signaling domain of CD3 ⁇ includes the amino acid sequence shown in SEQ ID NO: 79.
  • the intracellular signaling domain of CD137 includes the amino acid sequence shown in SEQ ID NO: 80.
  • the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 81.
  • the present invention provides a chimeric antigen receptor that can specifically bind to GPC3, wherein the chimeric antigen receptor comprises an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain from its N-terminus to its C-terminus.
  • the intracellular signaling domain is a co-stimulatory signaling domain from the N-terminus to the C-terminus. domain and primary signaling domain.
  • 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 CD3 ⁇ and the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB).
  • the chimeric antigen receptor comprises the signal peptide, antigen binding domain, spacer domain, transmembrane domain, intracellular signaling domain (co-stimulatory signaling domain and primary signaling domain from N-terminus to C-terminus) in sequence.
  • Methods for generating chimeric antigen receptors and immune effector cells comprising the chimeric antigen receptor are known in the art, and may include transfecting cells with at least one polynucleotide encoding CAR, and expressing the polynucleotides in cells.
  • the nucleic acid molecules encoding the CAR of the present invention may be included in an expression vector (e.g., a lentiviral vector), which can be expressed in a host cell such as a T cell to manufacture the CAR.
  • the sixth aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect.
  • nucleotide sequence encoding a chimeric antigen receptor of the present invention may have a variety of different sequences. Therefore, unless otherwise specified, 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 seventh aspect of the present invention also provides a nucleic acid construct comprising a nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect.
  • the eighth aspect of the present invention provides a vector comprising the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect.
  • the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, and a viral vector.
  • 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.
  • Toxic carrier 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-defective retrovirus or lentivirus.
  • the ninth aspect of the present invention provides a host cell, which comprises the isolated nucleic acid molecule as described in the sixth aspect, the nucleic acid construct as described in the seventh aspect, or the vector as described in the eighth aspect.
  • the vector as described above can be introduced into the host cell by various suitable means, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (such as liposome) or viral vector-mediated transfection (such as lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological means for transfer into host cells, 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 present invention.
  • the host cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct, and the host cell expresses the chimeric antigen receptor of the present invention and another biologically active molecule.
  • the host cell is selected from the immune cells of mammals (such as humans).
  • the immune cells are derived from patients or healthy donors.
  • 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 a cell expressing the chimeric antigen receptor of the present invention, comprising: (1) providing a host cell; (2) introducing the isolated nucleic acid molecule as described in the sixth aspect or a vector comprising the nucleic acid molecule into the host cell to obtain a host cell capable of expressing the chimeric antigen receptor.
  • a method for a cell co-expressing the chimeric antigen receptor of the present invention and another biologically active molecule comprising: (1) providing a host cell; (2) introducing the nucleic acid construct as described in the seventh aspect or a vector comprising the nucleic acid construct into the host cell to obtain a host cell capable of co-expressing the chimeric antigen receptor and another biologically active molecule.
  • the host cell is selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages and any combination thereof.
  • the immune cell is selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof.
  • the immune cells are provided from a patient or a healthy donor and are pretreated; the pretreatment includes sorting, activation and/or proliferation of the immune cells; in certain embodiments, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, 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 immune cell by viral infection. In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by non-viral vector transfection, such as by transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • non-viral vector transfection such as by transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.
  • the method further comprises: expanding the immune cells obtained in step (2).
  • immune cells derived from patients or healthy donors can be transformed into immune cells expressing CAR that specifically binds to GPC3 and optionally other biologically active molecules.
  • the eleventh aspect of the present invention also provides a modified immune cell, which expresses the CAR of the present invention that specifically binds to GPC3.
  • the engineered immune cell comprises the isolated nucleic acid molecule described in the sixth aspect or a vector comprising the nucleic acid molecule.
  • the engineered immune cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct.
  • the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells of patients or healthy donors and any combination thereof. These immune cells are introduced into the isolated nucleic acid molecules described in the sixth aspect, the nucleic acid construct described in the seventh aspect or the vector described in the eighth aspect by the method provided in the tenth aspect to prepare the transformed immune cells.
  • the transcription or expression of one or two target genes among genes related to immune exclusion (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B or HLA-C) and genes of immune co-inhibitory pathways or signaling molecules (e.g., PD-1, CTLA-4 or LAG-3) of the engineered immune cells is inhibited, so that the signal transduction mediated by the target genes is blocked in the engineered immune cells; preferably, the transcription or expression of the target genes is inhibited by a method selected from gene knockout (e.g., CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
  • gene knockout e.g., CRISPR, CRISPR/Cas9
  • homologous recombination e.g., interfering RNA.
  • the present invention also provides an immune cell composition, which includes the aforementioned modified immune cells, and optional unmodified and/or unsuccessfully modified immune cells, which do not express CAR specific for GPC3.
  • an immune cell composition which includes the aforementioned modified immune cells, and optional unmodified and/or unsuccessfully modified immune cells, which do not express CAR specific for GPC3.
  • the aforementioned modified immune cells and optional unmodified and/or unsuccessfully modified immune cells, which do not express CAR specific for GPC3.
  • the immune cell composition can contain immune cells that express and do not express CAR specific for GPC3, and the immune cell composition can still meet the needs of clinical applications.
  • the engineered immune cells expressing a CAR specific for GPC3 account for approximately 10%-100%, preferably 40%-80% of the total number of cells in the immune cell composition.
  • the immune cell composition is cultured into an immune cell line. Therefore, in another aspect, the present invention also provides an immune cell line containing the immune cell composition.
  • the present invention provides a kit for preparing a chimeric antigen receptor that specifically binds to GPC3, or for preparing a cell expressing the chimeric antigen receptor or an immune cell that co-expresses the chimeric antigen receptor and another biologically active molecule.
  • the kit includes 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, or the host cell described in the ninth aspect, and necessary solvents, such as sterile water, physiological saline, or cell culture fluid, such as LB culture fluid, such as EliteCell primary T lymphocyte culture system (Product No.: PriMed-EliteCell-024), and optionally, instructions for use.
  • necessary solvents such as sterile water, physiological saline, or cell culture fluid, such as LB culture fluid, such as EliteCell primary T lymphocyte culture system (Product No.: PriMed-EliteCell-024), and optionally, instructions for use.
  • the present invention provides use of the aforementioned kit for preparing a chimeric antigen receptor capable of specifically binding to GPC3, or a cell expressing the chimeric antigen receptor, or an immune cell co-expressing the chimeric antigen receptor and another bioactive molecule.
  • the present invention provides a pharmaceutical composition, which contains the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a bispecific chimeric antigen receptor or a CAR construct co-expressed with another biologically active molecule), the isolated nucleic acid molecule described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect or the immune cell composition described in the twelfth aspect, and a pharmaceutically acceptable carrier and/or excipient.
  • 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 antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect, or the immune cell composition of the twelfth aspect and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.
  • the pharmaceutical composition of the present invention comprises: 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, or the host cell described in the ninth aspect.
  • the pharmaceutical composition of the present invention comprises: the engineered immune cell or immune cell composition of the present invention.
  • any dosage form known in the medical field for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixir
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions.
  • a preferred dosage form is an injection.
  • Such an injection can be a sterile injection solution.
  • the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for easy storage and use.
  • Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as 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), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., 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
  • a solution containing a surfactant e.g., 0.01% polysorbate 20
  • a pH buffer solution e.g., phosphate buffer solution
  • Ringer's solution e.g., Ringer's solution, and any combination thereof.
  • the constructed immune cells or the immune cell composition described in the twelfth aspect can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (such as powder, ointment or drops), or nasal route.
  • parenteral administration such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection.
  • Technicians should understand that the route of administration and/or mode will change according to the intended purpose.
  • the antibody or its antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect, the isolated nucleic acid molecule described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the carrier described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect, or the immune cell composition described in the twelfth aspect is administered by intravenous injection or push injection.
  • the pharmaceutical composition of the present invention may include a "therapeutically effective amount” or a "prophylactic effective amount” of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the second or sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third or eighth aspect, the host cell of the fourth or ninth aspect, the modified immune cell of the eleventh aspect, or the immune cell composition of the twelfth aspect.
  • “Prophylactic effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease.
  • “Therapeutically effective amount” refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease.
  • the therapeutically effective amount of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the second or sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third or eighth aspect, the host cell of the fourth or ninth aspect, the modified immune cell of the eleventh aspect, or the immune cell composition of the twelfth aspect may vary according to the following factors: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.
  • the present invention provides a method for preventing and/or treating a disease associated with the expression of GPC3 in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect, the immune cell composition of the twelfth aspect, or the pharmaceutical composition of the present invention.
  • a subject e.g., a human
  • the disease associated with the expression of GPC3 is selected from a proliferative disease, such as a tumor. In certain embodiments, the disease associated with the expression of GPC3 is a non-tumor-related indication associated with the expression of GPC3.
  • the tumor is a GPC3-positive tumor.
  • the tumor is selected from solid tumors.
  • the tumor is selected from liver cancer (e.g., hepatocellular carcinoma), melanoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma and ovarian cancer (e.g., ovarian clear cell carcinoma).
  • the tumor is selected from blood tumors; preferably, the blood tumor is selected from leukemia and lymphoma.
  • the method comprises administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of the first aspect.
  • the method comprises administering to the subject an effective amount of the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the eighth aspect, the host cell of the ninth aspect, the modified immune cell of the eleventh aspect, or the immune cell composition of the twelfth aspect.
