WO2024114404A1 - Chimeric antigen receptor specifically binding to gpc3 and use thereof - Google Patents

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

Info

Publication number
WO2024114404A1
WO2024114404A1 PCT/CN2023/132244 CN2023132244W WO2024114404A1 WO 2024114404 A1 WO2024114404 A1 WO 2024114404A1 CN 2023132244 W CN2023132244 W CN 2023132244W WO 2024114404 A1 WO2024114404 A1 WO 2024114404A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
cdr
variant
variable region
Prior art date
Application number
PCT/CN2023/132244
Other languages
French (fr)
Chinese (zh)
Inventor
蔡珍珍
侯攀燕
周阳
葛均友
卫立辛
Original Assignee
成都科伦精准生物科技有限公司
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 成都科伦精准生物科技有限公司 filed Critical 成都科伦精准生物科技有限公司
Publication of WO2024114404A1 publication Critical patent/WO2024114404A1/en

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine, and in particular, the present invention relates to single-chain antibodies and chimeric antigen receptors (CARs) that specifically bind to GPC3.
  • CARs chimeric antigen receptors
  • the present invention also relates to immune cells expressing the CAR, nucleic acid molecules encoding such CARs or co-expressed molecules, and methods for preparing the modified immune cells.
  • the present invention also relates to methods for using these CARs and immune cells to prevent and/or treat GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma.
  • HCC hepatocellular carcinoma
  • melanoma melanoma
  • ovarian clear cell carcinoma GPC3-positive tumors
  • Glypican-3 (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.
  • a large number of patients with hepatocellular carcinoma still lack accurate and effective clinical treatment methods.
  • Most patients with liver cancer are already in the advanced or advanced stage when diagnosed. Only 30% of patients have the opportunity for surgical resection.
  • the metastasis and recurrence rate within 5 years after resection is as high as 60% to 70%, and the overall 5-year survival rate is low, only 7% to 10%.
  • Chimeric antigen receptor (CAR)-T cell therapy is considered to be one of the most promising cancer treatment methods and has become a new hope for human beings to fight cancer. It cultivates immune cells collected from patients in vitro, transduces specific exogenous genes in vitro, and then amplifies them in vitro and then infuses them back into the patient's body to achieve the purpose of treating tumors in a non-MHC restricted manner.
  • CAR-T cell therapy has achieved remarkable therapeutic effects in the treatment of hematological malignancies, with a complete remission rate of over 90% for relapsed and refractory B-cell leukemia. Solid tumors account for about 90% of all malignant tumors, and there is a large demand for therapeutic drugs. However, the therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly because solid tumors have complex tumor microenvironments and high tumor heterogeneity.
  • the present invention provides a CAR targeting GPC3 or a modified immune cell co-expressing a CAR targeting GPC3.
  • the modified immune cell can be used for the treatment of GPC3-positive hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma, etc.
  • HCC hepatocellular carcinoma
  • melanoma melanoma
  • ovarian clear cell carcinoma etc.
  • the inventors first developed a fully human antibody with low immunogenicity that can specifically recognize/bind to GPC3.
  • the present invention further designs and constructs a CAR targeting GPC3.
  • the CAR of the present invention can direct the specificity and reactivity of immune effector cells to cells expressing GPC3 (such as hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma) in a non-MHC restricted manner so that they can be eliminated. Therefore, the CAR targeting GPC3 of the present invention has the potential to be used for the prevention and/or treatment of GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma, and has great clinical value.
  • HCC hepatocellular carcinoma
  • melanoma and ovarian clear cell carcinoma a non-MHC restricted manner
  • the substitutions are conservative substitutions.
  • the CDRs are defined according to the Kabat, IMGT, Chothia, or AbM numbering systems.
  • the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:
  • VH heavy chain variable region
  • CDR-H2 having a sequence of SEQ ID NO: 10 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: 25 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
  • 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; preferably, the substitutions are conservative substitutions.
  • the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:
  • VH heavy chain variable region
  • 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 having a sequence of SEQ ID NO: 31 or a variant thereof
  • CDR-H3 having 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; preferably, The substitutions described are conservative substitutions.
  • the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:
  • VH heavy chain variable region
  • CDR-H2 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; preferably, the substitutions are conservative substitutions.
  • the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VH).
  • VH heavy chain variable region
  • VH light chain variable region
  • VL Chain variable region
  • 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
  • CDR-H2 having a sequence of SEQ ID NO: 67 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 (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 antigen binding molecules of the invention comprise:
  • 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; preferably, the substitutions are conservative substitutions.
  • the antigen binding molecule described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.
  • the heavy chain of the antigen binding molecule comprises a heavy chain constant region from or derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4).
  • the heavy chain of the antigen binding molecule comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region, which has an effector function (e.g., enhanced ADCC activity) that is changed compared to a wild-type Fc region.
  • the light chain of the antigen binding molecule comprises a light chain constant region from or derived from a human immunoglobulin (eg, kappa or lambda).
  • a human immunoglobulin eg, kappa or lambda
  • the antigen binding molecule described in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the antigen binding molecule is selected from a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab)' 2 fragment, a F(ab)' 3 fragment, a single-chain antibody (e.g., scFv, di-scFv or (scFv) 2 ), a miniantibody, a disulfide-stabilized Fv protein (dsFv) and a single domain antibody (sdAb, nanobody).
  • a single-chain antibody e.g., scFv, di-scFv or (scFv) 2
  • dsFv disulfide-stabilized Fv protein
  • sdAb single domain antibody
  • the VH and VL of the antigen binding molecules of the invention are linked by one or more linkers.
  • the linker is typically a peptide linker, 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 antigen binding molecules 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 a sequence as shown in SEQ ID NO: 1 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof; or, a 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;
  • VH-linker comprising a sequence as shown in SEQ ID NO: 3 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 4 or a variant thereof; or, a 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;
  • VH-linker comprising a sequence as shown in SEQ ID NO: 5 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 6 or a variant thereof; or, a 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;
  • VH-linker comprising a sequence as shown in SEQ ID NO: 7 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 8 or a variant thereof; or, a 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, 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 single-chain antibody comprises a single-chain antibody as shown in any one of SEQ ID NOs: 68, 69, 70, 71.
  • the antigen binding molecules of the present invention further comprise a constant region derived from human immunoglobulin.
  • the heavy chain of the antigen binding molecules comprises a heavy chain constant region derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antigen binding molecules comprises a light chain constant region derived from human immunoglobulin (e.g., ⁇ or ⁇ ).
  • the heavy chain of the antigen binding molecule 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 antigen-binding molecule 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., 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.
  • 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 antigen binding molecules of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, a DNA molecule encoding the antigen binding molecule is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and then transfected into a host cell. Then, the host cell after transfection is cultured under specific conditions, and the antigen binding molecules of the present invention are expressed.
  • 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 these antigen-binding fragments are fully known to those skilled in the art.
  • the second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antigen binding molecule of the present invention.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:89, (ii) a sequence substantially identical to SEQ ID NO:89 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:89, 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:90, (v) a sequence substantially identical to SEQ ID NO:90 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:90, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:91, (ii) a sequence substantially identical to SEQ ID NO:91 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to 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 higher sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:92), or (vi) a degenerate sequence of the above (iv) or (v).
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:93, (ii) a sequence substantially identical to SEQ ID NO:93 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:93, 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:94, (v) a sequence substantially identical to SEQ ID NO:94 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO:94, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
  • the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
  • the nucleic acid molecule encoding the antibody heavy chain variable region 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 higher sequence identity compared to SEQ ID NO:95, 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:96, (v) a sequence substantially identical to SEQ ID NO:96 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO:96, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
  • the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:72, (ii) a sequence substantially identical to SEQ ID NO:72 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:72, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
  • the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:73, (ii) a sequence substantially identical to SEQ ID NO:73 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:73, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
  • the isolated nucleic acid molecule comprises: (i) a nucleotide sequence as shown in SEQ ID NO: 74;
  • the invention relates to a sequence of the invention that is a sequence of the invention that is substantially identical to SEQ ID NO:74 (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:74), or (iii) a degenerate sequence of (i) or (ii) above.
  • the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:75, (ii) a sequence substantially identical to SEQ ID NO:75 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:75, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
  • the third aspect of the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above.
  • the vector of the present invention is, for example, a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector or a viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.
  • the fourth aspect of the present invention provides a host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention.
  • the host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli).
  • Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).
  • the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T).
  • the isolated nucleic acid molecule contained in the host cell may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antigen binding molecule (eg, ScFv) of the present invention.
  • the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen binding molecule (eg, scFv) of the present invention.
  • the present invention also relates to a method for preparing the antigen-binding molecule of the present invention, comprising culturing the host cell as described above under conditions allowing protein expression, and recovering the antigen-binding molecule 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, which comprises an antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain.
  • the antigen binding domain contained in the chimeric antigen receptor of the present invention confers the ability of the CAR to recognize GPC3.
  • the antigen binding domain comprises the antigen binding molecule described in the first aspect.
  • the antigen binding domain comprises the antigen binding molecule as a first antigen binding domain, and further comprises a second antigen binding domain that does not bind to GPC3.
  • the antigen bound by the second antigen binding domain is selected from: PD-1, PD-L1, CTLA-, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
  • the antigen binding domain is a single chain antibody.
  • the first antigen binding domain comprises the sequence shown in any one of SEQ ID NO:68,69,70,71 or a variant thereof, wherein the variant has a sequence that is 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% identical to any one of SEQ ID NO:68,69,70,71, or has 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); preferably, the substitutions are conservative substitutions.
  • 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 a transmembrane region selected from the group consisting of the ⁇ , ⁇ or ⁇ chains of T cell receptors, CD28, CD45, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1, and any combination thereof.
  • the transmembrane domain is a transmembrane region selected from the following proteins: CD8, CD28, CD4, PD-1, CD152 and CD154.
  • the transmembrane domain comprises a CD8 transmembrane region whose sequence is 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 of a naturally occurring protein or a portion thereof.
  • the spacer domain is selected from a hinge domain and/or a CH2 and CH3 region of an immunoglobulin (e.g., IgG1 or IgG4).
  • the hinge domain comprises a hinge region of CD8, IgG4, PD-1, CD152, or CD154.
  • the hinge domain comprises a CD8 hinge region having a sequence as 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 can also be co-expressed with another biologically active molecule.
  • the other biologically active molecule can have its own signal peptide, which is named signal peptide to distinguish it from the signal peptide in the previous paragraph.
  • the signal peptide-2 guides the transport of other biologically active molecules to a specific site in the cell or outside the cell membrane.
  • the signal peptide-2 may be the same as or different from the signal peptide described in the previous paragraph.
  • the signal peptide-2 may be different from the signal peptide described in the previous paragraph.
  • the signal peptide-2 is an IL2 signal peptide (e.g., the amino acid sequence is 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 selected from the following proteins: 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(4-1BB).
  • the intracellular signaling domain of CD3 ⁇ includes the amino acid sequence shown in SEQ ID NO:79.
  • the intracellular signaling domain of CD137(4-1BB) 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 capable of specifically binding 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 and a primary signaling domain from the N-terminus to the C-terminus.
  • the spacer domain comprises the hinge region of CD8 (e.g., CD8 ⁇ ) (e.g., the hinge region whose sequence is shown in SEQ ID NO:78).
  • the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8 ⁇ ) (e.g., a transmembrane region whose sequence is shown in SEQ ID NO:77).
  • CD8 e.g., CD8 ⁇
  • SEQ ID NO:77 a transmembrane region whose sequence is shown in SEQ ID NO:77.
  • the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3 ⁇ (e.g., the sequence shown in SEQ ID NO:79), and the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO:80); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:81.
  • 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.
  • the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8 ⁇ signal peptide (e.g., a signal peptide having a sequence as shown in SEQ ID NO: 82).
  • the CAR of the present invention comprises a sequence as shown in any one of SEQ ID NO: 85, 86, 87, 88, or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity 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; preferably, the substitution is a conservative substitution.
  • the CAR described in the fifth aspect of the present invention can also be co-expressed with other biologically active molecules.
  • the self-cleaving peptide can prevent the amino acids from forming covalent bonds during the translation process and maintain the translation to continue, so that the translation product is "self-cut", thereby separating the chimeric antigen receptor of the present invention from other biologically active molecules.
  • the chimeric antigen receptor that can specifically bind to GPC3 becomes an independent CAR having an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, while other biologically active molecules can be secreted outside the cell or expressed as membrane-forming chimeric polypeptides or proteins.
  • the immune cells expressing CAR amplify and enrich in the tumor microenvironment
  • other biologically active molecules are enriched in the tumor microenvironment and synergize with CAR to exert anti-tumor effects.
  • the nucleic acid sequence encoding CAR is connected to the nucleic acid sequence of another biologically active molecule through the nucleic acid sequence of a self-cleaving peptide.
  • CAR can be at the N-terminus or C-terminus of another biologically active molecule.
  • CAR is at the 5' end of another biologically active molecule.
  • Any self-cleaving peptide that can cause the fusion protein to cleave into two independent proteins can be applied to the present invention.
  • the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 130, and its nucleotide sequence can be optimized according to the needs of genetic recombination.
  • the fusion protein comprising CAR and another biologically active molecule has the following structure:
  • N'-signal peptide--extracellular antigen binding domain that specifically binds to GPC3--spacer domain--transmembrane domain--intracellular signal transduction domain--self-cleaving peptide--signal peptide-2--another biologically active molecule-C', wherein the signal peptide-2 is the same as or different from the N-terminal signal peptide.
  • the signal peptide-2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (e.g., as shown in SEQ ID NO:84).
  • the method of generating a chimeric antigen receptor and an immune effector cell (eg, T cell) comprising the chimeric antigen receptor is known in the art, and may include transfecting cells with at least one polynucleotide encoding CAR, and expressing the polynucleotide in the cell.
  • the nucleic acid molecule encoding the CAR of the present invention may be included in an expression vector (eg, 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 according to the fifth aspect.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.
  • the nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect is selected from: (1) a sequence shown in any one of SEQ ID NOs: 72, 73, 74, 75, or a degenerate variant thereof; (2) a sequence substantially identical to the sequence described in (1), for example, 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 compared to (1), or a sequence having one or more nucleotide substitutions compared to the sequence described in (1); and the sequence substantially retains at least one biological activity of the nucleotide sequence from which it is derived (for example, being able to encode the ability to direct the specificity and reactivity of immune effect
  • the CAR of the present invention can also be co-expressed with other biologically active molecules to synergistically exert anti-tumor effects.
  • the seventh aspect of the present invention also provides a nucleic acid construct, which comprises a first nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect, and further comprises a second nucleic acid sequence encoding another biologically active molecule.
  • the additional biologically active molecule encoded by the second nucleic acid sequence has anti-tumor activity.
  • the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide-2 at its N-terminus.
  • the first nucleotide sequence is located upstream of the second nucleotide sequence.
  • the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof).
  • a self-cleaving peptide e.g., P2A, E2A, F2A, T2A or any combination thereof.
  • the sequence encoding the self-cleaving peptide is connected to the 3' end of the first nucleotide sequence and to the 5' end of the second nucleotide sequence.
  • the self-cleaving peptide is P2A (e.g., as shown in SEQ ID NO:83).
  • the nucleic acid construct of the seventh aspect comprises, from its 5' end to its 3' end, in order: a nucleotide sequence encoding the signal peptide, a nucleotide sequence encoding the antigen binding domain, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, a nucleotide sequence encoding the signal peptide-2 Acid sequences, nucleotide sequences encoding other biologically active molecules.
  • the eighth aspect of the present invention provides a vector comprising the isolated nucleic acid molecule described in the second aspect or 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.
  • 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 a chimeric antigen receptor of the present invention, comprising: (1) providing a host cell; (2) introducing the isolated nucleic acid molecule according to the sixth aspect or a vector comprising the nucleic acid molecule into a host cell;
  • the invention also provides a method for producing a cell that co-expresses 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 of the seventh aspect or a vector comprising the nucleic acid construct into the host cell to obtain a host cell that co-expresses 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 host cells are provided from a patient or a healthy donor and are pretreated; the pretreatment includes sorting, activation and/or proliferation of 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 host cell by viral infection. In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host 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: amplifying the host cells obtained in step (2).
  • the eleventh aspect of the present invention further provides a modified immune cell, which comprises and expresses the isolated nucleic acid molecule described in the second aspect and the sixth aspect of the present invention.
  • the modified immune cell expresses the chimeric antigen receptor described in the fifth aspect.
  • the twelfth aspect of the present invention further provides a modified immune cell, which comprises and expresses the nucleic acid construct of the seventh aspect of the present invention.
  • the modified immune cell expresses the chimeric antigen receptor of the fifth aspect and the additional biologically active molecule.
  • the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are obtained from patients; alternatively, the immune cells are obtained from healthy donors; preferably, the immune cells are derived from T lymphocytes or NK cells.
  • the engineered immune cells express genes related to immune rejection (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) is inhibited; preferably, the transcription or expression of the target gene 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 a method for preparing modified immune cells, which comprises: (1) providing immune cells from a patient or a healthy donor; (2) introducing the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1) to obtain immune cells capable of expressing and optionally other biologically active molecules.
  • the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of the immune cells; more preferably, 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 cells by viral infection.
  • the nucleic acid molecule or vector in step (2), is introduced into the immune cell by non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.
  • non-viral vector transfection such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.
  • the method further comprises the step of expanding the immune cells obtained in step (2) after step (2).
  • the present invention also provides an immune cell composition, which includes the modified immune cells of any of the foregoing aspects, and optional unmodified and/or unsuccessfully modified immune cells, which do not express the target CAR.
  • an immune cell composition which includes the modified immune cells of any of the foregoing aspects, and optional unmodified and/or unsuccessfully modified immune cells, which do not express the target CAR.
  • the engineered immune cells expressing the target CAR account for approximately 10%-100%, preferably 40%-80% of the total cell number of the immune cell composition.
  • the immune cell composition is cultured into an immune cell line. Therefore, in another aspect, the present invention also provides an immune cell line containing the immune cell composition.
  • the present invention provides a pharmaceutical composition, which contains the antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which a chimeric antigen receptor is 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 twelfth aspect, or the immune cell composition described in the thirteenth 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 pharmaceutical composition of the present invention comprises: the antigen binding molecule described in the first aspect.
  • the pharmaceutical composition of the present invention comprises: the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, or a vector comprising the same.
  • the pharmaceutical composition of the present invention comprises: the modified immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect.
  • the host cell described in the first or ninth aspect, the modified immune cell described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical 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 antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including the CAR construct co-expressed by the chimeric antigen receptor and another bioactive molecule), the nucleic acid molecules 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 transformed immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, leaf sheath, endocytoplasmic reticulum groove, groin, bladder, local (such as, powder, ointment or drops), or nasal route.
  • parenteral administration e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection. It should be understood by the technician that the route of administration and/or mode will change according to the intended purpose.
  • the pharmaceutical composition of the present invention may include a "therapeutically effective amount” or a "prophylactically effective amount” of the antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct co-expressing a chimeric antigen receptor and another biologically active molecule), 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 twelfth aspect, or the immune cell of the thirteenth aspect. Immune cell composition.
  • Preventive 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 patients with a disease.
  • the antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressed with a chimeric antigen receptor and 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 twelfth aspect, or the therapeutically effective amount of the immune cell composition described in the thirteenth aspect may vary according to the following factors: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the
  • 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 antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), 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 twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect.
  • a subject e.g., a human
  • the method comprising administering to a subject in need thereof an effective amount of the antigen binding molecule of the first aspect of the present invention, the chimeric
  • 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 (e.g., liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma, one or a combination thereof.
  • the tumor is selected from blood tumors (e.g., leukemia, lymphoma, etc.).
  • the method comprises administering to the subject an effective amount of the antigen binding molecule of the first aspect.
  • the method comprises administering to the subject an effective amount of the engineered immune cell of the eleventh or twelfth aspect, or the immune cell composition of the thirteenth aspect.
  • 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 second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1); (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 second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1)
  • administering the immune cells obtained in step (2) to the subject for treatment.
  • the method administers immune cells expressing a CAR of interest to the subject by dividing the dose into fractions, e.g., one, two, three or more times, administering a partial dose separately, 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.
  • a partial dose separately, 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.
  • 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) treatment day.
  • 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, and 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 target CAR-positive immune cells, for example, comprises (1-5) ⁇ 10 7 to (5-10) ⁇ 10 8 target 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 other agents include (i) increasing cells containing CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, modified immune cells of the present invention). (ii) an agent for improving the efficacy of cells (e.g., immune cells expressing the CAR of the present invention, modified immune cells or immune cell compositions) comprising CAR nucleic acids or CAR polypeptides; (iii) another pharmaceutically active agent with anti-tumor activity.
  • CAR nucleic acids or CAR polypeptides e.g., immune cells expressing CAR of the present invention, modified immune cells of the present invention.
  • an agent for improving the efficacy of cells e.g., immune cells expressing the CAR of the present invention, modified immune cells or immune cell compositions
  • another pharmaceutically active agent with anti-tumor activity e.g., another pharmaceutically active agent with anti-tumor activity.
  • agents can be administered before, simultaneously or after the antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a chimeric antigen receptor and a CAR construct co-expressed with another biologically active molecule), the isolated nucleic acid molecules 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 twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect.
  • 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 antigen binding molecule of the first aspect of the present invention the chimeric antigen receptor of the fifth aspect (including the CAR construct co-expressing the chimeric antigen receptor and another biologically active molecule), 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 twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect are provided for the preparation of a drug for preventing and/or treating a disease associated with the expression of GPC3.
  • the dosage, dosage form, administration route, indication, combination therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.
  • the present invention provides the antigen binding molecule of the first aspect, the chimeric antigen receptor of the fifth aspect (including the CAR construct co-expressing the chimeric antigen receptor and another biologically active molecule), 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 twelfth aspect, or the immune cell composition of the thirteenth aspect, or the drug of the fifteenth aspect.
  • the drug composition is used for preventing and/or treating diseases related to the expression of GPC3.
  • the dosage, dosage form, administration route, indications, combined therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.
  • antigen binding molecule is an antibody molecule or an antigen binding fragment thereof.
  • antibody refers to an immunoglobulin molecule that can specifically bind to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • 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 specific 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 specific 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 antibodies can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form an antigen binding site, respectively.
  • the allocation of amino acids to each region or domain can follow the definitions 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 According to the definition in 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.
  • 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 bound by the full-length antibody 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 ed., 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 techniques 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 , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (“dsFv”) and single domain antibodies (sdAb, nanobodies) and polypeptides that contain 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 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, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • 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.
  • a disulfide bond may also be present between the VH and VL of the scFv.
  • the VH and VL domains may be positioned relative to each other in any suitable arrangement.
  • scFv comprising NH 2 -VH-VH-COOH, NH 2- VL-VL-COOH.
  • the scFv can form any possible engineering 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.
  • single-domain antibody has the meaning generally understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody.
  • sdAb single-domain antibody
  • Single domain antibodies are also called nanobodies.
  • 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 “specifically directed against” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against 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 refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the 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 speed of formation and dissociation of antigen binding sites/antigen complexes.
  • association rate constants ka or kon
  • dissociation rate constants kdis or koff
  • K D dissociation constant
  • 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 using 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 two compared sequences is occupied by the same base or amino acid monomer subunit (for example, 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 ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (a total of 100). 6 positions match).
  • the two sequences are compared when aligned for maximum identity.
  • Such an alignment can be achieved 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. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol 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 either the Blossum 62 matrix or the 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 of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues 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 acids with basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • 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, histidine
  • 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 broadly used to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes a virally derived nucleic acid element that typically facilitates transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer.
  • the 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 LTRs).
  • the terms "lentiviral vector”, “lentiviral expression vector” may be used to refer to a lentiviral transfer plasmid and/or an infectious lentiviral particle.
  • elements e.g., cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.
  • an "integration-defective" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that cannot integrate the viral genome into the genome of a host cell.
  • the integrase protein is mutated to specifically reduce its integrase activity.
  • An integration-defective lentiviral vector can be obtained by modifying the pol gene encoding the integrase protein to produce a mutant pol gene encoding an integration-defective integrase.
  • the integration-defective viral vector has been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.
  • the term "host cell” refers to a cell that can be used to introduce a vector, 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 antibody-based specificity for a target antigen (e.g., GPC3) with an immune effector cell activation intracellular domain to exhibit specific immune activity for cells expressing the target antigen (e.g., GPC3).
  • a target antigen e.g., GPC3
  • an immune effector cell activation intracellular domain 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 incorporated herein by reference in their 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 the portion of a protein that transmits effector signaling function signals and directs cells to perform specialized functions.
  • the effector function of the T cell can be cytolytic activity or helper 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 the subject and the 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, 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 generally understood by those skilled in the art, which are capable of stabilizing 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
  • the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • such sterile injectable liquids are 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., a 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 surfactant e.g., 0.01% polysorbate 20
  • a pH buffer solution e.g., a phosphate buffer solution
  • Ringer's solution e.g., Ringer's solution, and any combination thereof.
  • 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. For example, 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 gender, 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 xenogeneic).
  • 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
  • xenogeneic refers to cells from a different subject that are genetically identical to the comparison cells
  • xenogeneic refers to cells from a different subject that are genetically identical to the comparison cells
  • “of” refers to cells that are from a different species than the comparison cells.
  • the cells of the invention are allogeneic.
  • T lymphocytes and/or NK cells.
  • T cell or “T lymphocyte” is 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 expressing any CAR described herein, or an immune cell into which any isolated nucleic acid or vector described herein is introduced.
  • the CAR polypeptide can also be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods 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 a polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell.
  • a virus-mediated method can be used.
  • Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc.
  • Transient transformation methods include, for example, but not limited to microinjection, electroporation, or microparticle bombardment.
  • Polynucleotides 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 therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly because solid tumors have complex tumor microenvironments and high tumor heterogeneity.
  • the present invention provides a CAR targeting GPC3 or an immune cell comprising the CAR, which improves the killing of cells expressing tumor antigens and reduces their off-target toxicity to a certain extent by specifically targeting GPC3, thereby enhancing the tumor killing effect of CAR-T cells.
  • Figure 1A shows the results of the killing activity test of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T) against HepG2 target cells.
  • Figure 1B shows the results of the killing activity test of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T) against Huh7 target cells.
  • Figure 2A shows the results of the detection of IFN- ⁇ secretion levels in HepG2 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
  • Figure 2B shows the results of the detection of TNF- ⁇ secretion levels in HepG2 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
  • Figure 2C shows the results of the IFN- ⁇ secretion level detection in Huh7 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
  • Figure 2D shows the results of the detection of TNF- ⁇ secretion levels in Huh7 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, and AB9-T).
  • the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention.
  • 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.
  • a total of 4 fully human anti-GPC3 monoclonal antibodies CB6, AB9, CE3 and CH6 were screened. After sequencing and analyzing the above monoclonal antibodies, the sequences of VH and VL were obtained, and the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 were obtained according to the Kabat, IMGT, Chothia and AbM numbering systems (see Tables 1 and 2 for specific sequences).
  • 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 main peak area of the four candidate scFv 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 (for related sequences, see WO2017020812) 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.
  • Human PBMC cells were isolated using lymphocyte separation medium (GE), cultured in an incubator at 37°C and 5% CO2 , 100 ⁇ L/mL of CD3 antibody and CD28 antibody were added, mixed thoroughly, and incubated at room temperature for 15 minutes.
  • GE lymphocyte separation medium
  • the tube remains in the magnet and is gently inverted to pour out the cells.
  • the cell density was adjusted to 1 ⁇ 10 6 cells/mL, and cytokine and antibody complexes (configured at a final concentration of 300 U/mL of IL-2, 10 ng/mL of IL-7, 5 ng/mL of IL-15, 500 ng/mL of Anti-CD3 (OKT3), and 2 ⁇ g/mL of Anti-CD28) were added and cultured for 48 hours.
  • 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 the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS test results are shown in Table 7 below.
  • HEPG2-luc cells 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 were inoculated in a 96-well plate at 100 ⁇ L/well, and the cells were placed in a 5% CO 2 37°C incubator for 30 minutes. CAR-T was 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 a 96-well plate containing HEPG2-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 culture was completed, the well plate was removed from the incubator, 20ul of fluorescence detection reagent was added, and the fluorescence reading was detected using an enzyme reader.
  • the results of the CAR-T killing activity test are shown in Figure 1.
  • 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 is as high as about 99%.
  • HepG2-luc and Huh7-luc cells were collected, and the cell density was adjusted to 1 ⁇ 10 5 /mL using culture medium.
  • the target cells were inoculated in a 96-well plate at 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 in a 5% CO 2 37°C incubator overnight.
  • E/T effector cell/target cell
  • CB6-T, AB9-T, CE3-T, and CH6-T The four types of CAR-T cells constructed in the present application can kill tumor cells to varying degrees and release IFN- ⁇ (with HepG2 as the target cell, the test results are shown in FIG. 2A ; with Huh7 as the target cell, the test results are shown in FIG. 2C ) and TNF- ⁇ (with HepG2 as the target cell, the test results are shown in FIG. 2B ; with Huh7 as the target cell, the test results are shown in FIG. 2D ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

A chimeric antigen receptor specifically binding to GPC3, a modified immune cell expressing the chimeric antigen receptor, and a method for preparing the modified immune cell. The present invention also relates to a method of using said chimeric antigen and immune cell for preventing and/or treating diseases related to GPC3 expression.

Description

特异性结合GPC3的嵌合抗原受体及其应用Chimeric antigen receptor specifically binding to GPC3 and its application 技术领域Technical Field
本发明涉及生物医药领域，具体而言，本发明涉特异性结合GPC3的单链抗体及嵌合抗原受体(CAR)。本发明还涉及表达所述CAR的免疫细胞，编码这种CAR或共表达分子的核酸分子以及制备所述经改造的免疫细胞的方法。本发明还涉及这些CAR和免疫细胞用于预防和/或治疗肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌等GPC3阳性肿瘤的方法。The present invention relates to the field of biomedicine, and in particular, the present invention relates to single-chain antibodies and chimeric antigen receptors (CARs) that specifically bind to GPC3. The present invention also relates to immune cells expressing the CAR, nucleic acid molecules encoding such CARs or co-expressed molecules, and methods for preparing the modified immune cells. The present invention also relates to methods for using these CARs and immune cells to prevent and/or treat GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma.
背景技术Background technique
磷脂酰肌醇蛋白聚糖3(Glypican-3，GPC3也称为DGSX，GTR2-2，MXR7，OCI-5，SDYS，SGB，SGBS和SGBS1)为硫酸乙酰肝素蛋白多糖家族一员，通过糖基化的磷脂酰肌醇锚定在细胞表面，是目前临床前研究中具有代表性的肝癌标志物之一。GPC3在许多人类恶性肿瘤细胞及血清中表达，包括肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌，在其他癌症和正常组织中表达很少。GPC3是HCC的潜在生物标志物，其与WNT形成复合体，激活下游信号通路，促进肝癌细胞的增殖，参与多个与肿瘤发生和发展密切相关的信号通路的调节。Glypican-3 (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.