  • the host cell is an immune cell (e.g., a human immune cell).
  • the method comprises the following steps: (1) providing the subject with the immune cells required (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 sixth aspect of the present invention, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect into the immune cells described in step (1) to obtain cells expressing the chimeric antigen receptor and optionally additional biologically active molecules; (3) administering the immune cells obtained in step (2) to the subject for treatment.
  • the immune cells required e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells
  • the isolated nucleic acid molecule described in the sixth aspect of the present invention, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect into the immune cells described in step (1) to obtain cells expressing the chimeric antigen receptor and optionally additional biologically active molecules; (3) administering the
  • the method administers immune cells expressing the CAR of the present invention to the subject by dose fractionation, e.g., one, two, three or more separate administrations of partial doses, e.g., administering a first percentage of the total dose on the first day of treatment, administering a second percentage of the total dose on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day, e.g., administering a third percentage (e.g., the remaining percentage) of the total dose on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth day or later) treatment day.
  • dose fractionation e.g., one, two, three or more separate administrations of partial doses, e.g., administering a first percentage of the total dose on the first day of treatment, administering a second percentage of the total dose on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day, e
  • 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 50% of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh or later) day of treatment.
  • 1/3 of the total dose of cells is administered on the first day of treatment
  • 1/3 of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day
  • 1/3 of the total dose of cells is administered on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth day or later) treatment day.
  • the total cell dose comprises 1 ⁇ 10 7 to 10 ⁇ 10 8 CAR-positive immune cells, for example, comprises (1-5) ⁇ 10 7 to (5-10) ⁇ 10 8 CAR-positive immune cells.
  • the physician may adjust the dosage or treatment regimen based on clinical circumstances such as the patient's condition, tumor size and stage, or combination therapy drugs.
  • the antibody or its antigen binding fragment described in the first aspect of the present invention is administered in combination with another agent.
  • the chimeric antigen receptor described in the fifth aspect is administered in combination with another agent.
  • the isolated nucleic acid molecule described in the second aspect or the sixth aspect is administered in combination with another agent.
  • the nucleic acid construct described in the seventh aspect is administered in combination with another agent.
  • the other agent includes (i) an agent for increasing the efficacy of cells containing CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, modified immune cells or immune cell compositions of the present invention); (ii) an agent for improving one or more side effects associated with the administration of cells containing CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, modified immune cells or immune cell compositions of the present invention); (iii) another pharmaceutically active agent with anti-tumor activity.
  • an agent for increasing the efficacy of cells containing CAR nucleic acids or CAR polypeptides e.g., immune cells expressing CAR of the present invention, modified immune cells or immune cell compositions of the present invention
  • an agent for improving one or more side effects associated with the administration of cells containing CAR nucleic acids or CAR polypeptides e.g., immune cells expressing CAR of the present invention, modified immune cells or immune
  • agents can be administered before, simultaneously with, or after administration of the antibody or antigen-binding fragment thereof described in the first aspect, the chimeric antigen receptor described in the fifth aspect, the isolated nucleic acid molecule described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect, the immune cell composition described in the twelfth aspect, or the pharmaceutical composition of the present invention.
  • the above method further comprises administering to the subject a second therapy, which can be any therapy known for 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 can be any therapy known for 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 can be applied separately or in combination with the above-described method; or, the second therapy can be applied simultaneously or sequentially with the above-described method.
  • the subject can be a mammal, such as a human.
  • the dosage, dosage form, administration route, indications, combination therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.
  • antibody refers to an immunoglobulin molecule that can specifically bind to a target (such as a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc.
  • the term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') 2 , Fv), single chains (such as scFv, di-scFv, (scFv) 2 ) and domain antibodies (including, for example, shark and camel antibodies), as well as fusion proteins including antibodies, and immunoglobulin molecules of any other modified configuration including antigen recognition sites.
  • the antibodies of the present invention are not limited by any particular method for producing antibodies.
  • Antibodies include antibodies of any type, such as IgG, IgA or IgM (or its subclass), and antibodies do not need to belong to any particular type.
  • immunoglobulins can be assigned to different types. There are five major 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 ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Antibody light chains can be classified as ⁇ (kappa) and ⁇ (lambda) light chains.
  • the heavy chain constant region consists of four 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 binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions of an antibody can also be subdivided into highly variable regions called complementarity determining regions (CDRs) interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of the following sequence: FR1, CDR2, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form antigen binding sites respectively.
  • the distribution of amino acids in each region or domain can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 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 and light chains each contain three CDRs, designated 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), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDRs contained in the antibodies or antigen-binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody 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 polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".
  • an antigen-binding portion See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes.
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include camelid Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab)' 3 fragment, Fd, Fv, scFv, di-scFv, (scFv) 2 , miniantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, Disulfide-stabilized Fv proteins ("dsFv”) and single domain antibodies (sdAb, nanobodies) and polypeptides comprising 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.
  • 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 comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • the term “Fab'fragment” means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.
  • the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • the Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.
  • Fc means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via a disulfide bond.
  • the Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are 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, ed., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have a 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.
  • GGGGS linker having the amino acid sequence
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
  • a disulfide bond may also be present between the VH and VL of the scFv.
  • the VH and VL domains may be in any suitable The arrangement is relative to each other.
  • a scFv comprising 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 single scFvs connected in series to form an antibody.
  • scFv can form (scFv) 2 , which refers to two or more single scFvs connected in parallel to form an antibody.
  • bifunctional antibody refers to an antibody fragment with two antigen binding sites, the fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • Bifunctional antibodies can be bivalent or bispecific. Bifunctional antibodies are more fully described in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90:6444-6448 (1993).
  • Trifunctional antibodies and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and/or the ability to compete with the full-length antibody for specific binding to the antigen.
  • Antibody antigen-binding fragments can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the expression “specific binding” or “specific for” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed.
  • the strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction.
  • KD 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 method involves measuring the rate of formation and dissociation of the antigen binding site/antigen complex.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be determined by the concentration and the actual rate of association and dissociation. Calculated (see Malmqvist M, Nature, 1993, 361: 186-187).
  • the ratio of kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473).
  • KD, kon and kdis values can be measured by any effective method.
  • the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR).
  • the dissociation constant can be measured by bioluminescence interferometry or Kinexa.
  • identity is used to refer to the matching 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 (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine)
  • the molecules are identical at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the two sequences are compared when they are aligned to produce maximum identity.
  • Such an alignment can be achieved by 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.).
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • conservative substitution means an amino acid substitution that does not adversely affect or change 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 substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • amino acid residues 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, etc.
  • amino acids with nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector may include a sequence that replicates directly and autonomously in a cell, or may include a sequence sufficient to allow integration into the host cell DNA.
  • an expression vector When a vector enables the expression of a protein encoded by an inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and viral vectors, etc.
  • plasmids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • bacteriophages 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • a vector may contain a variety of elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain a replication initiation site.
  • the term "episomal vector” means that the vector is capable of replicating without being integrated into the host's chromosomal DNA and being gradually lost by dividing host cells, and also means that the vector replicates extrachromosomally or episomally.
  • viral vector is used broadly to refer to a vector that typically facilitates transfer of nucleic acid molecules.
  • the invention relates to a nucleic acid molecule (e.g., a transfer plasmid) containing a viral-derived nucleic acid element that is transferred or integrated into the genome of a cell, or a viral particle that mediates nucleic acid transfer.
  • a viral particle typically will include various viral components and sometimes host cell components.
  • viral vector can 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 primarily derived from viruses.
  • retroviral vector refers to a viral vector or plasmid that contains structural and functional genetic elements primarily derived from retroviruses, or portions thereof.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from a lentivirus or portions thereof (including LTR).
  • lentiviral vector lentiviral expression vector
  • elements e.g., cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.
  • sequences of these elements are present in the lentiviral particles of the present invention in the form of RNA and in the DNA plasmids of the present invention in the form of DNA.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, 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, NK cells, monocytes, macrophages or dendritic cells, etc.).
  • Host cells can include single cells or cell groups.
  • chimeric antigen receptor refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, a spacer domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain”), which combines the antibody-based specificity for the target antigen (e.g., GPC3) with the immune effector cell activation intracellular domain to exhibit specific immune activity for cells expressing the target antigen (e.g., GPC3).
  • target antigen e.g., GPC3
  • the expression "immune effector cell expressing CAR” refers to an immune effector cell expressing CAR and having an antigen-specificity determined by the targeting domain of the CAR.