原发性肝癌是我国第4位的常见恶性肿瘤和第3位的肿瘤致死原因，严重威胁我国人民的生命健康。我国每年肝癌新发病例约46.6万，约占全球每年新发病例的55％,我国每年约42.2万人因肝癌而死亡。大量肝细胞癌患者依然缺乏精准有效的临床治疗手段。肝癌患者诊断时多已处于进展期或晚期，仅30％的患者有手术切除机会，切除后5年内转移、复发率高达60％～70％，总体5年生存率低，仅7％～10％。嵌合抗原受体(CAR)-T细胞疗法被认为是最有前途的癌症治疗方法之一，成为了人类对抗癌症的新希望。它通过从病人体内采集的免疫细胞进行体外培养，在体外转导特定的外源基因，体外扩增后回输到病人体内，以非MHC限制性的方式达到治疗肿瘤的目的。CAR-T细胞疗法在血液恶行肿瘤治疗中取得了显著的疗效，对复发难治性B细胞白血病的完全缓解率超过90％。实体瘤约占所有恶行肿瘤的90％，其治疗药物需求量大。但是，CAR-T细胞治疗目前在实体瘤中的治疗效果仍不足，其主要原因是实体瘤有复杂的肿瘤微环境和肿瘤异质性高。Primary liver cancer is the fourth most common malignant tumor and the third leading cause of cancer-related death in my country, which seriously threatens the life and health of the Chinese people. There are about 466,000 new cases of liver cancer in my country each year, accounting for about 55% of the new cases worldwide each year. About 422,000 people die of liver cancer in my country each year. A large number of patients with hepatocellular carcinoma still lack accurate and effective clinical treatment methods. Most patients with liver cancer are already in the advanced or advanced stage when diagnosed. Only 30% of patients have the opportunity for surgical resection. The metastasis and recurrence rate within 5 years after resection is as high as 60% to 70%, and the overall 5-year survival rate is low, only 7% to 10%. Chimeric antigen receptor (CAR)-T cell therapy is considered to be one of the most promising cancer treatment methods and has become a new hope for human beings to fight cancer. It cultivates immune cells collected from patients in vitro, transduces specific exogenous genes in vitro, and then amplifies them in vitro and then infuses them back into the patient's body to achieve the purpose of treating tumors in a non-MHC restricted manner. CAR-T cell therapy has achieved remarkable therapeutic effects in the treatment of hematological malignancies, with a complete remission rate of over 90% for relapsed and refractory B-cell leukemia. Solid tumors account for about 90% of all malignant tumors, and there is a large demand for therapeutic drugs. However, the therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly because solid tumors have complex tumor microenvironments and high tumor heterogeneity.
本发明提供一种靶向GPC3的CAR或者共表达靶向GPC3的CAR的经改造的免疫细胞，修饰后的免疫细胞可以用于GPC3阳性的肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌等的治疗。 The present invention provides a CAR targeting GPC3 or a modified immune cell co-expressing a CAR targeting GPC3. The modified immune cell can be used for the treatment of GPC3-positive hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma, etc.
发明内容Summary of the invention
在本申请中，发明人首先开发了免疫原性低的能够特异性识别/结合GPC3的全人源抗体。在此基础上，本发明进一步设计构建了的靶向GPC3的CAR。本发明的CAR能够以非MHC限制的方式将免疫效应细胞特异性和反应性指向表达GPC3的细胞(例如肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌)从而使其被清除。因此，本发明的靶向GPC3的CAR具有用于预防和/或治疗例如肝细胞癌(HCC)、黑色素瘤和卵巢透明细胞癌等GPC3阳性肿瘤的潜力，具有重大的临床价值。In the present application, the inventors first developed a fully human antibody with low immunogenicity that can specifically recognize/bind to GPC3. On this basis, the present invention further designs and constructs a CAR targeting GPC3. The CAR of the present invention can direct the specificity and reactivity of immune effector cells to cells expressing GPC3 (such as hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma) in a non-MHC restricted manner so that they can be eliminated. Therefore, the CAR targeting GPC3 of the present invention has the potential to be used for the prevention and/or treatment of GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma, and has great clinical value.
抗原结合分子Antigen Binding Molecules
本发明第一方面提供了一种抗原结合分子，其包含能够与GPC3特异性结合的结合结构域；所述抗原结合分子包含如下的互补决定区(CDRs)：The first aspect of the present invention provides an antigen binding molecule comprising a binding domain capable of specifically binding to GPC3; the antigen binding molecule comprises the following complementarity determining regions (CDRs):
(a)SEQ ID NO:1所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:2所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(a) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:1; and/or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:2;
或，or,
(b)SEQ ID NO:3所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:4所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(b) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:3; and/or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:4;
或，or,
(c)SEQ ID NO:5所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:6所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(c) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:5; and/or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:6;
或，or,
(d)SEQ ID NO:7所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(d) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:7; and/or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:8;
或，or,
(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)。(e) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and/or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein the heavy chain variable region (VH) and/or or light chain variable region (VL) compared to any one of the heavy chain variable region and/or light chain variable region described in (a) to (d), at least one CDR contains a mutation, and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids).
在某些实施方案中，所述的置换为保守置换。In certain embodiments, the substitutions are conservative substitutions.
在某些实施方案中，所述CDR根据Kabat、IMGT、Chothia或AbM编号系统定义。In certain embodiments, the CDRs are defined according to the Kabat, IMGT, Chothia, or AbM numbering systems.
在某些实施方案中，本发明的抗原结合分子包含重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：In certain embodiments, the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:
(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：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；(1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 9 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 10 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 11 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 12 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 14 or a variant thereof;
或，or,
(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；(1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 24 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 25 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 26 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 27 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 29 or a variant thereof;
或，or,
(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，(1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 38 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 39 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 40 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 41 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 42 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 43 or a variant thereof; or,
(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； (1d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 53 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 56 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 57 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 58 or a variant thereof;
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)；优选地，所述的置换是保守置换。Wherein, 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; preferably, the substitutions are conservative substitutions.
在某些实施方案中，本发明的抗原结合分子包含重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：In certain embodiments, the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:
(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：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；(2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 15 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 18 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 19 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 14 or a variant thereof;
或，or,
(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；(2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 30 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 31 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 32 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 33 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 34 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 29 or a variant thereof;
或，or,
(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，(2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 44 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 45 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 46 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 47 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 48 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 43 or a variant thereof; or,
(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；(2d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 59 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 62 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 63 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 58 or a variant thereof;
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)；优选地，所 述的置换是保守置换。Wherein, 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; preferably, The substitutions described are conservative substitutions.
在某些实施方案中，本发明的抗原结合分子包含重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：In certain embodiments, the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:
(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：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；(3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 20 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 12 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 14 or a variant thereof;
或，or,
(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；(3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 35 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 27 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 29 or a variant thereof;
或，or,
(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，(3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 49 or a variant thereof; 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; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 41 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 42 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 43 or a variant thereof; or,
(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；(3d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 64 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 56 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 57 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 58 or a variant thereof;
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)；优选地，所述的置换是保守置换。Wherein, 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; preferably, the substitutions are conservative substitutions.
在某些实施方案中，本发明的抗体或其抗原结合片段包含重链可变区(VH)和/或轻 链可变区(VL)，其中CDR按AbM编号系统定义：In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VH). Chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:
(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：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；(4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 22 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 12 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 14 or a variant thereof;
或，or,
(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；(4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 36 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 27 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 29 or a variant thereof;
或，or,
(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；(4c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 51 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 41 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 42 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 43 or a variant thereof;
或，or,
(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；(4d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 66 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 67 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 55 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 56 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 57 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 58 or a variant thereof;
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。Wherein, 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. In certain embodiments, the substitutions are conservative substitutions.
在某些实施方案中，本发明的抗原结合分子包含：In certain embodiments, the antigen binding molecules of the invention comprise:
(a)包含如SEQ ID NO:1所示的序列或其变体的VH和/或包含如SEQ ID NO:2所示的序列或其变体的VL； (a) a VH comprising 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;
或，or,
(b)包含如SEQ ID NO:3所示的序列或其变体的VH和/或包含如SEQ ID NO:4所示的序列或其变体的VL；(b) a VH comprising the sequence shown in SEQ ID NO:3 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO:4 or a variant thereof;
或，or,
(c)包含如SEQ ID NO:5所示的序列或其变体的VH和/或包含如SEQ ID NO:6所示的序列或其变体的VL；(c) a 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;
或or
(d)包含如SEQ ID NO:7所示的序列或其变体的VH和/或包含如SEQ ID NO:8所示的序列或其变体的VL；(d) a 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;
其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Wherein, 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; preferably, the substitutions are conservative substitutions.
在某些实施方案中，上述任一实施方案中所述的抗原结合分子可以包含来自或源自人免疫球蛋白的恒定区。In certain embodiments, the antigen binding molecule described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.
在某些实施方案中，所述抗原结合分子的重链包含来自或源自人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区。在某些实施方案中，所述抗原结合分子的重链包含野生型Fc区，或者包含突变的或化学修饰的Fc区，其与野生型Fc区相比具有改变的效应子功能(例如增强的ADCC活性)。In certain embodiments, the heavy chain of the antigen binding molecule comprises a heavy chain constant region from or derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the heavy chain of the antigen binding molecule comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region, which has an effector function (e.g., enhanced ADCC activity) that is changed compared to a wild-type Fc region.
在某些实施方案中，所述抗原结合分子的轻链包含来自或源自人免疫球蛋白(例如κ或λ)的轻链恒定区。In certain embodiments, the light chain of the antigen binding molecule comprises a light chain constant region from or derived from a human immunoglobulin (eg, kappa or lambda).
在某些实施方案中，上述任一实施方案中所述的抗原结合分子为鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。In certain embodiments, the antigen binding molecule described in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
在某些实施方案中，所述抗原结合分子选自全长抗体、Fab片段、Fab'片段、F(ab)'2片段、F(ab)'3片段、单链抗体(例如scFv、di-scFv或(scFv)2)、微型抗体、二硫键稳定的Fv蛋白(dsFv)和单结构域抗体(sdAb，纳米抗体)。In certain embodiments, the antigen binding molecule is selected from a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab)' 2 fragment, a F(ab)' 3 fragment, a single-chain antibody (e.g., scFv, di-scFv or (scFv) 2 ), a miniantibody, a disulfide-stabilized Fv protein (dsFv) and a single domain antibody (sdAb, nanobody).
在某些实施方案中，本发明的抗原结合分子的VH和VL通过一个或多个连接子连 接。连接子通常是肽接头，例如柔性和/或可溶性肽接头，例如富含甘氨酸、丝氨酸和/或苏氨酸的肽接头。在一些实施方案中，连接子还包括带电荷的残基(如赖氨酸和/或谷氨酸)，其可以改善溶解性。在一些实施方案中，连接子还包括一个或多个脯氨酸。In certain embodiments, the VH and VL of the antigen binding molecules of the invention are linked by one or more linkers. The linker is typically a peptide linker, such as a flexible and/or soluble peptide linker, such as a peptide linker rich in glycine, serine and/or threonine. In some embodiments, the linker also includes charged residues (such as lysine and/or glutamic acid), which can improve solubility. In some embodiments, the linker also includes one or more proline.
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。In certain embodiments, 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. In certain embodiments, the linker has a sequence of SEQ ID NO: 76.
在某些实施方案中，本发明的抗原结合分子是单链抗体，例如scFv、di-scFv或(scFv)2。In certain embodiments, the antigen binding molecules of the invention are single chain antibodies, such as scFv, di-scFv or (scFv)2.
在某些实施方案中，所述单链抗体从其N端至C端依次包括：In certain embodiments, the single-chain antibody comprises, from its N-terminus to its C-terminus:
(1)包含如SEQ ID NO:1所示的序列或其变体的VH-连接子-包含如SEQ ID NO:2所示的序列或其变体的VL；或，包含如SEQ ID NO:2所示的序列或其变体的VL-连接子-包含如SEQ ID NO:1所示的序列或其变体的VH；(1) a VH-linker comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof; or, a 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;
或or
(2)包含如SEQ ID NO:3所示的序列或其变体的VH-连接子-包含如SEQ ID NO:4所示的序列或其变体的VL；或，包含如SEQ ID NO:4所示的序列或其变体的VL-连接子-包含如SEQ ID NO:3所示的序列或其变体的VH；(2) a VH-linker comprising a sequence as shown in SEQ ID NO: 3 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 4 or a variant thereof; or, a 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;
或or
(3)包含如SEQ ID NO:5所示的序列或其变体的VH-连接子-包含如SEQ ID NO:6所示的序列或其变体的VL；或，包含如SEQ ID NO:6所示的序列或其变体的VL-连接子-包含如SEQ ID NO:5所示的序列或其变体的VH；(3) a VH-linker comprising a sequence as shown in SEQ ID NO: 5 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 6 or a variant thereof; or, a 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;
或or
(4)包含如SEQ ID NO:7所示的序列或其变体的VH-连接子-包含如SEQ ID NO:8所示的序列或其变体的VL；或，包含如SEQ ID NO:8所示的序列或其变体的VL-连接子-包含如SEQ ID NO:7所示的序列或其变体的VH；(4) a VH-linker comprising a sequence as shown in SEQ ID NO: 7 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 8 or a variant thereof; or, a 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;
其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；在某些实施方案中，所述的置换是保守置换。Wherein, 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.
在某些实施方案中，所述单链抗体包含SEQ ID NO:68,69,70,71任一项所示的 序列或其变体，所述变体与SEQ ID NO:68,69,70,71相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列，或具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；在某些实施方案中，所述的置换是保守置换。In certain embodiments, the single-chain antibody comprises a single-chain antibody as shown in any one of SEQ ID NOs: 68, 69, 70, 71. A sequence or a variant thereof, wherein the variant has a sequence that is 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% identical to SEQ ID NO: 68, 69, 70, 71, or has a substitution, deletion or addition of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids); in certain embodiments, the substitution is a conservative substitution.
在某些实施方案中，本发明的抗原结合分子进一步包含来源于人免疫球蛋白的恒定区。在某些实施方案中，所述抗原结合分子的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗原结合分子的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。In certain embodiments, the antigen binding molecules of the present invention further comprise a constant region derived from human immunoglobulin. In certain embodiments, the heavy chain of the antigen binding molecules comprises a heavy chain constant region derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antigen binding molecules comprises a light chain constant region derived from human immunoglobulin (e.g., κ or λ).
在某些实施方案中，所述抗原结合分子的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，In certain embodiments, the heavy chain of the antigen binding molecule comprises 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,
所述抗原结合分子的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。The light chain of the antigen-binding molecule 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., 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.
在某些实施方案中，所述重链恒定区是IgG、IgM、IgE、IgD或IgA重链恒定区。在某些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。In certain embodiments, the heavy chain constant region is an IgG, IgM, IgE, IgD or IgA heavy chain constant region. In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.
在某些实施方案中，所述轻链恒定区是κ或λ轻链恒定区。在某些优选的实施方案中，所述轻链恒定区是人κ轻链恒定区。In certain embodiments, the light chain constant region is a kappa or lambda light chain constant region. In certain preferred embodiments, the light chain constant region is a human kappa light chain constant region.
抗原结合分子的制备Preparation of antigen-binding molecules
本发明的抗原结合分子可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码该抗原结合分子的DNA分子，将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗原结合分子。 The antigen binding molecules of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, a DNA molecule encoding the antigen binding molecule is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and then transfected into a host cell. Then, the host cell after transfection is cultured under specific conditions, and the antigen binding molecules of the present invention are expressed.
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。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)). For example, 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)). In addition, Fv, Fab or F(ab')2 fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.
因此，本发明第二方面提供了一种分离的核酸分子，其包含编码本发明的抗原结合分子的核苷酸序列。在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：Therefore, the second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antigen binding molecule of the present invention. In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO: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，The nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:89, (ii) a sequence substantially identical to SEQ ID NO:89 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:89, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above; and/or,
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。The nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence shown in SEQ ID NO:90, (v) a sequence substantially identical to SEQ ID NO:90 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:90, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO: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，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 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to 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,
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。 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 higher sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:92), or (vi) a degenerate sequence of the above (iv) or (v).
在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO: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，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 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:93, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above; and/or,
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。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 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity with SEQ ID NO:94, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
在某些实施方案中，所述分离的核酸分子包含编码抗体重链可变区的核酸分子，和/或编码抗体轻链可变区的核酸分子，其中：In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein:
所述编码抗体重链可变区的核酸分子包含：(i)SEQ ID NO: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，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 higher sequence identity compared to SEQ ID NO:95, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above; and/or,
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。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 higher sequence identity with SEQ ID NO:96, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:72所示的核苷酸序列、(ii)与SEQ ID NO:72基本上相同的序列(例如，与SEQ ID NO:72相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:72, (ii) a sequence substantially identical to SEQ ID NO:72 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:72, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:73所示的核苷酸序列、(ii)与SEQ ID NO:73基本上相同的序列(例如，与SEQ ID NO:73相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:73, (ii) a sequence substantially identical to SEQ ID NO:73 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:73, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:74所示的核苷酸序 列、(ii)与SEQ ID NO:74基本上相同的序列(例如，与SEQ ID NO:74相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) a nucleotide sequence as shown in SEQ ID NO: 74; The invention relates to a sequence of the invention that is a sequence of the invention that is substantially identical to SEQ ID NO:74 (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:74), or (iii) a degenerate sequence of (i) or (ii) above.
在某些实施方案中，所述分离的核酸分子包含：(i)SEQ ID NO:75所示的核苷酸序列、(ii)与SEQ ID NO:75基本上相同的序列(例如，与SEQ ID NO:75相比，具有至少大约85％、90％、95％、99％或更高序列同一性的序列，或具有一个或更多个核苷酸取代的序列)、或(iii)上述(i)或(ii)的简并序列。In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence shown in SEQ ID NO:75, (ii) a sequence substantially identical to SEQ ID NO:75 (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:75, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above.
本发明第三方面提供了一种载体(例如克隆载体或表达载体)，其包含如上所述的分离的核酸分子。在某些实施方案中，本发明的载体是例如DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体或病毒载体；优选的，所述载体是表达载体；优选地，所述载体是游离型载体；优选地，所述载体是病毒载体；更优选地，所述病毒载体是慢病毒载体、腺病毒载体或逆转录病毒载体。The third aspect of the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vector of the present invention is, for example, a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector or a viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.
本发明第四方面提供了一种宿主细胞，其包含本发明的分离的核酸分子或本发明的载体。宿主细胞可以是真核细胞(例如哺乳动物细胞、昆虫细胞、酵母细胞)或原核细胞(例如大肠杆菌)。合适的真核细胞包括但不限于NS0细胞、Vero细胞、Hela细胞、COS细胞、CHO细胞、ExpiCHO细胞、HEK293细胞、Expi293细胞、BHK细胞、和MDCKII细胞。适宜的昆虫细胞包括但不限于Sf9细胞。在某些实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DXB11、ExpiCHO、CHO DG44)。The fourth aspect of the present invention provides a host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention. The host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).
在某些实施方案中，本发明的宿主细胞可以是嵌合抗原受体T细胞(CAR-T)。在此类实施方案中，所述宿主细胞所包含的分离的核酸分子可以包含编码嵌合抗原受体的核苷酸序列，所述编码嵌合抗原受体的核苷酸序列进一步包含编码本发明的抗原结合分子(例如ScFv)的核苷酸序列。在某些实施方案中，所述宿主细胞所包含的分离的核酸分子编码包含本发明抗原结合分子(例如scFv)的嵌合抗原受体。In certain embodiments, the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antigen binding molecule (eg, ScFv) of the present invention. In certain embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen binding molecule (eg, scFv) of the present invention.
在另一个方面，本发明还涉及制备本发明的抗原结合分子的方法，其包括，在允许蛋白表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗原结合分子。 In another aspect, the present invention also relates to a method for preparing the antigen-binding molecule of the present invention, comprising culturing the host cell as described above under conditions allowing protein expression, and recovering the antigen-binding molecule from the cultured host cell culture.
嵌合抗原受体Chimeric Antigen Receptor
本发明涉及靶向GPC3的CAR，其特征包括非MHC限制的GPC3识别能力，其赋予表达该CAR的免疫细胞(例如，T细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞)不依赖于抗原加工及提呈而识别表达GPC3的细胞(例如肿瘤细胞)的能力。The present invention relates to a CAR targeting GPC3, 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.
因此，本发明第五方面提供了一种嵌合抗原受体，其包含抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域。Therefore, the fifth aspect of the present invention provides a chimeric antigen receptor, which comprises an antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain.
I.胞外抗原结合结构域I. Extracellular antigen binding domain
本发明的嵌合抗原受体中所包含的抗原结合结构域赋予所述CAR识别GPC3的能力。The antigen binding domain contained in the chimeric antigen receptor of the present invention confers the ability of the CAR to recognize GPC3.
在某些实施方案中，所述抗原结合结构域包含第一方面所述的抗原结合分子。In certain embodiments, the antigen binding domain comprises the antigen binding molecule described in the first aspect.
在某些实施方案中，所述抗原结合结构域包含所述抗原结合分子作为第一抗原结合结构域，并且进一步包含不结合GPC3的第二抗原结合结构域。在某些实施方案中，所述第二抗原结合结构域结合的抗原选自：PD-1、PD-L1、CTLA-、CD3、ASGPR1、CD19、MSLN、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2。In certain embodiments, the antigen binding domain comprises the antigen binding molecule as a first antigen binding domain, and further comprises a second antigen binding domain that does not bind to GPC3. In certain embodiments, the antigen bound by the second antigen binding domain is selected from: PD-1, PD-L1, CTLA-, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
在某些实施方案中，所述抗原结合结构域是单链抗体。In certain embodiments, the antigen binding domain is a single chain antibody.
在某些实施方案中，所述第一抗原结合结构域包含SEQ ID NO:68,69,70,71任一项所示的序列或其变体，所述变体与SEQ ID NO:68,69,70,71任一项相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％同一性的序列，或具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。In certain embodiments, the first antigen binding domain comprises the sequence shown in any one of SEQ ID NO:68,69,70,71 or a variant thereof, wherein the variant has a sequence that is 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% identical to any one of SEQ ID NO:68,69,70,71, or has 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); preferably, the substitutions are conservative substitutions.
II.跨膜结构域II. Transmembrane domain
本发明的嵌合抗原受体所包含的跨膜结构域可以是本领域已知的任何蛋白结构，只要其能够在细胞膜(特别是真核细胞膜)中热力学稳定。适用于本发明的CAR的跨膜结构域可衍生自天然来源。在此类实施方案中，所述跨膜结构域可衍生自任何膜结合的或跨膜的蛋白质。或者，所述跨膜结构域可为合成的非天然存在的蛋白质区段，例如主要包含疏水残基例如亮氨酸和缬氨酸的蛋白质区段。The transmembrane domain 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. In such embodiments, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Alternatively, 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.
在某些实施方案中，所述跨膜结构域是选自下列蛋白的跨膜区：T细胞受体的α、β或ζ链、CD28、CD45、CD3ε、CD3ζ、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD152、CD154和PD-1及其任意组合。在某 些优选的实施方案中，所述跨膜结构域是选自下列蛋白的跨膜区：CD8、CD28、CD4、PD-1、CD152和CD154。在某些实施方案中，所述跨膜结构域包含序列如SEQ ID NO:77所示的CD8跨膜区。In certain embodiments, the transmembrane domain is a transmembrane region selected from the group consisting of the α, β or ζ chains of T cell receptors, CD28, CD45, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1, and any combination thereof. In some preferred embodiments, the transmembrane domain is a transmembrane region selected from the following proteins: CD8, CD28, CD4, PD-1, CD152 and CD154. In certain embodiments, the transmembrane domain comprises a CD8 transmembrane region whose sequence is shown in SEQ ID NO:77.
III.间隔结构域III. Spacer Domain
本发明的嵌合抗原受体所包含间隔结构域位于胞外抗原结合结构域与跨膜结构域之间。The chimeric antigen receptor of the present invention comprises a spacer domain located between the extracellular antigen binding domain and the transmembrane domain.
在某些实施方案中，所述间隔结构域包含免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区。在此类实施方案中，不受特定理论的约束，认为CH2和CH3使所述CAR的抗原结合结构域从表达CAR的细胞的细胞膜延伸出去，并且可更精确地模拟天然TCR的大小和结构域结构。In certain embodiments, the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4). In such embodiments, without being bound by a particular theory, it is believed that CH2 and CH3 extend the antigen binding domain of the CAR from the cell membrane of the cell expressing the CAR, and can more accurately mimic the size and domain structure of a natural TCR.
在某些实施方案中，所述间隔结构域包含铰链结构域。铰链结构域可以是通常在蛋白质的两个结构域之间发现的氨基酸区段，其可以允许蛋白质具有柔性并且允许一个或两个结构域相对于彼此的运动。因此，所述铰链结构域可以是任何氨基酸序列，只要其能够提供胞外抗原结合结构域的这种柔性以及其相对于跨膜结构域的这种运动性。In certain embodiments, the spacer domain comprises a hinge domain. A hinge domain 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.
在某些实施方案中，所述铰链结构域是天然存在的蛋白质的铰链区或其部分。在某些实施方案中，所述间隔结构域选自铰链结构域和/或免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区。在某些实施方案中，所述铰链结构域包含CD8、IgG4、PD-1、CD152或CD154的铰链区。在某些实施方案中，所述铰链结构域包含序列如SEQ ID NO:78所示的CD8铰链区。In certain embodiments, the hinge domain is a hinge region of a naturally occurring protein or a portion thereof. In certain embodiments, the spacer domain is selected from a hinge domain and/or a CH2 and CH3 region of an immunoglobulin (e.g., IgG1 or IgG4). In certain embodiments, the hinge domain comprises a hinge region of CD8, IgG4, PD-1, CD152, or CD154. In certain embodiments, the hinge domain comprises a CD8 hinge region having a sequence as shown in SEQ ID NO: 78.
IV.信号肽IV. Signal Peptide
在某些实施方案中，本发明的CAR可进一步在其N端包含信号肽。通常，信号肽是将与其连接的序列靶向至所需位点的多肽序列。在某些实施方案中，所述信号肽可以将与其连接的CAR靶向至细胞的分泌途径，并允许该CAR进一步整合并锚定到脂质双分子层中。可用于CAR的信号肽是本领域技术人员已知的。在某些实施方案中，所述信号肽包含重链信号肽(例如IgG1的重链信号肽)、粒细胞-巨噬细胞集落刺激因子受体2(GM-CSFR2)信号肽、IL2信号肽、或CD8α信号肽。在某些优选的实施方案中，所述信号肽选自CD8α信号肽。在某些示例性实施方案中，所述信号肽包含SEQ ID NO:82所示的氨基酸序列。In certain embodiments, the CAR of the present invention may further include a signal peptide at its N-terminus. Typically, a signal peptide is a polypeptide sequence that targets the sequence connected thereto to the desired site. In certain embodiments, 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. In certain embodiments, 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 CD8α signal peptide. In certain preferred embodiments, the signal peptide is selected from a CD8α signal peptide. In certain exemplary embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:82.
在某些实施方案中，本发明的CAR还可以与另外的生物活性分子共表达。所述另外的生物活性分子可以有其专有的信号肽，为与上一段的信号肽区别，此信号肽命名为信 号肽-2。该信号肽-2引导另外的生物活性分子转运到细胞内特定的位点或细胞膜外。该述信号肽-2可与上一段所述的信号肽相同或不同。在某些实施方案中，该信号肽-2可与上一段所述的信号肽不同。在某些实施方案中，所述信号肽-2是IL2信号肽(例如，氨基酸序列如SEQ ID NO:84所示)。In certain embodiments, the CAR of the present invention can also be co-expressed with another biologically active molecule. The other biologically active molecule can have its own signal peptide, which is named signal peptide to distinguish it from the signal peptide in the previous paragraph. The signal peptide-2 guides the transport of other biologically active molecules to a specific site in the cell or outside the cell membrane. The signal peptide-2 may be the same as or different from the signal peptide described in the previous paragraph. In certain embodiments, the signal peptide-2 may be different from the signal peptide described in the previous paragraph. In certain embodiments, the signal peptide-2 is an IL2 signal peptide (e.g., the amino acid sequence is shown in SEQ ID NO: 84).
V.胞内信号传导结构域V. Intracellular signaling domain
本发明的CAR中所包含的胞内信号传导结构域参与将本发明的CAR与GPC3的结合所产生的信号传导进免疫效应细胞内部，激活表达CAR的免疫效应细胞的至少一种正常效应子功能，或增强表达CAR的免疫效应细胞的至少一种细胞因子的分泌(例如IL-2，IFN-γ)。The intracellular signaling domain contained in the CAR of the present invention 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-γ) of the immune effector cell expressing CAR.
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域和/或共刺激信号传导结构域。In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and/or a co-stimulatory signaling domain.
在某些实施方案中，所述初级信号传导结构域可以是包含免疫受体酪氨酸活化基序(ITAM)的任何胞内信号传导结构域。在某些实施方案中，所述初级信号传导结构域包含免疫受体酪氨酸活化基序(ITAM)。在某些实施方案中，所述初级信号传导结构域包含选自下列蛋白的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CDS、CD22、CD79a、CD79b或CD66d。在某些实施方案中，所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域。In certain embodiments, the primary signaling domain 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: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b or CD66d. In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of CD3ζ.
在某些实施方案中，所述共刺激信号传导结构域可以是来自共刺激分子的胞内信号传导结构域。在某些实施方案中，所述共刺激信号传导结构域包含选自下列蛋白的胞内信号传导结构域：CARD11、CD2、CD7、CD27、CD28、CD30、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)或DAP10。In certain embodiments, the costimulatory signaling domain may be an intracellular signaling domain from a costimulatory molecule. In certain embodiments, the costimulatory signaling domain comprises an intracellular signaling domain selected from the following proteins: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10.
在某些实施方案中，所述共刺激信号传导结构域选自CD28的胞内信号传导结构域、或CD137(4-1BB)的胞内信号传导结构域、或二者片段的组合。In certain embodiments, the 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.
在某些实施方案中，所述胞内信号传导结构域包含一个共刺激信号传导结构域。在某些实施方案中，所述胞内信号传导结构域包含两个或更多个共刺激信号传导结构域。在此类实施方案中，所述两个或更多个共刺激信号传导结构域可以是相同的，也可以是不同的。In certain embodiments, the intracellular signaling domain comprises a 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.
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域以及至少一个共刺激信号传导结构域。所述初级信号传导结构域以及至少一个共刺激信号传导结构域可以以任意顺序串联至跨膜结构域的羧基端。 In certain embodiments, the intracellular signaling domain 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.
在某些实施方案中，所述胞内信号传导结构域可包含CD3ζ的胞内信号传导结构域和CD137(4-1BB)的胞内信号传导结构域。在某些示例性实施方案中，所述CD3ζ的胞内信号传导结构域包含SEQ ID NO:79所示的氨基酸序列。在某些示例性实施方案中，所述CD137(4-1BB)的胞内信号传导结构域包含SEQ ID NO:80所示的氨基酸序列。In certain embodiments, the intracellular signaling domain may include the intracellular signaling domain of CD3ζ and the intracellular signaling domain of CD137(4-1BB). In certain exemplary embodiments, the intracellular signaling domain of CD3ζ includes the amino acid sequence shown in SEQ ID NO:79. In certain exemplary embodiments, the intracellular signaling domain of CD137(4-1BB) includes the amino acid sequence shown in SEQ ID NO:80.
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。In certain exemplary embodiments, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:81.
VI.全长CARVI. Full-length CAR
本发明提供了能够特异性地结合GPC3的嵌合抗原受体，所述嵌合抗原受体从其N端至C端依次包含抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域。在某些优选实施方案中，其中所述胞内信号传导结构域从N端到C端为共刺激信号传导结构域和初级信号传导结构域。The present invention provides a chimeric antigen receptor capable of specifically binding 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. In certain preferred embodiments, the intracellular signaling domain is a co-stimulatory signaling domain and a primary signaling domain from the N-terminus to the C-terminus.
在某些实施方案中，优选地，所述间隔结构域包含CD8(例如CD8α)的铰链区(例如，序列如SEQ ID NO:78所示的铰链区)。In certain embodiments, preferably, the spacer domain comprises the hinge region of CD8 (e.g., CD8α) (e.g., the hinge region whose sequence is shown in SEQ ID NO:78).
在某些实施方案中，所述跨膜结构域包含CD8(例如CD8α)的跨膜区(例如，序列如SEQ ID NO:77所示的跨膜区)。In certain embodiments, the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8α) (e.g., a transmembrane region whose sequence is shown in SEQ ID NO:77).
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域(例如，如SEQ ID NO:79所示序列)，所述共刺激信号传导结构域包含CD137(4-1BB)的胞内信号传导结构域(例如，如SEQ ID NO:80所示序列)；更优选地，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO:81所示序列。In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO:79), and the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO:80); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:81.