  • Methods for making CARs are known in the art, see, for example, 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 U.S. Patent Publication 2012/0213783, all of which are disclosed by The reference is incorporated herein in its entirety.
  • extracellular antigen binding domain refers to a polypeptide that is capable of specifically binding to an antigen or receptor of interest.
  • the domain will be capable of interacting with cell surface molecules.
  • an extracellular antigen binding domain can be selected to recognize an antigen that is a target cell surface marker associated with a particular disease state.
  • intracellular signaling domain refers to a protein portion that conducts effector signaling function signals and guides cells to perform specialized functions. Therefore, the intracellular signaling domain has the ability to activate at least one normal effector function of an immune effector cell expressing a CAR.
  • the effector function of a T cell can be a cytolytic activity or an auxiliary activity, including the secretion of cytokines.
  • primary signaling domain refers to a portion of a protein that is capable of regulating the primary activation of a TCR complex in a stimulatory or inhibitory manner.
  • Primary signaling domains that act in a stimulatory manner typically contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , 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 that provide the second signal required for the efficient activation and function of T lymphocytes after binding to an antigen, except for antigen receptors or Fc receptors.
  • Non-limiting examples of the 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” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: sterile water, physiological saline, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives.
  • pH regulators include, but are not limited to, phosphate buffers.
  • 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, and the like.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, 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), etc.
  • 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 meanings commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, 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 dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.
  • the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • 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), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., 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
  • a solution containing a surfactant e.g., 0.01% polysorbate 20
  • a pH buffer solution e.g., phosphate buffer solution
  • Ringer's solution e.g., Ringer's solution
  • prevention refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject.
  • treatment refers to a method implemented in order to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable.
  • treatment can also refer to, compared to the expected survival period (if not receiving treatment), extending the survival period.
  • the term “subject” refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the term “subject” refers to a living organism in which an immune response can be elicited. In certain embodiments, the subject (e.g., a human) has a tumor (e.g., a tumor associated with GPC3), or is at risk of having the above-mentioned disease.
  • a tumor e.g., a tumor associated with GPC3
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., a tumor
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art.
  • an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.
  • immune cell refers to a cell involved in an immune response, such as a cell involved in promoting immune effector functions.
  • immune cells include T cells (e.g., ⁇ / ⁇ T cells and ⁇ / ⁇ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.
  • the immune cells of the present invention can be self/autologous ("self") or non-self ("non-self", e.g., allogeneic, isogenic, or allogeneic).
  • self refers to cells from the same subject
  • allogeneic refers to cells of the same species that are genetically different from the comparison cells
  • isogenic refers to cells from a different subject that are genetically identical to the comparison cells
  • allogeneic refers to cells from a different species than the comparison cells.
  • the cells of the present invention are allogeneic.
  • T lymphocytes and/or NK cells.
  • T cell or “T lymphocyte” is well known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.
  • T cells can 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; CD4T cells) CD4T cells, cytotoxic T cells (CTL; CD8T cells), CD4CD8T cells, CD4CD8T cells or any other T cell subsets.
  • T cells may include primary 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 in vivo or in vitro to differentiate into immune cells.
  • immune cells include progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained in CD34+ cell populations derived from umbilical cord blood, bone marrow or flowing peripheral blood, which differentiate into mature immune cells after administration in a subject, or they can be induced in vitro to differentiate into mature immune cells.
  • HSCs hematopoietic stem cells
  • modified immune cell refers to an immune cell that expresses any of the CARs described herein, or into which any of the isolated nucleic acids or vectors described herein have been introduced.
  • the CAR polypeptide can be synthesized in situ in the cell after the polynucleotide encoding the CAR polypeptide has been introduced into the cell by a variety of methods. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, a stable transformation method can be used to integrate the polynucleotide construct into the genome of the cell.
  • a transient transformation method can be used to transiently express the polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell.
  • a virus-mediated method can be used.
  • the polynucleotide can be introduced into the cell by any suitable method, such as a recombinant virus. Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or microprojectile bombardment.
  • the polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.
  • immune effector function refers to the function or response of immune effector cells to enhance or promote immune attack on target cells (e.g., killing of target cells, or inhibiting their growth or proliferation).
  • target cells e.g., killing of target cells, or inhibiting their growth or proliferation.
  • the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • the terms "about” or “approximately” when used with a numerical variable generally means that the value of the variable is within experimental error (e.g., within a 95% confidence interval for the mean) or within ⁇ 10% or wider of the specified value.
  • the present invention provides an antibody or an antigen-binding fragment thereof targeting GPC3, which can be used to prepare a chimeric antigen receptor.
  • Immune effector cells expressing the chimeric antigen receptor of the present invention have improved effector functions (eg, tumor killing activity and cytokine release activity).
  • Figure 1 shows the results of the cytotoxic activity test of CAR-T cells (CB6-T, AB9-T, CE3-T, CH6-T, P7D4-T, blank T) on target cells;
  • Figure 1A is the result of the target cell HepG2, and
  • Figure 1B is the result of the target cell Huh7.
  • Figures 2A and 2B show the results of the detection of IFN- ⁇ and TNF- ⁇ secretion by target HepG2 cells after activation of CAR-T cells (CB6-T, AB9-T, CE3-T, CH6-T, P7D4-T, blank T).
  • Figures 2C and 2D show the results of the detection of IFN- ⁇ and TNF- ⁇ secretion by target Huh7 cells after activation of CAR-T cells (CB6-T, AB9-T, CE3-T, CH6-T, P7D4-T, blank T).
  • the full human phage library was screened using biotinylated GPC3 and SV magnetic beads, and the screening products were titrated by phage plating.
  • the first round of panning products were mixed with PBST and the second and third rounds of panning were performed according to the above steps.
  • VH and VL of the above-mentioned fully human antibody were connected through a linker (SEQ ID NO: 76) to obtain scFv.
  • the sequence information of each scFv is shown in the following table.
  • the candidate scFv sequences and the positive control scFv sequence (P7D4) were constructed in the TGEX-KAL vector, and then transfected into expi293 cells for expression and purification of scFv-Fc protein.
  • the results of SEC analysis experiments showed (Table 4) that the monomer peak (main peak area) of the four candidate sequences (CB6, AB9, CE3, CH6) was greater than 90%.
  • GPC3 scFv-Fc protein a cell line expressing GPC3 (293T/GPC3+) was selected for cell binding assay.
  • Mouse IgG isotype antibody (Mouse IgG Isotype Control, from Thermo Fisher Sci.) was used as a negative control, and anti-P7D4 antibody (see WO2017020812 for related sequences) was used as a positive control.
  • a CAR lentiviral expression vector was further constructed.
  • the intracellular domain of CD137 (4-1BB) and the ITAM region of CD3 ⁇ were used as activation signals, fused with the above scFv, and CD8 ⁇ signal peptide, CD8 hinge region, CD8 transmembrane region were added to construct a chimeric antigen receptor expression vector.
  • the chimeric antigen receptor structure is shown in Table 6 below.
  • Isolation of primary T cells Human PBMC cells were isolated using lymphocyte separation fluid (GE), cultured in an incubator at 37°C and 5% CO 2 , and 100 ⁇ l/mL of CD3 antibody and CD28 antibody were added. After fully mixing, incubated at room temperature for 15 minutes. Remove the magnetic beads and pipette up and down at least 5 times to mix thoroughly. Pipette 50 ⁇ l of magnetic beads/mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes. Add complete culture medium to a total volume of 2.5 mL in the tube, insert the tube (opened) into the magnetic pole, and let stand at room temperature for 5 minutes.
  • GE lymphocyte separation fluid
  • the tube After incubation, the tube continues to remain in the magnetic pole, gently inverted, and the cells in the tube are poured out. 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.
  • the cell density was adjusted to 1 ⁇ 10 6 cells/mL, and cytokine and antibody complexes (300 U/mL of IL-2, 10 ng/mL IL-7, 5 ng/mL IL-15, 500 ng/mL Anti-CD3 (OKT3), 2 ⁇ g/mL Anti-CD28) were added to the six-well plate and cultured continuously for 48 hours.
  • cytokine and antibody complexes 300 U/mL of IL-2, 10 ng/mL IL-7, 5 ng/mL IL-15, 500 ng/mL Anti-CD3 (OKT3), 2 ⁇ g/mL Anti-CD28
  • CAR-T cells expressing the CARs described in Example 2 (CB6-T, AB9-T, CE3-T, and CH6-T) were obtained by the above method.
  • the nucleic acid sequence encoding CAR is expressed under the drive of the promoter, and the T cells transfected with lentivirus are labeled with GPC3 antigen and measured by flow cytometry to reflect the expression level of CAR on the surface of T cells.
  • the CAR positive rate of CAR-T cells obtained in Example 3 was detected by the above method, and the FACS test results are shown in Table 7 below. The results show that the CAR positive rate of all CAR-T cells is greater than 10%, indicating that the lentivirus is successfully expressed after transfecting the effector cells.
  • CAR was developed and four types of T cells expressing GPC3-CAR chimeric antigen receptor (CB6-T, AB9-T, CE3-T and CH6-T) as well as a control chimeric antigen receptor T cell (P7D4-T) were successfully constructed.
  • HEPG2-luc and Huh7-luc cells were digested with 0.25% trypsin, and digestion was terminated with 1640 medium containing 10% FBS. After centrifugation, the cells were resuspended, and the cell density was adjusted to 1 ⁇ 10 5 /mL.
  • the target cells HEPG2-luc and Huh7-luc were inoculated in 96-well plates at 100 ⁇ L/well, and the cells were kept in a 5% CO 2 37°C incubator for 30 minutes. CAR-T cells were collected by centrifugation and resuspended in 10% FBS 1640 medium.
  • GPC3-CAR and blank T cells (UTD) without CAR transfection were used as effector cells, and then added to 96-well plates containing HEPG2-luc or Huh7-luc at different E/T (effector cell/target cell) ratios, 100 ⁇ L/well, and the final volume was supplemented to 200 ⁇ L/well, and cultured in a 5% CO 2 37°C incubator for 18 to 24 hours. After the incubation, take the well plate out of the incubator, add 20ul of fluorescence detection reagent, and use an enzyme reader to detect the fluorescence reading.
  • the results of the CAR-T killing activity test are shown in Figures 1A and 1B.
  • the four CAR-T cells (CB6-T, AB9-T, CE3-T, CH6-T) constructed in this application can effectively lyse tumor cells in different cell lines (HepG2, Huh7) and different E/T ratios.
  • the effector cell/target cell ratio is 1, the lysis rate of tumor cells by the CAR-T cells constructed in this application is as high as about 99%.
  • HepG2-luc and Huh7-luc cells were collected, and the cell density was adjusted to 1 ⁇ 10 5 cells/mL using culture medium.
  • the target cells were inoculated in a 96-well plate at a volume of 100 ⁇ L/well, and the CAR-T cells, GPC3-CAR, and blank T cells without CAR transfection were resuspended in culture medium as effector cells, and then added to the 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 was supplemented to 200 ⁇ L/well, and cultured overnight in a 5% CO 2 37°C incubator.
  • E/T effector cell/target cell
  • Kit IFN- ⁇ , TNF- ⁇ detects cytokine release.
  • the four CAR-T cells CB6-T, AB9-T, CE3-T, CH6-T constructed in this application can kill tumor cells to varying degrees and release IFN- ⁇ (with HepG2 as the target cell, the test results are shown in Figure 2A; with Huh7 as the target cell, the test results are shown in Figure 2C) and TNF- ⁇ (with HepG2 as the target cell, the test results are shown in Figure 2B; with Huh7 as the target cell, the test results are shown in Figure 2D).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

特异性结合GPC3的抗体或其抗原结合片段以及包含其的药物组合物和试剂盒。抗体以及包含其的药物组合物和试剂盒用于预防和/或治疗GPC3的表达相关的疾病，例如肝癌、黑色素瘤、卵巢癌等癌症的用途以及预防和/或治疗肝癌、黑色素瘤、卵巢癌等GPC3阳性肿瘤的方法。

Description

靶向GPC3的抗体及其用途 技术领域
本申请涉及生物医药领域，具体而言，本申请涉及特异性结合GPC3的抗体或其抗原结合片段。本发明还涉及这些抗体或其抗原结合片段用于预防和/或治疗GPC3的表达相关的疾病的用途，例如肝癌、黑色素瘤、卵巢癌等癌症，以及预防和/或治疗肝癌、黑色素瘤、卵巢癌等GPC3阳性肿瘤的方法。
背景技术
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。
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％。
基于GPC3的表达特异性，针对GPC3的靶向治疗有希望成为攻克GPC3阳性肿瘤的方式之一。因此，提供一种靶向GPC3的抗体或其抗原结合片段对于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是迫切而必要的。
发明内容
在本申请中，发明人开发了免疫原性低且对GPC3具有高度特异性的能够特异性识别/结合GPC3的全人源抗体，其具有预防和/或治疗GPC3的表达相关的疾病，例如肝癌、黑色素瘤、卵巢癌等癌症的潜力，具有重大的临床价值。
本发明的抗体
在第一方面，本发明提供了一种能够特异性结合GPC3的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含如下的互补决定区(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；
(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；
(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；
(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；或
(e)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)。在某些实施方案中，所述的置换为保守置换。
在某些实施方案中，所述CDR根据IMGT、Kabat、Chothia或AbM编号系统定义。
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Kabat编号系统定义：
(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：9或其变体的CDR-H1；序列为SEQ ID NO：10或其变体的CDR-H2；序列为SEQ ID NO：11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：12或其变体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：14或其变体的CDR-L3；
(1b)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：26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：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：29或其变体的CDR-L3；
(1c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：38或其变体的CDR-H1；序列为SEQ ID NO：39或其变体的CDR-H2；序列为SEQ ID NO：40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：41或其变体的CDR-L1；序列为SEQ ID NO：42或其变体的CDR-L2；序列为SEQ ID NO：43或其变体的CDR-L3；或，
(1d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：53或其变体的CDR-H1；序列为SEQ ID NO：54或其变体的CDR-H2；序列为SEQ ID NO：55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：56或其变体的CDR-L1；序列为SEQ ID NO：57或其变体的CDR-L2；序列为SEQ ID NO：58或其变体的CDR-L3；
其中，(1a)-(1d)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。
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IMGT编号系统定义：
(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：15或其变体的CDR-H1；序列为SEQ ID NO：16或其变体的CDR-H2；序列为SEQ ID NO：17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：18或其变体的CDR-L1；序列为SEQ ID NO：19或其变体的CDR-L2；序列为SEQ ID NO：14或其变体的CDR-L3；
(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：30或其变体的CDR-H1；序列为SEQ ID NO：31或其变体的CDR-H2；序列为SEQ ID NO：32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：33或其变体的CDR-L1；序列为SEQ ID NO：34或其变体的CDR-L2；序列为SEQ ID NO：29或其变体的CDR-L3；
(2c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：44或其变体的CDR-H1；序列为SEQ ID NO：45或其变体的CDR-H2；序列为SEQ ID NO：46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：47或其变体的CDR-L1；序列为SEQ ID NO：48或其变体的CDR-L2；序列为SEQ ID NO：43或其变体的CDR-L3；或，
(2d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：59或其变体的CDR-H1；序列为SEQ ID NO：60或其变体的CDR-H2；序列为SEQ ID NO：61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：62或其变体的CDR-L1；序列为SEQ ID NO：63或其变体的CDR-L2；序列为SEQ ID NO：58或其变体的CDR-L3；
其中，(2a)-(2d)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。
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Chothia编号系统定义：
(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：20或其变体的CDR-H1；序列为SEQ ID NO：21或其变体的CDR-H2；序列为SEQ ID NO：11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：12或其变体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：14或其变体的CDR-L3；
(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：35或其变体的CDR-H1；序列为SEQ ID NO：21或其变体的CDR-H2；序列为SEQ ID NO：26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：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：29或其变体的CDR-L3；
(3c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：49或其变体的CDR-H1；序列为SEQ ID NO：50或其变体的CDR-H2；序列为SEQ ID NO：40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：41或其变体的CDR-L1；序列为SEQ ID NO：42或其变体的CDR-L2；序列为SEQ ID NO：43或其变 体的CDR-L3；或，
(3d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：64或其变体的CDR-H1；序列为SEQ ID NO：65或其变体的CDR-H2；序列为SEQ ID NO：55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：56或其变体的CDR-L1；序列为SEQ ID NO：57或其变体的CDR-L2；序列为SEQ ID NO：58或其变体的CDR-L3；
其中，(3a)-(3d)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。
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：
(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：22或其变体的CDR-H1；序列为SEQ ID NO：23或其变体的CDR-H2；序列为SEQ ID NO：11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：12或其变体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：14或其变体的CDR-L3；
(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：36或其变体的CDR-H1；序列为SEQ ID NO：37或其变体的CDR-H2；序列为SEQ ID NO：26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：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：29或其变体的CDR-L3；
(4c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：51或其变体的CDR-H1；序列为SEQ ID NO：52或其变体的CDR-H2；序列为SEQ ID NO：40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：41或其变体的CDR-L1；序列为SEQ ID NO：42或其变体的CDR-L2；序列为SEQ ID NO：43或其变体的CDR-L3；或，
(4d)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：55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：56或其变 体的CDR-L1；序列为SEQ ID NO：57或其变体的CDR-L2；序列为SEQ ID NO：58或其变体的CDR-L3；
其中，(4a)-(4d)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。
在某些实施方案中，所述抗体或其抗原结合片段包括来自人的免疫球蛋白的构架区(FRs)。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(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；
(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；
(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；或，
(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；或，
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。
在某些实施方案中，本发明的抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。
在某些实施方案中，本发明的抗体或其抗原结合片段为选自全长抗体、单链抗体(例如scFv、di-scFv或(scFv)2)、Fab、Fab'、Fab'-SH、F(ab')2、F(ab)'3片段、Fv片段、微型抗体、二硫键连接的Fv(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。
在某些实施方案中，本发明的抗体或其抗原结合片段的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。
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：76的序列。
在某些实施方案中，本发明的抗体或其抗原结合片段是单链抗体，例如scFv、di-scFv或(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：
(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；
(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；
(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；
(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；
(5)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；
(6)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；
(7)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；或，
(8)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；
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。
在某些实施方案中，所述单链抗体包含选自下列的氨基酸序列：(1)SEQ ID NOs:68、69、70、71任一项所示的氨基酸序列；(2)与SEQ ID NOs:68、69、70、71任一项所示的氨基酸序列相比具有至少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(3)与SEQ ID NOs:68、69、70、71任一项所示的氨基酸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，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。
在某些实施方案中，本发明的抗体或其抗原结合片段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，所述抗体或其抗原结合片段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，所述抗体或其抗原结合片段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。
在某些实施方案中，所述抗体或其抗原结合片段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，
所述抗体或其抗原结合片段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)。
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。
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。
衍生的抗体
本发明的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会不利影响其对GPC3(特别是人GPC3)的结合。因此，本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本发明的抗体或其抗原结合片段功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。
作为抗体的衍生物之一，本发明提供一种偶联物，其包括本发明的抗体或其抗原结合片段以及偶联部分。
在某些实施方案中，所述偶联部分选自可检测的标记。本发明所述的可检测标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如，)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，所述可检测标记选自放射性同位素、荧光物质、发光物质、有色物质或酶。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
在某些实施方案中，所述偶联部分选自治疗剂。在某些实施方案中，所述治疗剂优选地为抗肿瘤剂，例如细胞毒剂、细胞因子、毒素或放射性核素。
在某些实施方案中，所述偶联部分选自能够改善抗体的生物学特性(例如增加血清半衰期)的物质，例如可以是化学基团，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。
作为抗体的衍生物之一，本发明提供一种多特异性抗体，其包含本发明的抗体或其抗原结合片段。
在某些实施方案中，所述多特异性抗体包含本发明的抗体或其抗原结合片段作为第一抗原结合结构域，并且还包含至少一种针对其他靶标的第二抗原结合结构域。
在某些实施方案中，所述多特异性抗体的各个抗原结合结构域保持各自的原结合特异性。
在某些实施方案中，所述多特异性抗体为双特异性抗体或三特异性抗体或四特异性抗体。
抗体的制备
本发明的抗体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扩增获得编码本发明抗体的重链和轻链基因的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，并表达本发明的抗体。
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.,Science229:81(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。
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体或其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的核苷酸序列：
(a)所述编码抗体重链可变区的核酸分子包含：(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；和/或，所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:90所示的核苷酸序列、(v)与SEQ ID NO:90基本上相同的序列(例如，与SEQ ID NO:90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；
或