在某些优选的实施方案中，所述嵌合抗原受体从其N端至C端依次包含所述信号肽、抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域(从N端到C端为共刺激信号传导结构域和初级信号传导结构域)。In certain preferred embodiments, the chimeric antigen receptor 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.
在某些实施方案中，所述信号肽包含IgG1的重链信号肽或CD8α信号肽(例如，序列如SEQ ID NO:82所示的信号肽)。在某些示例性实施方案中，本发明的CAR包含SEQ ID NO:85、86、87、88任一项所示的序列或其变体，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换为保守置换。 In certain embodiments, the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (e.g., a signal peptide having a sequence as shown in SEQ ID NO: 82). In certain exemplary embodiments, the CAR of the present invention comprises a sequence as shown in any one of SEQ ID NO: 85, 86, 87, 88, or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity 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; preferably, the substitution is a conservative substitution.
VII.共表达的CAR和另外的生物活性分子VII. Co-expression of CAR and Additional Biologically Active Molecules
在一些情况下，本发明第五方面所述的CAR还可以与另外的生物活性分子共表达。自裂解肽能阻止翻译过程中氨基酸形成共价键并维持翻译继续进行，这样一来，翻译产物就被“自切割”，从而使本发明的嵌合抗原受体和另外的生物活性分子分离。因此，当本发明第五方面所述的CAR还可以与另外的生物活性分子共表达时，能够特异性地结合GPC3的嵌合抗原受体成为独立的具有胞外抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域的CAR，而另外的生物活性分子则能够分泌到细胞外或者表达成膜嵌合型的多肽或蛋白。随着表达CAR的免疫细胞在肿瘤微环境的扩增和富集，另外的生物活性分子富集在肿瘤微环境，与CAR协同发挥抗肿瘤效应。In some cases, the CAR described in the fifth aspect of the present invention can also be co-expressed with other biologically active molecules. The self-cleaving peptide can prevent the amino acids from forming covalent bonds during the translation process and maintain the translation to continue, so that the translation product is "self-cut", thereby separating the chimeric antigen receptor of the present invention from other biologically active molecules. Therefore, when the CAR described in the fifth aspect of the present invention can also be co-expressed with other biologically active molecules, the chimeric antigen receptor that can specifically bind to GPC3 becomes an independent CAR having an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, while other biologically active molecules can be secreted outside the cell or expressed as membrane-forming chimeric polypeptides or proteins. As the immune cells expressing CAR amplify and enrich in the tumor microenvironment, other biologically active molecules are enriched in the tumor microenvironment and synergize with CAR to exert anti-tumor effects.
在某些实施方案中，编码CAR的核酸序列通过自裂解肽的核酸序列与另外的生物活性分子的核酸序列连接。CAR可以在另外的生物活性分子的N端或C端。在某些示例的实施方案中，CAR在另外的生物活性分子的5’端。任何能够引起融合蛋白裂解成两个独立的蛋白的自裂解肽可以应用到本发明中。在某些示例的实施方案中，所述自裂解肽为P2A，优选地具有SEQ ID NO:130所示的序列，其核苷酸序列可以根据基因重组的需要进行优化。在此类实施方案中，包含CAR和另外的生物活性分子的融合蛋白具有如下的结构：In certain embodiments, the nucleic acid sequence encoding CAR is connected to the nucleic acid sequence of another biologically active molecule through the nucleic acid sequence of a self-cleaving peptide. CAR can be at the N-terminus or C-terminus of another biologically active molecule. In certain exemplary embodiments, CAR is at the 5' end of another biologically active molecule. Any self-cleaving peptide that can cause the fusion protein to cleave into two independent proteins can be applied to the present invention. In certain exemplary embodiments, the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 130, and its nucleotide sequence can be optimized according to the needs of genetic recombination. In such embodiments, the fusion protein comprising CAR and another biologically active molecule has the following structure:
N’-信号肽--特异地结合GPC3的胞外抗原结合结构域--间隔结构域—跨膜结构域-胞内信号传导结构域-自裂解肽-信号肽-2--另外的生物活性分子-C’。其中信号肽-2与N末端信号肽相同或不同。N'-signal peptide--extracellular antigen binding domain that specifically binds to GPC3--spacer domain--transmembrane domain--intracellular signal transduction domain--self-cleaving peptide--signal peptide-2--another biologically active molecule-C', wherein the signal peptide-2 is the same as or different from the N-terminal signal peptide.
在某些实施方案中，所述另外的生物活性分子N端的信号肽-2是IL2信号肽(例如，如SEQ ID NO:84所示)。In certain embodiments, the signal peptide-2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (e.g., as shown in SEQ ID NO:84).
嵌合抗原受体的制备Preparation of chimeric antigen receptors
生成嵌合抗原受体以及包含该嵌合抗原受体的免疫效应细胞(例如T细胞)的方法是本领域已知的，可包括用至少一种编码CAR的多核苷酸转染细胞，并在细胞中表达多核苷酸。例如，可将编码本发明的CAR的核酸分子包含于表达载体(例如，慢病毒载体)中，所述表达载体能够在宿主细胞例如T细胞中表达，以制造所述CAR。The method of generating a chimeric antigen receptor and an immune effector cell (eg, T cell) comprising the chimeric antigen receptor is known in the art, and may include transfecting cells with at least one polynucleotide encoding CAR, and expressing the polynucleotide in the cell. For example, the nucleic acid molecule encoding the CAR of the present invention may be included in an expression vector (eg, a lentiviral vector), which can be expressed in a host cell such as a T cell to manufacture the CAR.
因此，本发明第六方面提供了一种分离的核酸分子，其包含编码第五方面所述的嵌合抗原受体的核苷酸序列。Therefore, the sixth aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor according to the fifth aspect.
本领域技术人员理解，由于遗传密码的简并性，编码一种本发明的嵌合抗原受体的 核苷酸序列可以具有多种不同的序列。因此，除非另有说明，否则“编码氨基酸序列的核苷酸序列”包括作为彼此的简并形式且编码相同氨基酸序列的所有核苷酸序列。It is understood by those skilled in the art that due to the degeneracy of the genetic code, a chimeric antigen receptor of the present invention may be encoded by A nucleotide sequence 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.
在某些示例性实施方案中，所述编码第五方面所述的嵌合抗原受体的核苷酸序列选自：(1)SEQ ID NO:72、73、74、75任一项所示的序列或其简并变体；(2)与(1)所述的序列相比基本上相同的序列，例如，与(1)所述相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)所述的序列相比具有一个或更多个核苷酸取代的序列；并且所述序列基本保留了其所源自的核苷酸序列的至少一种生物学活性(例如，能够编码具有以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达GPC3的细胞的能力)。In certain exemplary embodiments, the nucleotide sequence encoding the chimeric antigen receptor described in the fifth aspect is selected from: (1) a sequence shown in any one of SEQ ID NOs: 72, 73, 74, 75, or a degenerate variant thereof; (2) a sequence substantially identical to the sequence described in (1), for example, 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 compared to (1), or a sequence having one or more nucleotide substitutions compared to the sequence described in (1); and the sequence substantially retains at least one biological activity of the nucleotide sequence from which it is derived (for example, being able to encode the ability to direct the specificity and reactivity of immune effector cells to cells expressing GPC3 in a non-MHC restricted manner).
如上文第五方面中所述的，本发明的CAR还可以与另外的生物活性分子共表达，以协同发挥抗肿瘤作用。As described in the fifth aspect above, the CAR of the present invention can also be co-expressed with other biologically active molecules to synergistically exert anti-tumor effects.
因此，本发明第七方面还提供了一种核酸构建体，其包含编码第五方面所述的嵌合抗原受体的第一核酸序列，并且进一步包含编码另外的生物活性分子的第二核酸序列。Therefore, the seventh aspect of the present invention also provides a nucleic acid construct, which comprises a first nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect, and further comprises a second nucleic acid sequence encoding another biologically active molecule.
在某些实施方案中，所述第二核酸序列所编码的另外的生物活性分子具有抗肿瘤活性。In certain embodiments, the additional biologically active molecule encoded by the second nucleic acid sequence has anti-tumor activity.
在某些实施方案中，所述第二核苷酸序列所编码的另外的生物活性分子在其N端进一步包含信号肽-2。In certain embodiments, the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide-2 at its N-terminus.
在某些实施方案中，所述第一核苷酸序列位于所述第二核苷酸序列的上游。In certain embodiments, the first nucleotide sequence is located upstream of the second nucleotide sequence.
在某些实施方案中，所述第一核酸序列和第二核酸序列通过编码自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)的核苷酸序列连接。在某些示例性实施方案中，所述编码自裂解肽的序列连接至所述第一核苷酸序列的3’端，并且连接至所述第二核苷酸序列的5’端。In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof). In certain exemplary embodiments, the sequence encoding the self-cleaving peptide is connected to the 3' end of the first nucleotide sequence and to the 5' end of the second nucleotide sequence.
在某些实施方案中，所述自裂解肽是P2A(例如，如SEQ ID NO:83所示)。In some embodiments, the self-cleaving peptide is P2A (e.g., as shown in SEQ ID NO:83).
在某些示例性实施方案中，第七方面所述的核酸构建体从其5’端至3’端依次包含：编码所述信号肽的核苷酸序列、编码所述抗原结合结构域的核苷酸序列、编码所述间隔结构域的核苷酸序列、编码所述跨膜结构域的核苷酸序列、编码所述胞内信号传导结构域的核苷酸序列、编码所述自裂解肽序列的核苷酸序列、编码所述信号肽-2的核苷 酸序列、编码另外的生物活性分子的核苷酸序列。In certain exemplary embodiments, the nucleic acid construct of the seventh aspect comprises, from its 5' end to its 3' end, in order: a nucleotide sequence encoding the signal peptide, a nucleotide sequence encoding the antigen binding domain, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, a nucleotide sequence encoding the signal peptide-2 Acid sequences, nucleotide sequences encoding other biologically active molecules.
本发明第八方面提供了一种载体，其包含第二方面、第六方面所述的分离的核酸分子，或第七方面所述的核酸构建体。The eighth aspect of the present invention provides a vector comprising the isolated nucleic acid molecule described in the second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect.
在某些实施方案中，所述载体选自DNA载体，RNA载体，质粒，转座子载体，CRISPR/Cas9载体，病毒载体。In certain embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, and a viral vector.
在某些实施方案中，所述载体是表达载体。In certain embodiments, the vector is an expression vector.
在某些实施方案中，所述载体是游离型载体。In certain embodiments, the vector is an episomal vector.
在某些实施方案中，所述载体是病毒载体。In certain embodiments, the vector is a viral vector.
在某些示例性实施方案中，所述病毒载体是慢病毒载体、腺病毒载体或逆转录病毒载体。In certain exemplary embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.
在某些实施方案中，所述载体是游离型或非整合病毒载体，例如整合缺陷型逆转录病毒或慢病毒。In certain embodiments, the vector is an episomal or non-integrating viral vector, such as an integration-defective retrovirus or lentivirus.
本发明第九方面提供了一种宿主细胞，其包含如上第六方面所述的分离的核酸分子、第七方面所述的核酸构建体或第八方面所述的载体。可以通过各种合适的方式将如上所述的载体引入宿主细胞，例如磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、电穿孔、TALEN方法、ZFN方法、非病毒载体介导的转染(例如脂质体)或病毒载体介导的转染(如慢病毒感染，逆转录病毒感染，腺病毒感染)，以及其他用于转移入宿主细胞的物理、化学或生物学手段，如转座子技术，CRISPR-Cas9等技术。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.
在某些实施方案中，所述宿主细胞包含第六方面所述的分离的核酸分子或包含所述核酸分子的载体，所述宿主细胞表达本发明的嵌合抗原受体。In certain embodiments, the host cell comprises the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule, and the host cell expresses the chimeric antigen receptor of the present invention.
在某些实施方案中，所述宿主细胞包含第七方面所述的核酸构建体或包含所述核酸构建体的载体，所述宿主细胞表达本发明的嵌合抗原受体以及另外的生物活性分子。In certain embodiments, the host cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct, and the host cell expresses the chimeric antigen receptor of the present invention and another biologically active molecule.
在某些实施方案中，所述宿主细胞选自哺乳动物(如人)的免疫细胞。在某些实施方案中，所述免疫细胞来源于患者或健康供体。在某些实施方案中，所述免疫细胞选自T淋巴细胞、自然杀伤(NK)细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合；优选地，所述免疫细胞来源于T淋巴细胞或NK细胞。In certain embodiments, the host cell is selected from the immune cells of mammals (such as humans). In certain embodiments, the immune cells are derived from patients or healthy donors. In certain embodiments, the immune cells are selected from T lymphocytes, natural killer (NK) cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are derived from T lymphocytes or NK cells.
本发明第十方面提供了制备表达本发明的嵌合抗原受体的细胞的方法，其包括：(1)提供宿主细胞；(2)将如第六方面所述的分离的核酸分子或包含所述核酸分子的载体引 入所述宿主细胞，以获得能够表达所述嵌合抗原受体的宿主细胞。还提供了共表达本发明的嵌合抗原受体以及另外的生物活性分子的细胞的方法，其包括：(1)提供宿主细胞；(2)将第七方面所述的核酸构建体或包含所述核酸构建体的载体引入所述宿主细胞，获得能够共表达所述嵌合抗原受体和另外的生物活性分子的宿主细胞。The tenth aspect of the present invention provides a method for preparing a cell expressing a chimeric antigen receptor of the present invention, comprising: (1) providing a host cell; (2) introducing the isolated nucleic acid molecule according to the sixth aspect or a vector comprising the nucleic acid molecule into a host cell; The invention also provides a method for producing a cell that co-expresses 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 of the seventh aspect or a vector comprising the nucleic acid construct into the host cell to obtain a host cell that co-expresses the chimeric antigen receptor and another biologically active molecule.
在某些实施方案中，所述宿主细胞选自免疫细胞，例如T淋巴细胞、NK细胞、单核细胞、树突状细胞、巨噬细胞及其任意组合。在某些实施方案中，所述免疫细胞选自T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及这些细胞的任意组合。In certain embodiments, the host cell is selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages and any combination thereof. In certain embodiments, the immune cell is selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof.
在某些实施方案中，在步骤(1)中，所述宿主细胞提供自患者或者健康供体，并且经过预处理；所述预处理包括免疫细胞的分选、激活和/或增殖；在某些实施方案中，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞。In certain embodiments, in step (1), the host cells are provided from a patient or a healthy donor and are pretreated; the pretreatment includes sorting, activation and/or proliferation of 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.
在某些实施方案中，在步骤(2)中，将核酸分子或载体通过病毒感染引入宿主细胞。在某些实施方案中，在步骤(2)中将核酸分子或载体通过非病毒载体转染的方式引入宿主细胞，如通过转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法、电穿孔方法、磷酸钙转染、DEAE-葡聚糖介导的转染或显微注射等方法。In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host cell by viral infection. In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host 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.
在某些实施方案中，在步骤(2)之后，所述方法还包括：扩增步骤(2)获得的宿主细胞。In certain embodiments, after step (2), the method further comprises: amplifying the host cells obtained in step (2).
经改造的免疫细胞Engineered immune cells
本发明第十一方面还提供了一种经改造的免疫细胞，其包含表达本发明第二方面、第六方面所述的分离的核酸分子。所述经改造的免疫细胞表达第五方面所述的嵌合抗原受体。The eleventh aspect of the present invention further provides a modified immune cell, which comprises and expresses the isolated nucleic acid molecule described in the second aspect and the sixth aspect of the present invention. The modified immune cell expresses the chimeric antigen receptor described in the fifth aspect.
本发明第十二方面还提供了一种经改造的免疫细胞，其包含表达本发明第七方面所述的核酸构建体。所述经改造的免疫细胞表达第五方面所述的嵌合抗原受体以及所述另外的生物活性分子。The twelfth aspect of the present invention further provides a modified immune cell, which comprises and expresses the nucleic acid construct of the seventh aspect of the present invention. The modified immune cell expresses the chimeric antigen receptor of the fifth aspect and the additional biologically active molecule.