(b)所述编码抗体重链可变区的核酸分子包含：(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；和/或，所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:92所示的核苷酸序列、(v)与SEQ ID NO:92基本上相同的序列(例如，与SEQ ID NO:92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；
或
(c)所述编码抗体重链可变区的核酸分子包含：(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；和/或，所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:94所示的核苷酸序列、(v)与SEQ ID NO:94基本上相同的序列(例如，与SEQ ID NO:94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；
或
(d)所述编码抗体重链可变区的核酸分子包含：(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；和/或，所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:96所示的核苷酸序列、(v)与SEQ ID NO:96基本上相同的序列(例如，与SEQ ID NO:96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。
在某些实施方案中，所述分离的核酸分子包含选自下列的核苷酸序列：(1)SEQ ID NO：72、73、74和75任一项所示的核苷酸序列；(2)与SEQ ID NO：72、73、74和75 任一项所示的核苷酸序列相比具有至少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。
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。在某些实施方案中，所述载体是表达载体。在某些实施方案中，所述载体是游离型载体。在某些实施方案中，所述载体是病毒载体。在某些实施方案中，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。
本发明第四方面提供了一种宿主细胞，其包含如上所述的分离的核酸分子或载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
在另一个方面，本发明还涉及制备本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，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体或其抗原结合片段。
嵌合抗原受体(CAR)
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。
因此，本发明第五方面提供了一种能够特异性结合GPC3的嵌合抗原受体(CAR)，其包含胞外抗原结合结构域(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。
I.胞外抗原结合结构域
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。
在某些实施方案中，所述抗原结合结构域包含抗原结合结构域，所述抗原结合结构域包含能够特异性结合GPC3(例如人GPC3)的抗体或其抗原结合片段。在某些实施方案中，所述抗体或其抗原结合片段选自第一方面所述的抗体或其抗原结合片段。
在某些实施方案中，所述抗体或其抗原结合片段是单链抗体，例如scFv、di-scFv或(scFv)2。
在某些实施方案中，所述抗体或其抗原结合片段的VH和VL通过连接子连接。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。在某些实施方案中，m为3、4、或5。在某些实施方案中，n为1或2。在某些实施方案中，所述连接子具有SEQ ID NO：76的序列。
II.跨膜结构域
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。
在某些实施方案中，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，及其任意组合。在某些优选的实施方案中，所述跨膜结构域选自下列蛋白的跨膜区：CD8、CD4、PD-1、CD152和CD154。在某些优选的实施方案中，所述跨膜结构域包含CD8的跨膜区。在某些优选的实施方案中，所述跨膜结构域包含序列如SEQ ID NO：77所示的CD8跨膜区。
III.间隔结构域
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。
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。
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。
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，所述铰链结构域包含CD8的铰链区或其部分，例如含有CD8的铰链区的至少15个(例如20、25、30、35或40个)连续氨基酸的片段。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。在某些示例性实施方案中，所述间隔结构域包含SEQ ID NO:78所示的氨基酸序列。
IV.信号肽
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:82所示的氨基酸序列。
在某些实施方案中，本发明的CAR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。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。所述信号肽-2可与上一段所述的信号肽相同或不同。优选地，所述信号肽-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:84所示)。
V.胞内信号传导结构域
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)。
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。
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。
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。
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。
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。
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。
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:79所示的氨基酸序列。在某些示例性实施方案中，所述CD137的胞内信号传导结构域包含SEQ ID NO:80所示的氨基酸序列。
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：81所示序列。
VI.全长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。
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，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。
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)。
嵌合抗原受体的制备
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。
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。
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。
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核酸序列。
本发明第八方面提供了一种载体，其包含第六所述的分离的核酸分子，或第七方面所述的核酸构建体。
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。
在某些实施方案中，所述载体是表达载体。
在某些实施方案中，所述载体是游离型载体。
在某些实施方案中，所述载体是病毒载体。
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。
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。
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。
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。
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。
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。
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。
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。
在某些实施方案中，在步骤(1)中，所述免疫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。
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免疫细胞。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免疫细胞，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。
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)获得的免疫细胞。
经改造的免疫细胞
通过本发明提供的上述制备方法可将来源于患者或健康供体的免疫细胞改造为表达特异性结合GPC3的CAR以及可选的另外的生物活性分子的免疫细胞。
因此，本发明第十一方面还提供了一种经改造的免疫细胞，其表达本发明的特异性结合GPC3的CAR。
在某些实施方案中，所述经改造的免疫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。
在某些实施方案中，所述经改造的免疫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。
在某些实施方案中，所述免疫细胞来源于患者或健康供体的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。这些免疫细胞被通过第十方面所提供的方法导入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从而制备为经改造的免疫细胞。
在某些实施方案中，经改造的免疫细胞的免疫排除有关的基因(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，使得靶基因介导的信号传导在所述的经改造的免疫细胞中被阻断；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。
免疫细胞组合物
在第十二方面，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特异性针对GPC3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特异性针对GPC3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特异性针对GPC3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。
在某些实施方案中，经改造的表达特异性针对GPC3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％。
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。
在另一个方面，本发明提供了制备特异性结合GPC3的嵌合抗原受体，或用于制备表达所述嵌合抗原受体的细胞或者共表达所述嵌合抗原受体以及另外的生物活性分子的免疫细胞的试剂盒。在某些实施方案中，所述试剂盒包括如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，或第九方面所述的宿主细胞，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。
在另一个方面，本发明提供了前述试剂盒用于制备能够特异性结合GPC3的嵌合抗原受体、或表达所述嵌合抗原受体的细胞、或共表达所述嵌合抗原受体以及另外的生物活性分子的免疫细胞的应用。
药物组合物
在第十三方面，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体或其抗原结合片段、第五方面所述的嵌合抗原受体(包括双特异性嵌合抗原受体或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、第十一方面所述的经改造的免疫细胞或第十二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。
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)。
在某些实施方案中，本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二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。
在某些实施方案中，本发明的药物组合物包含：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、或第九方面所述的宿主细胞。
在某些实施方案中，本发明的药物组合物包含：本发明的经改造的免疫细胞或免疫细胞组合物。
本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二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。
本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改 造的免疫细胞或第十二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体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物通过静脉注射或推注给予。
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体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二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体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二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。
治疗方法及用途
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体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或药物组合物。
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。
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。在某些实施方案中，所述肿瘤选自肝癌(例如，肝细胞癌)、黑色素瘤、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、结直肠癌、胃癌、神经胶质瘤和卵巢癌(例如，卵巢透明细胞癌)。在某些实施方案中，所述肿瘤选自血液肿瘤；优选地，所述血液肿瘤选自白血病和淋巴瘤。
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体或其抗原结合片段。
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五方面所述的嵌合抗原受体、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、第八方面所述的载体、第九方面所述的宿主细胞、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物。在某些实施方案中，所述宿主细胞是免疫细胞(例如人免疫细胞)。
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)将包含本发明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导入步骤(1)所述的免疫细胞，以获得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。
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本发明的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)。
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。
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。
在某些实施方案中，总细胞剂量包含1×107至10×108个CAR阳性免疫细胞，例如包含(1-5)×107至(5-10)×108个CAR阳性免疫细胞。
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。
在某些实施方案中，将本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或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体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或药物组合物之前、同时或之后施用。
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。
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。
在某些实施方案中，所述受试者可以为哺乳动物，例如人。
在另一个方面，提供了本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或药物组合物在制备用于预防和/或治疗肿瘤的药物中的用途。
在另一个方面，提供了本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的嵌合抗原受体、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、第十一方面所述的经改造的免疫细胞或第十二方面所述的免疫细胞组合物、或药物组合物用于预防和/或治疗肿瘤。
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。
缩略词

术语定义
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，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA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。
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。
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。
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)。
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。
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。
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。
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。
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。
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。
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,Science242:423-426(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。
如本文中所使用的，术语“双功能抗体”是指具有两个抗原结合位点的抗体片段，所述片段在同一多肽链(VH-VL)中包含连接到轻链可变结构域(VL)的重链可变结构域(VH)。通过使用过短以使得同一链上的两个结构域之间不能配对的连接子，迫使结构域与另一链的互补结构域配对，并且产生两个抗原结合位点。双功能抗体可以是二价的或双特异性的。双功能抗体更全面描述于例如EP 404,097；WO 1993/01161；Hudson等人,自然医学(Nat.Med.)9:129-134(2003)；和Hollinger等人,PNAS USA 90:6444-6448(1993)中。三功能抗体和四功能抗体也描述于Hudson等人,自然医学9:129-134(2003)中。
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的能力。
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。
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。
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。
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。
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。
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 USA 94:412-417(1997)，其通过引用并入本文)。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
如本文中所使用的，当术语“约”或“大约”与数值变量并用时，通常指该变量的数值在实验误差内(例如对于平均值95％的置信区间内)或在指定数值的±10％或更宽范围内。
发明的有益效果
本发明提供了靶向GPC3的抗体或其抗原结合片段，其可用于制备嵌合抗原受体。表达本发明的嵌合抗原受体的免疫效应细胞具有提高的效应子功能(例如，肿瘤杀伤活性以及释放细胞因子活性)。
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显然。
附图说明
图1显示了CAR-T细胞(CB6-T、AB9-T、CE3-T、CH6-T、P7D4-T、空白T)对靶细胞的杀伤活性检测结果；其中，图1A为靶细胞HepG2的结果，图1B为靶细胞Huh7的结果。
图2A和图2B显示了CAR-T细胞(CB6-T、AB9-T、CE3-T、CH6-T、P7D4-T、空白T)激活后靶细胞HepG2分泌IFN-γ和TNF-α的检测结果。图2C和图2D显示了CAR-T细胞(CB6-T、AB9-T、CE3-T、CH6-T、P7D4-T、空白T)激活后靶细胞Huh7分泌IFN-γ和TNF-α的检测结果。
序列信息
本发明涉及的序列信息提供于下面的表1中。







具体实施方式
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。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。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。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1靶向人GPC3 scFv抗体的制备
1)噬菌体库筛选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。
2)ELISA检测单克隆噬菌体
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。
综上，共筛选得到4株全人源抗GPC3单克隆抗体CB6，AB9，CE3，CH6。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、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)。
表2.抗GPC3单克隆抗体的序列
3)scFv-Fc的构建与抗体聚体分析
将上述全人源抗体的VH和VL通过linker(SEQ ID NO:76)连接，获得scFv，各scFv的序列信息如下表所示。
表3：scFv的结构
将候选scFv序列和阳性对照scFv序列(P7D4)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)，4个候选序列(CB6，AB9，CE3，CH6)单体峰(main peak area)大于90％。
表4. 4个scFv-Fc蛋白SEC数据

4)GPC3细胞结合试验
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P7D4抗体(相关序列参见WO2017020812)作为阳性对照。表5结果表明CB6候选scFvs对GPC3阳性细胞293T/GPC3+的亲和力优于阳性对照组；CH6和AB9候选scFvs对GPC3阳性细胞293T/GPC3+的亲和力接近阳性对照组。
表5.GPC3细胞结合试验结果
实施例2嵌合抗原受体(CAR)慢病毒表达载体的构建及制备
1)慢病毒质粒的构建：
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。
表6.嵌合抗原受体的结构
2)病毒包装：
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(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。
实施例3 CAR-T细胞的制备
1)原代T细胞分离：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。取出磁珠，用移液枪上下吹打至少5次，充分混匀。吸取50μl磁珠/mL至上述样品中，充分混匀后，室温孵育10分钟。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。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。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。
2)T细胞的激活：
调整细胞密度至1×106细胞/mL，六孔板中加入细胞因子及抗体复合物(300U/mL的IL-2、10ng/mL IL-7、5ng/mL IL-15、500ng/mL Anti-CD3(OKT3)、2μg/mL Anti-CD28)，连续培养48小时。
3)病毒感染：
(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。
(2)从-80℃冰箱取出病毒后，迅速在37℃水浴锅中融化。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，使用封口膜将六孔板四边密封，800×g离心1小时。
(3)离心结束后，将六孔板置于37℃5％CO2的培养箱中，继续培养24小时。
(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。
通过上述方法分别获得表达实施例2中所述的CAR(CB6-T、AB9-T、CE3-T和CH6-T)的CAR-T细胞。
实施例4 CAR-T细胞的阳性率检测
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4种表达GPC3-CAR嵌合抗原受体的T细胞(CB6-T、AB9-T、CE3-T和CH6-T)以及对照嵌合抗原受体T细胞(P7D4-T)。
表7：CAR的阳性率检测结果
如表7所示，候选嵌合抗原受体T细胞CB6-T、AB9-T、CE3-T、CH6-T的阳性率都高于P7D4-T对照组，其中AB9-T阳性率最高。
实施例5 CAR-T对HepG2和Huh7靶细胞的杀伤活性评价
使用0.25％胰酶消化HEPG2-luc和Huh7-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/孔的量分别接种靶细胞HEPG2-luc和Huh7-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或Huh7-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。
CAR-T的杀伤活性检测结果如图1A和图1B所示，本申请构建的4种CAR-T细胞(CB6-T、AB9-T、CE3-T、CH6-T)在不同的细胞系(HepG2、Huh7)和不同E/T比例下都能有效裂解肿瘤细胞。在效应细胞/靶细胞比例为1时，本申请构建的CAR-T细胞对肿瘤细胞的裂解率高达约99％。
实施例6 CAR-T与HepG2和Huh7靶细胞共孵育时的细胞因子释放
收集HepG2-luc和Huh7-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(IFN-γ、TNF-α)检测细胞因子释放。本申请构建的4种CAR-T细胞(CB6-T、AB9-T、CE3-T、CH6-T)能够在不同程度上杀伤肿瘤细胞，并释放IFN-γ(以HepG2为靶细胞，检测结果如图2A所示；以Huh7为靶细胞，检测结果如图2C所示)和TNF-α(以HepG2为靶细胞，检测结果如图2B所示；以Huh7为靶细胞，检测结果如图2D所示)。
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。

Claims (23)