在某些实施方案中，所述免疫细胞来源于T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合；优选地，所述免疫细胞得自患者；可选地，所述免疫细胞得自健康供体；优选地，所述免疫细胞来源于T淋巴细胞或NK细胞。In certain embodiments, the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are obtained from patients; alternatively, the immune cells are obtained from healthy donors; preferably, the immune cells are derived from T lymphocytes or NK cells.
在某些实施方案中，所述经改造的免疫细胞的免疫排斥有关的基因(例如，TRAC、TRBC、B2M、HLA-A、HLA-B或HLA-C)和免疫共抑制通路或信号分子的基因(例如，PD- 1、CTLA-4或LAG-3)中的一种或两种靶基因的转录或表达被抑制；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。In certain embodiments, the engineered immune cells express genes related to immune rejection (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) is inhibited; preferably, the transcription or expression of the target gene is inhibited by a method selected from gene knockout (e.g., CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
本发明还提供了制备经改造的免疫细胞的方法，其包括：(1)提供来自患者或者健康供体的免疫细胞；(2)将第六方面所述的分离的核酸分子，或第七方面所述的核酸构建体、或包含它们的载体引入步骤(1)所述的免疫细胞，以获得能够表达以及任选的另外的生物活性分子的免疫细胞。The present invention also provides a method for preparing modified immune cells, which comprises: (1) providing immune cells from a patient or a healthy donor; (2) introducing the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1) to obtain immune cells capable of expressing and optionally other biologically active molecules.
在某些实施方案中，在步骤(1)中，所述免疫细胞经预处理，所述预处理包括免疫细胞的分选、激活和/或增殖；更优选地，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞。In certain embodiments, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of the immune cells; more preferably, 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.
在某些实施方案中，在步骤(2)中将所述核酸分子或载体通过病毒感染引入免疫细胞。In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cells by viral infection.
在某些实施方案中，在步骤(2)中将所述核酸分子或载体通过非病毒载体转染的方式引入免疫细胞，如通过磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法或电穿孔方法。In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.
在某些实施方案中，在步骤(2)之后还包括扩增步骤(2)获得的免疫细胞的步骤。In certain embodiments, the method further comprises the step of expanding the immune cells obtained in step (2) after step (2).
免疫细胞组合物Immune cell composition
在第十三方面，本发明还提供了免疫细胞组合物，所述免疫细胞组合物包括前述任一方面的经改造的免疫细胞，以及可选的未改造和/或未成功改造的免疫细胞，这些未改造和/或未成功改造的免疫细胞不表达目的的CAR。限制于当前的技术水平及一些未知的原因，并不是所有免疫细胞经过改造都能表达目的CAR。而且不表达CAR的免疫细胞也有一定的生物学活性，因此免疫细胞组合物可以含有表达和不表达目的CAR的免疫细胞，该免疫细胞组合物依然能够满足临床应用的需求。In the thirteenth aspect, the present invention also provides an immune cell composition, which includes the modified immune cells of any of the foregoing aspects, and optional unmodified and/or unsuccessfully modified immune cells, which do not express the target CAR. Limited by the current level of technology and some unknown reasons, not all immune cells can express the target CAR after modification. Moreover, immune cells that do not express CAR also have certain biological activities, so the immune cell composition can contain immune cells that express and do not express the target CAR, and the immune cell composition can still meet the needs of clinical applications.
在某些实施方案中，经改造的表达目的CAR的免疫细胞占免疫细胞组合物总细胞数的大约10％-100％，优选地40％-80％。In certain embodiments, the engineered immune cells expressing the target CAR account for approximately 10%-100%, preferably 40%-80% of the total cell number of the immune cell composition.
在某些实施方案中，免疫细胞组合物被培养成免疫细胞系，因此，另一方面，本发明还提供了含有免疫细胞组合物的免疫细胞系。In certain embodiments, the immune cell composition is cultured into an immune cell line. Therefore, in another aspect, the present invention also provides an immune cell line containing the immune cell composition.
在第十四方面，本发明提供了用于制备上述任一方面所述的经改造的免疫细胞的试剂盒。在某些实施方案中，所述试剂盒包括第六方面所述的分离的核酸分子，或第七 方面所述的核酸构建体、或包含它们的载体，和必要的溶剂，如无菌水，生理盐水，或细胞培养液，如LB培养液，如EliteCell原代T淋巴细胞培养体系(产品编号：PriMed-EliteCell-024)，以及可选的，还包括使用说明书。In a fourteenth aspect, the present invention provides a kit for preparing the modified immune cell described in any of the above aspects. In certain embodiments, the kit comprises the isolated nucleic acid molecule described in the sixth aspect, or the seventh aspect. The nucleic acid constructs described in the aspect, or vectors containing them, 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.
药物组合物Pharmaceutical composition
在第十五方面，本发明提供了一种药物组合物，其含有本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物，以及药学上可接受的载体和/或赋形剂。In the fifteenth aspect, the present invention provides a pharmaceutical composition, which contains the antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which a chimeric antigen receptor is 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 twelfth aspect, or the immune cell composition described in the thirteenth aspect, and a pharmaceutically acceptable carrier and/or excipient.
在某些实施方案中，所述药物组合物还包含另外的药学活性剂，例如具有抗肿瘤活性的药物(例如anti-PD1抗体、anti-PD-L1抗体、anti-CTLA-4抗体、anti-CD3抗体、anti-ASGPR1抗体、索拉菲尼或其衍生物、瑞格菲尼或其衍生物、培美曲塞、顺铂、紫杉醇、吉西他滨、卡培他滨或FOLFIRINOX)。In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug with anti-tumor activity (e.g., anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX).
在某些实施方案中，第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物与所述另外的药学活性剂可以同时、分开或相继施用。In certain embodiments, the antigen binding molecule described in the first aspect, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which a chimeric antigen receptor is 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 twelfth aspect, or the immune cell composition described in the thirteenth aspect and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.
在某些实施方案中，本发明的药物组合物包含：第一方面所述的抗原结合分子。In certain embodiments, the pharmaceutical composition of the present invention comprises: the antigen binding molecule described in the first aspect.
在某些实施方案中，本发明的药物组合物包含：第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、或包含它们的载体。In certain embodiments, the pharmaceutical composition of the present invention comprises: the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, or a vector comprising the same.
在某些实施方案中，本发明的药物组合物包含：第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物。In certain embodiments, the pharmaceutical composition of the present invention comprises: the modified immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect.
本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方 面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。The antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including the CAR construct co-expressing the chimeric antigen receptor and another biologically active molecule), 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 vector of the fourth aspect The host cell described in the first or ninth aspect, the modified immune cell described in the eleventh or twelfth aspect, or the immune cell composition described in the thirteenth aspect can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical 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. In addition, 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.
本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物通过静脉注射或推注给予。The antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including the CAR construct co-expressed by the chimeric antigen receptor and another bioactive molecule), the nucleic acid molecules 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 transformed immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, leaf sheath, endocytoplasmic reticulum groove, groin, bladder, local (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route of administration/mode is parenteral administration (e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It should be understood by the technician that the route of administration and/or mode will change according to the intended purpose. In certain embodiments, the antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which a chimeric antigen receptor is 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 twelfth aspect, or the immune cell composition described in the thirteenth aspect is administered by intravenous injection or push injection.
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免 疫细胞组合物。“预防有效量”是指，足以预防，阻止，或延迟疾病的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct co-expressing a chimeric antigen receptor and another biologically active molecule), 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 twelfth aspect, or the immune cell of the thirteenth aspect. Immune cell composition. "Preventive 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 patients with a disease. The antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressed with a chimeric antigen receptor and 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 twelfth aspect, or the therapeutically effective amount of the immune cell composition described in the thirteenth aspect may vary according to the following factors: the severity of the disease to be treated, the overall 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.
治疗方法及用途Treatment methods and uses
在另一方面，本发明提供了一种用于在受试者(例如人)中预防和/或治疗与GPC3的表达相关的疾病的方法，所述方法包括向有此需要的受试者施用有效量的本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物。On the other hand, 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 antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), 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 twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect.
在某些实施方案中，所述与GPC3的表达相关的疾病选自增生性疾病，例如肿瘤。在某些实施方案中，所述与GPC3的表达相关的疾病是与GPC3的表达相关的非肿瘤相关的适应症。In certain embodiments, the disease associated with 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.
在某些实施方案中，所述肿瘤是GPC3阳性肿瘤。在某些实施方案中，所述肿瘤选自实体瘤(例如肝癌、肝细胞癌、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、卵巢癌、卵巢透明细胞癌、黑色素瘤、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、直结肠癌、胃癌、神经胶质瘤中的一种或其组合。在某些实施方案中，所述肿瘤选自血液肿瘤(例如白血病、淋巴瘤等)。In certain embodiments, the tumor is a GPC3-positive tumor. In certain embodiments, the tumor is selected from solid tumors (e.g., liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma, one or a combination thereof. In certain embodiments, the tumor is selected from blood tumors (e.g., leukemia, lymphoma, etc.).
在某些实施方案中，所述方法包括向所述受试者施用有效量的第一方面所述的抗原结合分子。In certain embodiments, the method comprises administering to the subject an effective amount of the antigen binding molecule of the first aspect.
在某些实施方案中，所述方法包括向所述受试者施用有效量的第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物。 In certain embodiments, the method comprises administering to the subject an effective amount of the engineered immune cell of the eleventh or twelfth aspect, or the immune cell composition of the thirteenth aspect.
在某些实施方案中，所述方法包括以下步骤：(1)提供所述受试者所需的免疫细胞(例如T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突状细胞、或这些细胞的任意组合)；(2)将第二方面或第六方面所述的分离的核酸分子，或第七方面所述的核酸构建体、或包含它们的载体引入步骤(1)所述的免疫细胞；(3)将步骤(2)中获得的免疫细胞施用至所述受试者以进行治疗。In certain embodiments, the method 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 second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1); (3) administering the immune cells obtained in step (2) to the subject for treatment.
在某些实施方案中，所述方法通过剂量分次，例如一次，两次，三次或更多次分开施用部分剂量，向所述受试者施用表达目的CAR的免疫细胞，例如在治疗的第一天施用总剂量的第一百分比，在随后的(例如第二，第三，第四，第五，第六或第七天或更晚)治疗日施用总剂量的第二百分比，例如在随后的(例如第三，第四，第五，第六，第七，第八，第九，第十天或更晚)治疗日施用总剂量的第三百分比(例如，剩余百分比)。In certain embodiments, the method administers immune cells expressing a CAR of interest to the subject by dividing the dose into fractions, e.g., one, two, three or more times, administering a partial dose separately, 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.
在某些实施方案中，在治疗的第一天施用总剂量的10％的细胞，在第二天施用总剂量的30％的细胞，并且在第三天施用总剂量的剩余60％的细胞。In certain embodiments, 10% of the total dose of cells is administered on the first day of treatment, 30% of the total dose of cells is administered on the second day, and the remaining 60% of the total dose of cells is administered on the third day.
在某些实施方案中，在治疗的第一天施用总剂量的50％的细胞，在随后的(例如第二，第三，第四，第五，第六或第七或更晚)治疗日施用总剂量的50％的细胞。在某些实施方案中，在治疗的第一天施用总剂量的1/3的细胞，在随后的(例如第二，第三，第四，第五，第六或第七天或更晚)治疗日施用总剂量的1/3的细胞，在随后的(例如第三，第四，第五，第六，第七，第八，第九，第十天或更晚)施用总剂量的1/3的细胞。In certain embodiments, 50% of the total dose of cells is administered on the first day of treatment, and 50% of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh or later) treatment day. In certain embodiments, 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, and 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.
在某些实施方案中，总细胞剂量包含1×107至10×108个目的CAR阳性免疫细胞，例如包含(1-5)×107至(5-10)×108个目的CAR阳性免疫细胞。In certain embodiments, the total cell dose comprises 1×10 7 to 10×10 8 target CAR-positive immune cells, for example, comprises (1-5)×10 7 to (5-10)×10 8 target CAR-positive immune cells.
在某些实施方案中，医师可以根据病人的状态，肿瘤的大小和阶段，或联合治疗的药物等临床情况来调节剂量或治疗方案。In certain embodiments, the physician may adjust the dosage or treatment regimen based on clinical circumstances such as the patient's condition, tumor size and stage, or combination therapy drugs.
在某些实施方案中，将本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物与另外的试剂联合施用。在某些实施方案中，所述另外的试剂包括(i)增加包含CAR核酸或CAR多肽的细胞(例如表达本发明的CAR的免疫细胞，本发明的经改造的免 疫细胞或免疫细胞组合物)的功效的作用剂；(ii)改善与施用包含CAR核酸或CAR多肽的细胞(例如表达本发明的CAR的免疫细胞，本发明的经改造的免疫细胞或免疫细胞组合物)相关的一种或多种副作用的作用剂；(iii)具有抗肿瘤活性的另外的药学活性剂。这些试剂可以在施用本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物之前、同时或之后施用。In certain embodiments, the antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressed with a chimeric antigen receptor and 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 twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect is administered in combination with another agent. In certain embodiments, the other agents include (i) increasing cells containing CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, modified immune cells of the present invention). (ii) an agent for improving the efficacy of cells (e.g., immune cells expressing the CAR of the present invention, modified immune cells or immune cell compositions) comprising CAR nucleic acids or CAR polypeptides; (iii) another pharmaceutically active agent with anti-tumor activity. These agents can be administered before, simultaneously or after the antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a chimeric antigen receptor and a CAR construct co-expressed with another biologically active molecule), the isolated nucleic acid molecules 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 twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect.
在某些实施方案中，以上所述方法还包括向所述受试者施用第二疗法，所述第二疗法可以是已知用于肿瘤的任何疗法，例如手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其任意组合。In certain embodiments, the 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.
在某些实施方案中，所述第二疗法可以与以上所述的方法分开或联合应用；或，所述第二疗法可以与以上所述的方法同时或相继应用。In certain embodiments, the second therapy 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.
在某些实施方案中，所述受试者可以为哺乳动物，例如人。In certain embodiments, the subject can be a mammal, such as a human.
在另一个方面，提供了本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药物组合物在制备用于预防和/或治疗与GPC3的表达相关的疾病的药物中的用途。前述治疗方法中的剂量，剂型，给药途径，适应症，联合治疗等各个方面都可以应用到所述药物的用途中。In another aspect, the antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including the CAR construct co-expressing the chimeric antigen receptor and another biologically active molecule), 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 twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect are provided for the preparation of a drug for preventing and/or treating a disease associated with the expression of GPC3. The dosage, dosage form, administration route, indication, combination therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.
在另一个方面，提供了本发明第一方面所述的抗原结合分子、第五方面所述的嵌合抗原受体(包括嵌合抗原受体与另外的生物活性分子共表达的CAR构建体)、第二方面或第六方面所述的分离的核酸分子、第七方面所述的核酸构建体、第三方面或第八方面所述的载体、第四方面或第九方面所述的宿主细胞、第十一方面或第十二方面所述的经改造的免疫细胞、或第十三方面所述的免疫细胞组合物、或第十五方面所述的药 物组合物，用于预防和/或治疗与GPC3的表达相关的疾病。前述治疗方法中的剂量，剂型，给药途径，适应症，联合治疗等各个方面都可以应用到所述药物的用途中。In another aspect, the present invention provides the antigen binding molecule of the first aspect, the chimeric antigen receptor of the fifth aspect (including the CAR construct co-expressing the chimeric antigen receptor and another biologically active molecule), 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 twelfth aspect, or the immune cell composition of the thirteenth aspect, or the drug of the fifteenth aspect. The drug composition is used for preventing and/or treating diseases related to the expression of GPC3. The dosage, dosage form, administration route, indications, combined therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.
缩略词
Abbreviations
术语定义Definition of Terms
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、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。In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the following Provides definitions and explanations of relevant terms.
如本文中所使用的，术语“抗原结合分子”是抗体分子或其抗原结合片段。As used herein, the term "antigen binding molecule" is an antibody molecule or an antigen binding fragment thereof.