  1. 特异性结合GPC3的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含如下的互补决定区(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；
    (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；
    (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；
    (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；或
    (e)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(d)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；
    优选地，所述CDR根据IMGT、Kabat、Chothia或AbM编号系统定义。
  2. 权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (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：
    (1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：9或其变体的CDR-H1；序列为SEQ ID NO：10或其变体的CDR-H2；序列为SEQ ID NO：11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：12或其变 体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：14或其变体的CDR-L3；
    (1b)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：26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：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：29或其变体的CDR-L3；
    (1c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：38或其变体的CDR-H1；序列为SEQ ID NO：39或其变体的CDR-H2；序列为SEQ ID NO：40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：41或其变体的CDR-L1；序列为SEQ ID NO：42或其变体的CDR-L2；序列为SEQ ID NO：43或其变体的CDR-L3；或，
    (1d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：53或其变体的CDR-H1；序列为SEQ ID NO：54或其变体的CDR-H2；序列为SEQ ID NO：55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：56或其变体的CDR-L1；序列为SEQ ID NO：57或其变体的CDR-L2；序列为SEQ ID NO：58或其变体的CDR-L3；
    或，
    (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：
    (2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：15或其变体的CDR-H1；序列为SEQ ID NO：16或其变体的CDR-H2；序列为SEQ ID NO：17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：18或其变体的CDR-L1；序列为SEQ ID NO：19或其变体的CDR-L2；序列为SEQ ID NO：14或其变体的CDR-L3；
    (2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：30或其变体的CDR-H1；序列为SEQ ID NO：31或其变体的CDR-H2；序列为SEQ ID NO：32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：33或其变体的CDR-L1；序列为SEQ ID NO：34或其变体的CDR-L2；序列为SEQ ID NO：29或其变体的CDR-L3；
    (2c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：44或其变体的CDR-H1；序列为SEQ ID NO：45或其变体的CDR-H2；序列为SEQ ID NO：46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：47或其变体的CDR-L1；序列为SEQ ID NO：48或其变体的CDR-L2；序列为SEQ ID NO：43或其变体的CDR-L3；或，
    (2d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：59或其变体的CDR-H1；序列为SEQ ID NO：60或其变体的CDR-H2；序列为SEQ ID NO：61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：62或其变体的CDR-L1；序列为SEQ ID NO：63或其变体的CDR-L2；序列为SEQ ID NO：58或其变体的CDR-L3；
    或，
    (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：
    (3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：20或其变体的CDR-H1；序列为SEQ ID NO：21或其变体的CDR-H2；序列为SEQ ID NO：11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：12或其变体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：14或其变体的CDR-L3；
    (3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：35或其变体的CDR-H1；序列为SEQ ID NO：21或其变体的CDR-H2；序列为SEQ ID NO：26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：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：29或其变体的CDR-L3；
    (3c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：49或其变体的CDR-H1；序列为SEQ ID NO：50或其变体的CDR-H2；序列为SEQ ID NO：40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：41或其变体的CDR-L1；序列为SEQ ID NO：42或其变体的CDR-L2；序列为SEQ ID NO：43或其变体的CDR-L3；或，
    (3d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：64或其变体的CDR-H1；序列为SEQ ID NO：65或其变体的CDR-H2；序列为SEQ ID NO：55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：56或其变体的CDR-L1；序列为SEQ ID NO：57或其变体的CDR-L2；序列为SEQ ID NO：58或其变体的CDR-L3；
    或，
    (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：
    (4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：22或其变体的CDR-H1；序列为SEQ ID NO：23或其变体的CDR-H2；序列为SEQ ID NO：11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：12或其变体的CDR-L1；序列为SEQ ID NO：13或其变体的CDR-L2；序列为SEQ ID NO：14或其变体的CDR-L3；
    (4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：36或其变体的CDR-H1；序列为SEQ ID NO：37或其变体的CDR-H2；序列为SEQ ID NO：26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：27或其变体的CDR-L1；序列为SEQ ID NO：28或其变体的CDR-L2；序列为SEQ ID NO：29或其变体的CDR-L3；
    (4c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：51或其变体的CDR-H1；序列为SEQ ID NO：52或其变体的CDR-H2；序列为SEQ ID NO：40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：41或其变体的CDR-L1；序列为SEQ ID NO：42或其变体的CDR-L2；序列为SEQ ID NO：43或其变体的CDR-L3；或，
    (4d)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：55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：56或其变体的CDR-L1；序列为SEQ ID NO：57或其变体的CDR-L2；序列为SEQ ID NO：58或其变体的CDR-L3；
    其中，(1a)-(1d)、(2a)-(2d)、(3a)-(3d)、(4a)-(4d)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。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (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；
    (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；
    (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；或，
    (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；
    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。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自全长抗体、单链抗体(例如scFv、di-scFv或(scFv)2)、Fab、Fab'、Fab'-SH、F(ab')2、F(ab)'3片段、Fv片段、微型抗体、二硫键连接的Fv(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。
  5. 权利要求1-4任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段是单链抗体，例如scFv、di-scFv或(scFv)2；
    优选地，所述单链抗体从其N端至C端依次包括：
    (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；
    (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；
    (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；
    (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；
    (5)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；
    (6)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；
    (7)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；或，
    (8)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；
    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；
    优选地，所述单链抗体的VH和VL通过连接子连接；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：76的序列。
  6. 权利要求5所述的抗体或其抗原结合片段，其中，所述单链抗体包含选自下列的氨基酸序列：(1)SEQ ID NOs:68、69、70、71任一项所示的氨基酸序列；(2)与SEQ ID NOs:68、69、70、71任一项所示的氨基酸序列相比具有至少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(3)与SEQ ID NOs:68、69、70、71任一项所示的氨基酸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，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。
  7. 权利要求1-6任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结 合片段进一步包含重链恒定区(CH)和轻链恒定区(CL)；
    优选地，所述重链恒定区选自IgG、IgM、IgE、IgD和IgA；
    优选地，所述轻链恒定区选自κ或λ。
  8. 权利要求1-7任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段带有标记；优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  9. 分离的核酸分子，其包含编码权利要求1-8任一项所述的抗体或其抗原结合片段、其重链和/或轻链、或其重链可变区和/或轻链可变区的核苷酸序列。
  10. 权利要求9所述的分离的核酸分子，其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：
    (a)所述编码抗体重链可变区的核酸分子包含：(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；和/或，所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:90所示的核苷酸序列、(v)与SEQ ID NO:90基本上相同的序列(例如，与SEQ ID NO:90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；
    或
    (b)所述编码抗体重链可变区的核酸分子包含：(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；和/或，所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:92所示的核苷酸序列、(v)与SEQ ID NO:92基本上相同的序列(例如，与SEQ ID NO:92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；
    或
    (c)所述编码抗体重链可变区的核酸分子包含：(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；和/或，所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:94所示的核苷酸序列、(v)与SEQ ID NO:94基本上相同的序列(例如，与SEQ ID NO:94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；
    或
    (d)所述编码抗体重链可变区的核酸分子包含：(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；和/或，所述编码抗体轻链可变区的核酸分子包含：(iv)SEQ ID NO:96所示的核苷酸序列、(v)与SEQ ID NO:96基本上相同的序列(例如，与SEQ ID NO:96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。
  11. 权利要求9或10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：72、73、74和75任一项所示的核苷酸序列；(2)与SEQ ID NO：72、73、74和75任一项所示的核苷酸序列相比具有至少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。
  12. 载体，其包含权利要求9-11任一项所述的分离的核酸分子；
    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；
    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。
  13. 宿主细胞，其包含权利要求9-11任一项所述的分离的核酸分子，或权利要求12所述的载体。
  14. 制备权利要求1-8任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求13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体或其抗原结合片段。
  15. 偶联物，其包括权利要求1-8任一项所述的抗体或其抗原结合片段以及与其连接的偶联部分；
    优选地，所述偶联部分选自可检测标记(如放射性同位素、荧光物质、发光物质、有色物质或酶)或治疗剂(如细胞毒剂、细胞因子、毒素或放射性核素)。
  16. 多特异性抗体，其包含权利要求1-8任一项所述的抗体或其抗原结合片段；
    优选地，所述多特异性抗体包含权利要求1-8任一项所述的抗体或其抗原结合片段作为第一抗原结合结构域，并且还包含至少一种针对其他靶标的第二抗原结合结构域；
    优选的，所述多特异性抗体为双特异性抗体或三特异性抗体或四特异性抗体。
  17. 药物组合物，其含有权利要求1-8任一项所述的抗体或其抗原结合片段、或权利要求9-11任一项所述的分离的核酸分子、或权利要求12所述的载体、或权利要求13所述的宿主细胞、或权利要求15所述的偶联物、或权利要求16所述的多特异性抗体，以及药学上可接受的载体和/或赋形剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物；
    优选地，所述另外的药学活性剂选自：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、FOLFIRINOX或其任意组合；
    优选地，所述抗体或其抗原结合片段与所述另外的药学活性剂作为独立的组分或作为混合的组分提供。
  18. 试剂盒，所述试剂盒包括权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9-11任一项所述的分离的核酸分子，或权利要求12所述的载体，或权利要求13所述的宿主细胞，或权利要求15所述的偶联物，或权利要求16所述的多特异性抗体，或权利要求17所述的药物组合物，以及可选地使用说明书和/或给药装置。
  19. 权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9-11任一项所述的分离的核酸分子，或权利要求12所述的载体，或权利要求13所述的宿主细胞，或权利要求15所述的偶联物，或权利要求16所述的多特异性抗体，或权利要求17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；
    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；
    优选地，所述肿瘤是GPC3阳性肿瘤；
    优选地，所述肿瘤选自实体瘤；优选地，所述实体瘤选自肝癌(例如，肝细胞癌)、黑色素瘤、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、结直肠癌、胃癌、神经胶质瘤和卵巢癌(例如，卵巢透明细胞癌)；
    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和淋巴瘤；
    优选地，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、嵌合抗原受体或表达所述嵌合抗原受体的宿主细胞、偶联物、多特异性抗体、或药物组合物与另外的药学活性剂联合施用，例如同时、分开或相继施用；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物；
    优选地，所述另外的药学活性剂选自：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、FOLFIRINOX或其任意组合。
  20. 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权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9-11任一项所述的分离的核酸分子，或权利要求12所述的载体，或权利要求13所述的宿主细胞，或权利要求15所述的偶联物，或权利要求16所述的多特异性抗体，或权利要求17所述的药物组合物；
    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；
    优选地，所述肿瘤是GPC3阳性肿瘤；
    优选地，所述肿瘤选自实体瘤；优选地，所述实体瘤选自肝癌(例如，肝细胞癌)、黑色素瘤、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、结直肠癌、胃癌、神经胶质瘤和卵巢癌(例如，卵巢透明细胞癌)；
    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和淋巴瘤；
    优选地，所述方法还包括向所述受试者施用第二疗法，所述第二疗法选自手术、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。
  21. 一种检测样品中GPC3存在或其水平的方法，其包括在允许所述抗体或其抗原结合片段与GPC3之间形成复合物的条件下，使所述样品与权利要求1-8任一项所述的抗体或其抗原结合片段接触，和检测所述复合物的形成；
    优选地，所述方法用于诊断肿瘤，例如GPC3阳性肿瘤，例如肝癌(例如，肝细胞癌)、黑色素瘤、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、结直肠癌、胃癌、神经胶质瘤和卵巢癌(例如，卵巢透明细胞癌)、白血病、淋巴瘤或其任意组合；
    优选地，所述方法包括检测来自受试者的待测样品中GPC3的表达水平，和将该表达水平与参考值相比较，其中与参考值相比表达水平的提高是肿瘤的指征。
  22. 权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9-11任一项所述的分离的核酸分子，或权利要求12所述的载体，或权利要求13所述的宿主细胞，或权利要求15所述的偶联物，或权利要求16所述的多特异性抗体在制备检测试剂盒中的用途，所述试剂盒用于检测样品中GPC3存在或其水平和/或诊断肿瘤；
    优选地，所述肿瘤为GPC3阳性肿瘤；
    优选地，所述肿瘤选自肝癌(例如，肝细胞癌)、黑色素瘤、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、结直肠癌、胃癌、神经胶质瘤和卵巢癌(例如，卵巢透明细胞癌)、白血病、淋巴瘤或其任意组合。
  23. 权利要求1-8任一项所述的抗体或其抗原结合片段、或权利要求9-11任一项所述的分离的核酸分子，或权利要求12所述的载体，或权利要求13所述的宿主细胞，或权利要求15所述的偶联物，或权利要求16所述的多特异性抗体，或权利要求17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；
    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；
    优选地，所述肿瘤是GPC3阳性肿瘤；
    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；
    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。
PCT/CN2023/132296 2022-11-28 2023-11-17 靶向gpc3的抗体及其用途 WO2024114410A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202380069639.2A CN119968398A (zh) 2022-11-28 2023-11-17 靶向gpc3的抗体及其用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211500068.7 2022-11-28
CN202211500068 2022-11-28