如本文中所使用的，术语“抗体”指能够通过位于免疫球蛋白分子可变区的至少一个抗原识别位点特异性结合靶(如碳水化合物、多核苷酸、脂质、多肽等)的免疫球蛋白分子。如本文所用，该术语不仅包括完整的多克隆或单克隆抗体，而且包括其片段(例如Fab、Fab'、F(ab')2、Fv)、单链(例如scFv，di-scFv，(scFv)2)和结构域抗体(包括例如鲨鱼和骆驼抗体)、以及包括抗体的融合蛋白、以及包括抗原识别位点的任何其它修饰构型的免疫球蛋白分子。本发明的抗体不受任何特定的产生抗体的方法限制。抗体包括任何类型的抗体，例如IgG、IgA或IgM(或其亚类)，并且抗体不需要属于任何特定的类型。取决于抗体重链恒定区的氨基酸序列，免疫球蛋白可以分配到不同的类型。有五种主要类型的免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中几种可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类型的免疫球蛋白的重链恒定区分别被称为α、δ、ε、γ和μ。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。不同类型的免疫球蛋白的亚基结构和三维构型是众所周知的。重链恒定区由4个结构域(CH1、hinge region、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, 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 specific 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 specific type. Depending on the amino acid sequence of the constant region of the heavy chain of the antibody, 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 α, δ, ε, γ, and μ, respectively. Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known. The heavy chain constant region consists of 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.
抗体的VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDRs和4个FRs组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。The VH and VL regions of antibodies can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form an antigen binding site, respectively. The allocation of amino acids to each region or domain can follow the definitions 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.
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDRs，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可 按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as According to the definition in 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. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. In addition, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).
在本发明中，抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。In the present invention, the CDRs contained in the 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.
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
如本文中所使用的，术语抗体的“抗原结合片段”是指抗体的片段的多肽，例如全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括骆驼Ig、Ig NAR、Fab片段、Fab'片段、F(ab)'2片段、F(ab)'3片段、Fd、Fv、scFv、di-scFv、(scFv)2、微型抗体、双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白(“dsFv”)和单结构域抗体(sdAb，纳米抗体)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques 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 , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv") and single domain antibodies (sdAb, nanobodies) and polypeptides that contain 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.
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab'片段”意指还原连接F(ab')2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。 As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab') 2 fragment" means an antibody fragment 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.
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDRs赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDRs)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. 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 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.
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。As used herein, the term "Fc" means 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.
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在某些实施方案中，VH和VL结构域可以以任何合适的排列彼此相对定位。例如，包含NH2-VH-VH-COOH、NH2-VL-VL-COOH的scFv。所述scFv可以形成任何工程上可能的结构，单链抗体(scFv)，串联抗体(tandem di-scFvs)，双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白，骆驼Ig、IgNAR等。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv)2，其指的是两个或两个以上单个scFv并联而形成抗体。As used herein, the term "scFv" refers to a single polypeptide chain comprising 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, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). 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. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, scFv comprising NH 2 -VH-VH-COOH, NH 2- VL-VL-COOH. The scFv can form any possible engineering structure, single chain antibody (scFv), tandem antibody (tandem di-scFvs), bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond stabilized Fv protein, camel Ig, IgNAR, etc. In certain embodiments of the present invention, scFv can form di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In certain embodiments of the present invention, scFv can form (scFv) 2 , which refers to two or more single scFvs connected in parallel to form an antibody.
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的 能力(Holt,L.等人,生物技术趋势(Trends in Biotechnology),21(11):484-490，2003)。单域抗体也称为纳米抗体(nanobody)。As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody. Ability (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single domain antibodies are also called nanobodies.
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合的能力。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.
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。Antibody antigen-binding fragments (e.g., the antibody fragments described above) 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.
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。Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.
如本文中所使用的，表述“特异性结合”或“特异性针对”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。As used herein, the expression "specific binding" or "specifically directed against" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against 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. In the present invention, the term " KD " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites/antigen complexes. Both "association rate constants" (ka or kon) and "dissociation rate constants" (kdis or koff) can be calculated by concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis/kon is equal to the dissociation constant K D (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K D , kon and kdis values can be measured by any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共 6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (for example, 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), then 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 × 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (a total of 100). 6 positions match). Typically, the two sequences are compared when aligned for maximum identity. Such an alignment can be achieved 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. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol 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 either the Blossum 62 matrix or the 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.
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)。As used herein, the term "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. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues 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. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明 中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention In the present invention, amino acids are usually represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.
如本文中所使用的，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。载体可以包括在细胞中直接自主复制的序列，或可以包括足以允许整合到宿主细胞DNA中的序列。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及病毒载体等。病毒载体的非限制性实例包括，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. 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. 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. 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. In addition, the vector may also contain a replication initiation site.
如本文中所使用的，术语“游离型载体”中游离型是指载体能够复制而不整合到宿主的染色体DNA中并且不由分裂宿主细胞逐渐丧失，还意指所述载体在染色体外或游离地复制。As used herein, the term "episomal vector" means that the vector is capable of replicating without 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.
如本文中所使用的，术语“病毒载体”广泛用以指包括典型地促进核酸分子转移或整合到细胞的基因组中的病毒衍生的核酸元件的核酸分子(例如转移质粒)，或介导核酸转移的病毒颗粒。除了核酸之外，病毒颗粒典型地将包括各种病毒组分并且有时还包括宿主细胞组分。As used herein, the term "viral vector" is broadly used to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes a virally derived nucleic acid element that typically facilitates transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer. In addition to the nucleic acid, the viral particle typically will include various viral components and sometimes host cell components.
术语“病毒载体”可以指能够将核酸转移到细胞中的病毒或病毒颗粒，或指转移的核酸本身。病毒载体和转移质粒含有主要衍生自病毒的结构和/或功能遗传元件。The term "viral vector" 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.
如本文中所使用的，术语“逆转录病毒载体”是指含有主要衍生自逆转录病毒的结构和功能遗传元件或其部分的病毒载体或质粒。As used herein, the term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements primarily derived from retroviruses, or portions thereof.
如本文中所使用的，术语“慢病毒载体”是指含有主要衍生自慢病毒的结构和功能遗传元件或其部分(包括LTR)的病毒载体或质粒。在某些实施方案中，术语“慢病毒载体”、“慢病毒表达载体”可以用以指慢病毒转移质粒和/或感染性慢病毒颗粒。在本文提及元件(例如克隆位点、启动子、调节元件、异源核酸等)时，应理 解，这些元件的序列以RNA形式存在于本发明的慢病毒颗粒中并且以DNA形式存在于本发明的DNA质粒中。As used herein, the term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from a lentivirus or portions thereof (including LTRs). In certain embodiments, the terms "lentiviral vector", "lentiviral expression vector" may be used to refer to a lentiviral transfer plasmid and/or an infectious lentiviral particle. When referring to elements (e.g., cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.) herein, it should be understood that It should be understood that the sequences of these elements are present in the form of RNA in the lentiviral particles of the present invention and in the form of DNA in the DNA plasmid of the present invention.
如本文中所使用的，“整合缺陷型”逆转录病毒或慢病毒是指具有不能将病毒基因组整合到宿主细胞的基因组中的整合酶的逆转录病毒或慢病毒。在某些实施方案中，整合酶蛋白突变以特异性降低其整合酶活性。整合缺陷型慢病毒载体可以通过修饰编码整合酶蛋白的pol基因，产生编码整合缺陷型整合酶的突变pol基因而获得。所述整合缺陷型病毒载体已经描述于专利申请WO 2006/010834中，所述专利申请以全文引用的方式并入本文中。As used herein, an "integration-defective" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that cannot integrate the viral genome into the genome of a host cell. In certain embodiments, the integrase protein is mutated to specifically reduce its integrase activity. An integration-defective lentiviral vector can be obtained by modifying the pol gene encoding the integrase protein to produce a mutant pol gene encoding an integration-defective integrase. The integration-defective viral vector has been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞，免疫细胞(如T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞等)。宿主细胞可以包括单个细胞或细胞群体。As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, 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.
如本文中所使用的，术语“嵌合抗原受体”或“CAR”是指包含至少一个细胞外抗原结合结构域，间隔结构域，跨膜结构域和细胞质信号传导结构域(本文也称为“胞内信号传导结构域”)的重组多肽构建体，其将针对目的抗原(例如GPC3)的基于抗体的特异性与免疫效应细胞活化胞内结构域组合以展现针对表达该目的抗原(例如GPC3)细胞的特异性免疫活性。在本发明中，表述“表达CAR的免疫效应细胞”是指表达CAR并且具有由该CAR的靶向结构域决定的抗原特异性的免疫效应细胞。制造CAR(例如，用于癌症治疗)的方法是本领域已知的，可参见例如，Park等人,Trends Biotechnol.,29:550-557,2011；Grupp等人,N Engl J Med.,368:1509-1518,2013；Han等人,J.Hematol.Oncol.,6:47,2013；PCT专利公开文本WO2012/079000、WO2013/059593；和美国专利公开文本2012/0213783，其全部通过引用整体并入本文。As used herein, the term "chimeric antigen receptor" or "CAR" refers to a 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 antibody-based specificity for a target antigen (e.g., GPC3) with an immune effector cell activation intracellular domain to exhibit specific immune activity for cells expressing the target antigen (e.g., GPC3). In the present invention, 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 (e.g., for cancer treatment) 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 incorporated herein by reference in their entirety.
如本文中所使用的，术语“胞外抗原结合结构域”是指能够特异性结合目的抗原或受体的多肽。该结构域将能够与细胞表面分子相互作用。例如，可以选择胞外抗原结合结构域来识别作为与特定疾病状态相关的靶细胞细胞表面标志物的抗原。As used herein, the term "extracellular antigen binding domain" refers to a polypeptide that is capable of specifically binding to an antigen or receptor of interest. The domain will be capable of interacting with cell surface molecules. For example, 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.
如本文中所使用的，术语“胞内信号传导结构域”是指传导效应信号功能信号并引导细胞进行专门的功能的蛋白质部分。因此，胞内信号传导结构域具有激活表 达CAR的免疫效应细胞的至少一种正常效应子功能的能力。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。As used herein, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector signaling function signals and directs cells to perform specialized functions. The ability of the CAR to achieve at least one normal effector function of the immune effector cell. For example, the effector function of the T cell can be cytolytic activity or helper activity, including the secretion of cytokines.
如本文中所使用的，术语“初级信号传导结构域”是指能够以刺激方式或以抑制方式调节TCR复合物的初级活化的蛋白质部分。以刺激方式作用的初级信号传导结构域通常含有已知为基于免疫受体酪氨酸的活化基序(ITAM)的信号传导基序。含有特别用于本发明中的初级信号传导结构域的ITAM的非限制性实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。As used herein, the term "primary signaling domain" refers to a portion of a protein 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). Non-limiting examples of ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66d.
如本文中所使用的，术语“共刺激信号传导结构域”是指共刺激分子的胞内信号传导结构域。共刺激分子是除抗原受体或Fc受体以外的在结合到抗原后提供T淋巴细胞的高效活化和功能所需的第二信号的细胞表面分子。所述共刺激分子的非限制性实例包括CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)、DAP10。As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules 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.
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：无菌水，生理盐水，pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋 白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the 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, saline, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives. For example, 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 generally understood by those skilled in the art, which are capable of stabilizing 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 In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are 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., a phosphate buffer solution), Ringer's solution, and any combination thereof.
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, 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. In addition, "treatment" can also refer to, compared to the expected survival period (if not receiving treatment), extending the survival period.
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，术语“受试者”是指包括其中可以引出免疫应答的活生物体。在某些实施方式中，所述受试者(例如人)患有肿瘤(例如与GPC3相关的肿瘤)，或者，具有患有上述疾病的风险。As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the term "subject" 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.
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of 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. For example, 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 gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.
如本文中使用的，术语“免疫细胞”是指涉及免疫反应例如涉及促进免疫效应子功能的细胞。免疫细胞的实例包括T细胞(例如α/βT细胞和γ/δT细胞)、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞和骨髓来源巨噬细胞。As used herein, the term "immune cell" refers to a cell involved in an immune response, such as a cell involved in promoting immune effector functions. Examples of immune cells include T cells (e.g., α/β T cells and γ/δ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.
本发明所述的免疫细胞可以是自身的/自体的(“自我”)或非自身的(“非自我”，例如同种异体的、同基因的或异基因的)。如本文中使用的，“自身的”是指来自同一受试者的细胞；“同种异体的”是指与比较细胞遗传不同的同一物种的细胞；“同基因的”是指与比较细胞遗传相同的来自不同受试者的细胞；“异基因 的”是指与比较细胞来自不同物种的细胞。在优选实施例中，本发明的细胞是同种异体的。The immune cells of the present invention can be self/autologous ("self") or non-self ("non-self", e.g., allogeneic, isogenic, or xenogeneic). As used herein, "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; "xenogeneic" refers to cells from a different subject that are genetically identical to the comparison cells; and "xenogeneic" refers to cells from a different subject that are genetically identical to the comparison cells. "of" refers to cells that are from a different species than the comparison cells. In a preferred embodiment, the cells of the invention are allogeneic.
可用于本文所述的CAR的示例性免疫细胞包括T淋巴细胞和/或NK细胞。术语“T细胞”或“T淋巴细胞”是本领域公知的并且意图包括胸腺细胞、未成熟的T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化的T淋巴细胞。T细胞可以是T辅助(Th)细胞，例如T辅助1(Th1)或T辅助2(Th2)细胞。T细胞可以是辅助T细胞(HTL；CD4T细胞)CD4T细胞、细胞毒性T细胞(CTL；CD8T细胞)、CD4CD8T细胞、CD4CD8T细胞或任何其它T细胞子组。在某些实施方案中，T细胞可以包括原初T细胞和记忆T细胞。Exemplary immune cells that can be used for CAR described herein include T lymphocytes and/or NK cells. The term "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. In certain embodiments, T cells may include primary T cells and memory T cells.
本领域技术人员将理解，其它细胞也可以用作具有如本文所述的CAR的免疫细胞。具体来说，免疫细胞还包括NK细胞、单核细胞、巨噬细胞或树突状细胞、NKT细胞、嗜中性白细胞和巨噬细胞。免疫细胞还包括免疫细胞的祖细胞，其中所述祖细胞可以在体内或体外经诱导以分化成免疫细胞。因此，在某些实施方案中，免疫细胞包括免疫细胞的祖细胞，例如含于衍生自脐血、骨髓或流动周边血液的CD34+细胞群体内的造血干细胞(HSC)，其在受试者中投与后分化成成熟免疫细胞，或其可以在体外经诱导以分化成成熟免疫细胞。It will be appreciated by those skilled in the art that other cells may also be used as immune cells with CAR as described herein. Specifically, immune cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils and macrophages. Immune cells also include progenitor cells of immune cells, wherein the progenitor cells can be induced in vivo or in vitro to differentiate into immune cells. Therefore, in certain embodiments, 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.
如本文中使用的，术语“经改造的免疫细胞”是指，表达本文所述的任何一种CAR，或导入了本文所述的任何一种分离的核酸或载体的免疫细胞。可以用多种方法将编码CAR多肽的多核苷酸引入细胞后，也可以在细胞中原位合成CAR多肽。或者，可以在细胞外生产CAR多肽，然后将其引入细胞。将多核苷酸构建体引入细胞的方法是本领域已知的。在一些实施方案中，可以使用稳定的转化方法将多核苷酸构建体整合到细胞的基因组中。在其他实施方案中，瞬时转化方法可用于瞬时表达多核苷酸构建体，并且多核苷酸构建体未整合到细胞的基因组中。在其它实施方案中，可以使用病毒介导的方法。多核苷酸可以通过任何合适的方法引入细胞，例如重组病毒载体(例如逆转录病毒、腺病毒)，脂质体等。瞬时转化方法包括，例如但不限于显微注射、电穿孔或微粒轰击。多核苷酸可以包括在载体中，例如质粒载体或病毒载体。As used herein, the term "modified immune cell" refers to an immune cell expressing any CAR described herein, or an immune cell into which any isolated nucleic acid or vector described herein is introduced. After a polynucleotide encoding a CAR polypeptide is introduced into a cell in a variety of ways, the CAR polypeptide can also be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods 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. In other embodiments, a transient transformation method can be used to transiently express a polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, a virus-mediated method can be used. Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but not limited to microinjection, electroporation, or microparticle bombardment. Polynucleotides may be included in a vector, such as a plasmid vector or a viral vector.