Publications (1)

Publication Number Publication Date
WO2024114410A1 true WO2024114410A1 (zh) 2024-06-06

Family

ID=91322964

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/132296 WO2024114410A1 (zh) 2022-11-28 2023-11-17 靶向gpc3的抗体及其用途

Country Status (2)

Country Link
CN (1) CN119968398A (zh)
WO (1) WO2024114410A1 (zh)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103596985A (zh) * 2011-04-19 2014-02-19 美国政府(由卫生和人类服务部的部长所代表) 对磷脂酰肌醇蛋白聚糖3特异的人单克隆抗体及其用途
WO2017196847A1 (en) * 2016-05-10 2017-11-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Variable new antigen receptor (vnar) antibodies and antibody conjugates targeting tumor and viral antigens
CN109988240A (zh) * 2017-12-29 2019-07-09 安源生物科技(上海)有限公司 抗gpc-3抗体及其用途
CN111712520A (zh) * 2018-02-15 2020-09-25 西雅图基因公司 磷脂酰肌醇聚糖3抗体及其偶联物
CN112175085A (zh) * 2019-06-14 2021-01-05 厦门大学 抗gpc3抗体及其用途
WO2022002112A1 (en) * 2020-07-01 2022-01-06 Shandong Boan Biotechnology Co., Ltd. Anti-gpc3 antibody, anti-gpc3 chimeric antigen receptor and gpc3/cd3 bispecific antibody
WO2022121969A1 (zh) * 2020-12-10 2022-06-16 江苏先声药业有限公司 Gpc3抗体及其应用
WO2022171100A1 (zh) * 2021-02-10 2022-08-18 江苏先声药业有限公司 Gpc3人源化抗体及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103596985A (zh) * 2011-04-19 2014-02-19 美国政府(由卫生和人类服务部的部长所代表) 对磷脂酰肌醇蛋白聚糖3特异的人单克隆抗体及其用途
WO2017196847A1 (en) * 2016-05-10 2017-11-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Variable new antigen receptor (vnar) antibodies and antibody conjugates targeting tumor and viral antigens
CN109988240A (zh) * 2017-12-29 2019-07-09 安源生物科技(上海)有限公司 抗gpc-3抗体及其用途
CN111712520A (zh) * 2018-02-15 2020-09-25 西雅图基因公司 磷脂酰肌醇聚糖3抗体及其偶联物
CN112175085A (zh) * 2019-06-14 2021-01-05 厦门大学 抗gpc3抗体及其用途
WO2022002112A1 (en) * 2020-07-01 2022-01-06 Shandong Boan Biotechnology Co., Ltd. Anti-gpc3 antibody, anti-gpc3 chimeric antigen receptor and gpc3/cd3 bispecific antibody
WO2022121969A1 (zh) * 2020-12-10 2022-06-16 江苏先声药业有限公司 Gpc3抗体及其应用
WO2022171100A1 (zh) * 2021-02-10 2022-08-18 江苏先声药业有限公司 Gpc3人源化抗体及其应用

Also Published As

Publication number Publication date
CN119968398A (zh) 2025-05-09

Similar Documents

Publication Publication Date Title
KR102710963B1 (ko) 항b7-h3의 모노클로널 항체 및 그가 세포 치료 중에서의 응용
US12258418B2 (en) Claudin18.2 binding moieties and uses thereof
JP7425604B2 (ja) 抗ｃｔｌａ４－抗ｐｄ－１二機能性抗体、その医薬組成物および使用
JP7510246B2 (ja) 抗グリピカン３抗体およびその使用
CN114401989B (zh) 靶向bcma的抗体及嵌合抗原受体
WO2022078286A1 (zh) 特异性结合msln的嵌合抗原受体及其应用
CN108373504A (zh) Cd24特异性抗体和抗cd24-car-t细胞
CN115232209B (zh) 靶向gprc5d的抗体及其用途
CN113754780A (zh) 靶向cldn18.2的嵌合抗原受体、其组合物及用途
CN110799546B (zh) 重组双特异性抗体
CN111454358A (zh) 一种嵌合抗原受体及其应用
US20230357385A1 (en) Anti-gpc3 antibody, anti-gpc3 chimeric antigen receptor and gpc3/cd3 bispecific antibody
JP2021509820A (ja) 線維芽細胞活性化タンパク質αを標的とする結合ユニットとその応用
CN114437218A (zh) 靶向cd276的嵌合抗原受体以及包含其的免疫细胞
EP4421095A1 (en) Anti-mesothelin nanobodies and use thereof
KR20240046224A (ko) 이중특이성 항체 및 그 용도
CN109970859B (zh) Glypican-3特异性抗体及其特异性CAR-T细胞
WO2024114410A1 (zh) 靶向gpc3的抗体及其用途
WO2023045370A1 (zh) 靶向tigit的单克隆抗体
WO2023246574A1 (zh) 靶向gpc3的抗体及其用途
WO2024114404A1 (zh) 特异性结合gpc3的嵌合抗原受体及其应用
WO2023246578A1 (zh) 特异性结合gpc3的嵌合抗原受体及其应用
CN118234755A (zh) 特异性结合msln的嵌合抗原受体及其应用
CN114478767A (zh) 特异性结合cd33的嵌合抗原受体及其应用
WO2023274355A1 (zh) 改造的间充质干细胞和免疫效应细胞联合治疗肿瘤

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23896562

Country of ref document: EP

Kind code of ref document: A1