如本文中使用的，术语“免疫效应子功能”是指免疫效应细胞的增强或促进对靶细胞的免疫攻击(例如对靶细胞的杀伤，或者抑制其生长或增殖)的功能或反应。例如，T细胞的效应子功能可以是溶细胞活性或辅助活性，包括细胞因子的分泌。 As used herein, the term "immune effector function" refers to 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). For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
如本文中所使用的，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。As used herein, 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.
发明的有益效果Advantageous Effects of the Invention
CAR-T细胞治疗目前在实体瘤中的治疗效果仍不足，其主要原因是实体瘤有复杂的肿瘤微环境和肿瘤异质性高。本发明提供一种靶向GPC3的CAR或包含所述CAR的免疫细胞，通过特异性靶向GPC3提高对于表达肿瘤抗原细胞的杀伤并在一定程度上降低其脱靶毒性，增强CAR-T细胞的肿瘤杀伤效果。The therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly because solid tumors have complex tumor microenvironments and high tumor heterogeneity. The present invention provides a CAR targeting GPC3 or an immune cell comprising the CAR, which improves the killing of cells expressing tumor antigens and reduces their off-target toxicity to a certain extent by specifically targeting GPC3, thereby enhancing the tumor killing effect of CAR-T cells.
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。Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1A显示了CAR-T细胞(空白T，P7D4-T，CE3-T、CB6-T、CH6-T、AB9-T)对HepG2靶细胞的杀伤活性检测结果。Figure 1A shows the results of the killing activity test of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T) against HepG2 target cells.
图1B显示了CAR-T细胞(空白T，P7D4-T，CE3-T、CB6-T、CH6-T、AB9-T)对Huh7靶细胞的杀伤活性检测结果。Figure 1B shows the results of the killing activity test of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T) against Huh7 target cells.
图2A显示了CAR-T细胞(空白T，P7D4-T，CE3-T、CB6-T、CH6-T、AB9-T)激活后的HepG2靶细胞中IFN-γ分泌水平检测结果。Figure 2A shows the results of the detection of IFN-γ secretion levels in HepG2 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
图2B显示了CAR-T细胞(空白T，P7D4-T，CE3-T、CB6-T、CH6-T、AB9-T)激活后的HepG2靶细胞中TNF-α分泌水平检测结果。Figure 2B shows the results of the detection of TNF-α secretion levels in HepG2 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
图2C显示了CAR-T细胞(空白T，P7D4-T，CE3-T、CB6-T、CH6-T、AB9-T)激活后的Huh7靶细胞中IFN-γ分泌水平检测结果。Figure 2C shows the results of the IFN-γ secretion level detection in Huh7 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, AB9-T).
图2D显示了CAR-T细胞(空白T，P7D4-T，CE3-T、CB6-T、CH6-T、AB9-T)激活后的Huh7靶细胞中TNF-α分泌水平检测结果。 Figure 2D shows the results of the detection of TNF-α secretion levels in Huh7 target cells after activation of CAR-T cells (blank T, P7D4-T, CE3-T, CB6-T, CH6-T, and AB9-T).
序列信息Sequence information
本发明涉及的序列信息提供于下面的表1中。







The sequence information involved in the present invention is provided in Table 1 below.







具体实施方式Detailed ways
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention.
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。In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.
实施例1结合人类GPC3的特异性单链抗体(scFv)的制备Example 1 Preparation of specific single-chain antibody (scFv) binding to human GPC3
1)噬菌体库筛选GPC3 scFv1) Phage library screening of 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。 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.
2)ELISA检测单克隆噬菌体2) ELISA detection of monoclonal phage
从噬菌体淘筛产物滴定板中接种单个菌落到96深孔板中，ELISA检测单克隆噬菌体。Single colonies were inoculated from phage panning plates into 96-well plates and monoclonal phages were detected by ELISA.
共筛选得到4株全人源抗GPC3单克隆抗体CB6、AB9、CE3和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和、Chothia和AbM编号系统获得CDR-H1，CDR-H2，CDR-H3，CDR-L1，CDR-L2，CDR-L3的序列(具体序列参见表1和表2)。A total of 4 fully human anti-GPC3 monoclonal antibodies CB6, AB9, CE3 and CH6 were screened. After sequencing and analyzing the above monoclonal antibodies, the sequences of VH and VL were obtained, and the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 were obtained according to the Kabat, IMGT, Chothia and AbM numbering systems (see Tables 1 and 2 for specific sequences).
表2.抗GPC3单克隆抗体的序列
Table 2. Sequences of anti-GPC3 monoclonal antibodies
3)scFv-Fc的构建与抗体聚体分析3) Construction of scFv-Fc and analysis of antibody aggregates
将上述全人源抗体的VH和VL通过linker(SEQ ID NO:76)连接，获得scFv，各scFv的序列信息如下表所示。The 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.
表3：scFv的结构
Table 3: Structure of scFv
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scfv序列(CB6，AB9，CE3，CH6)单体峰(main peak area)大于90％。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 main peak area of the four candidate scFv sequences (CB6, AB9, CE3, CH6) was greater than 90%.
表4：scFv-Fc蛋白SEC数据
Table 4: SEC data of scFv-Fc protein
4)GPC3细胞结合试验4) GPC3 cell binding assay
为鉴定GPC3 scFv-Fc蛋白结合亲和力，选择表达GPC3的细胞系(293T/GPC3+)用于细胞结合测定。以鼠IgG同型抗体(Mouse IgG Isotype Control，来自Thermo Fisher Sci.)作为阴性对照，抗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；AB9候选scFvs对GPC3阳性细胞293T/GPC3+的亲和力接近阳性对照组。To identify the binding affinity of 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 (for related sequences, see WO2017020812) was used as a positive control. The results in Table 5 show that the affinity of CB6 candidate scFvs for GPC3 positive cells 293T/GPC3+ is better than that of the positive control group; the affinity of AB9 candidate scFvs for GPC3 positive cells 293T/GPC3+ is close to that of the positive control group.
表5.GPC3细胞结合试验结果
Table 5. GPC3 cell binding assay results
实施例2慢病毒质粒的构建及病毒包装Example 2 Construction of Lentivirus Plasmid and Virus Packaging
1)慢病毒质粒的构建：1) Construction of lentiviral plasmid:
基于上述实施例中的scFv序列，进一步构建CAR慢病毒表达载体。以CD137(4-1BB)的胞内结构域和CD3ζ的ITAM区作为激活信号，与上述scFv进行融合，同时加上CD8α信号肽，CD8铰链区，CD8跨膜区，构建嵌合抗原受体表达载体，构建的嵌合抗原受体结构如下表6所示。Based on the scFv sequence in the above embodiment, a CAR lentiviral expression vector was further constructed. The intracellular domain of CD137 (4-1BB) and the ITAM region of CD3ζ were used as activation signals, fused with the above scFv, and CD8α 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.
表6.嵌合抗原受体的结构

Table 6. Structure of chimeric antigen receptors

2)病毒包装：2) Virus packaging:
将以上构建的CAR慢病毒质粒与转染试剂混合液逐滴加入到293T(来源：ATCC)细胞中，轻轻晃动培养皿，充分混匀。将培养皿置于37℃、5％CO2培养箱；培养6～8小时后，将含有转染试剂的培养基去掉，更换为新鲜的完全培养基。连续培养48小时后，收集培养皿中含有病毒的培养基上清，用0.45μm的滤膜过滤，转至离心管中，配平后，20000×g 4℃离心2小时。离心结束后，在生物安全柜中，小心将离心管中的液体吸去，加入500μL PBS缓冲液将沉淀重悬，将病毒置于-80℃保存。Add the above-constructed CAR lentiviral plasmid and transfection reagent mixture dropwise to 293T (source: ATCC) cells, gently shake the culture dish and mix thoroughly. Place the culture dish in a 37°C, 5% CO2 incubator; after 6 to 8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium. After 48 hours of continuous culture, collect the supernatant of the culture medium containing the virus in the culture dish, filter it with a 0.45μm filter membrane, transfer it to a centrifuge tube, balance it, and centrifuge it at 20000×g 4°C for 2 hours. After the centrifugation is completed, in the biosafety cabinet, carefully aspirate the liquid in the centrifuge tube, add 500μL PBS buffer to resuspend the precipitate, and store the virus at -80°C.
实施例3 CAR-T细胞制备Example 3 CAR-T cell preparation
1)原代T细胞分离：1) Isolation of primary T cells:
(1)采用淋巴细胞分离液(GE)分离得到人的PBMC细胞，置于37℃，5％CO2的培养箱中培养，加入100μL/mL的CD3抗体和CD28抗体，充分混匀后，室温孵育15分钟。(1) Human PBMC cells were isolated using lymphocyte separation medium (GE), cultured in an incubator at 37°C and 5% CO2 , 100 μL/mL of CD3 antibody and CD28 antibody were added, mixed thoroughly, and incubated at room temperature for 15 minutes.
(2)取出磁珠，用移液枪上下吹打至少5次，充分混匀。(2) Remove the magnetic beads and mix thoroughly by pipetting up and down at least 5 times.
(3)吸取50μL磁珠/mL至上述样品中，充分混匀后，室温孵育10分钟。(3) Pipette 50 μL of magnetic beads/mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes.
(4)添加完全培养基至管内总体积为2.5mL，将管子(开盖)插入磁极中，室温静置5分钟。(4) Add complete medium to a total volume of 2.5 mL in the tube, insert the tube (with the lid open) into the magnet, and let it stand at room temperature for 5 minutes.
(5)孵育完毕后，管子继续留在磁极中，轻轻倒置，将管内的细胞倒出。(5) After incubation, the tube remains in the magnet and is gently inverted to pour out the cells.
(6)将细胞重悬于X-vivo 15培养基中，并添加10％FBS，300U/mL IL-2,5ng/mL IL-15和10ng/mL IL-7。(6) Resuspend the cells in X-vivo 15 medium and add 10% FBS, 300 U/mL IL-2, 5 ng/mL IL-15 and 10 ng/mL IL-7.
2)T细胞的激活：2) T cell activation:
调整细胞密度至1×106细胞/mL，加入细胞因子及抗体复合物(按终浓度为300U/mL的IL-2、10ng/mL IL-7、5ng/mL IL-15、500ng/mL Anti-CD3(OKT3)、2μg/mL Anti-CD28配置)，连续培养48小时。The cell density was adjusted to 1×10 6 cells/mL, and cytokine and antibody complexes (configured at a final concentration of 300 U/mL of IL-2, 10 ng/mL of IL-7, 5 ng/mL of IL-15, 500 ng/mL of Anti-CD3 (OKT3), and 2 μg/mL of Anti-CD28) were added and cultured for 48 hours.
3)病毒感染：3) Viral infection:
(1)按照MOI＝5，计算所需要的病毒量。计算公式如下：所需病毒量(mL)＝(MOI*细胞数量)/病毒滴度 (1) Calculate the required amount of virus according to MOI = 5. The calculation formula is as follows: Required amount of virus (mL) = (MOI * number of cells) / virus titer
(2)将病毒迅速复温到37℃。在六孔板中加入上述计算所得的病毒量，添加终浓度为5μg/mL的DEAE，充分混匀后，离心。(2) Rapidly rewarm the virus to 37°C. Add the amount of virus calculated above to a six-well plate, add DEAE at a final concentration of 5 μg/mL, mix thoroughly, and centrifuge.
(3)离心结束后，将六孔板置于37℃5％CO2的培养箱中，继续培养备用。(3) After centrifugation, place the six-well plate in a 37°C 5% CO 2 incubator and continue culturing for later use.
(4)250×g离心10分钟，去掉含有病毒的培养基上清，用新鲜培养基重悬细胞沉淀，将细胞转移至新的六孔板中，继续培养3-6天备用。(4) Centrifuge at 250 × g for 10 min, remove the supernatant containing the virus, resuspend the cell pellet with fresh culture medium, transfer the cells to a new six-well plate, and continue culturing for 3-6 days before use.
通过上述方法分别获得表达实施例2中所述的CAR(CB6-T、AB9-T、CE3-T和CH6-T)的CAR-T细胞。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.
实施例4 CAR-T细胞的阳性率检测Example 4 Detection of the positive rate of CAR-T cells
编码CAR的核酸序列在启动子的驱动下表达，使用GPC3抗原对慢病毒转染的T细胞进行标记并通过流式进行测定，反映CAR在T细胞表面的表达水平。通过如上方法检测实施例3获得的CAR-T细胞的CAR阳性率进行检测，FACS检测结果如下表7所示。结果显示，所有CAR-T细胞的CAR阳性率均大于10％，表明慢病毒转染效应细胞后，成功表达了CAR，成功构建了表达了4种GPC3-CAR嵌合抗原受体T细胞(CB6-T、AB9-T、CE3-T和CH6-T)及对照嵌合抗原受体T细胞(P7D4-T)。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 the 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 showed that the CAR positive rate of all CAR-T cells was greater than 10%, indicating that CAR was successfully expressed after lentivirus transfection of effector cells, and 4 GPC3-CAR chimeric antigen receptor T cells (CB6-T, AB9-T, CE3-T and CH6-T) and control chimeric antigen receptor T cells (P7D4-T) were successfully constructed.
表7：CAR的阳性率检测结果
Table 7: CAR positive rate test results
实施例5 CAR-T对HepG2靶细胞的杀伤活性评价Example 5 Evaluation of the killing activity of CAR-T on HepG2 target cells
使用0.25％胰酶消化HEPG2-luc细胞和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/孔的量接种靶细胞HEPG2-luc于96孔板中，5％CO2 37℃培养箱静置30min。收集CAR-T，离心收集并用10％FBS的1640培养基重悬CAR-T细胞，GPC3-CAR以及未转染CAR的空白T细胞(UTD)作为效应细胞，然后按照不同的E/T(效应细胞/靶细胞)比例加入到含有HEPG2-luc的96孔板中，100μL/孔，最终体积补至200μL/孔，5％CO2 37℃培养箱中培养18～24h。培养结束后，将孔板从培养箱中取出，加入20ul荧光检测试剂，使用酶标仪检测荧光读值。 HEPG2-luc cells 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 were inoculated in a 96-well plate at 100 μL/well, and the cells were placed in a 5% CO 2 37°C incubator for 30 minutes. CAR-T was 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 a 96-well plate containing HEPG2-luc at different E/T (effector cell/target cell) ratios, 100 μL/well, and the final volume was supplemented to 200 μL/well, and cultured in a 5% CO 2 37°C incubator for 18 to 24 hours. After the culture was completed, the well plate was removed from the incubator, 20ul of fluorescence detection reagent was added, and the fluorescence reading was detected using an enzyme reader.
CAR-T的杀伤活性检测结果如图1，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)和不同的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，对肿瘤细胞的裂解率高达约99％。The results of the CAR-T killing activity test are shown in Figure 1. 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. When the effector cell/target cell ratio is 1, the lysis rate of tumor cells is as high as about 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Example 6 Cytokine release when CAR-T cells are co-incubated with HepG2 and Huh7 target cells
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)。HepG2-luc and Huh7-luc cells were collected, and the cell density was adjusted to 1×10 5 /mL using culture medium. The target cells were inoculated in a 96-well plate at 100 μL/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 μL/well, and the final volume was supplemented to 200 μL/well, and cultured in a 5% CO 2 37°C incubator overnight. After the culture was completed, the well plate was removed from the incubator, centrifuged, and the supernatant was taken. The cytokine release was detected using an ELISA kit (IFN-γ, TNF-α). The four types of CAR-T cells (CB6-T, AB9-T, CE3-T, and CH6-T) constructed in the present application can kill tumor cells to varying degrees and release IFN-γ (with HepG2 as the target cell, the test results are shown in FIG. 2A ; with Huh7 as the target cell, the test results are shown in FIG. 2C ) and TNF-α (with HepG2 as the target cell, the test results are shown in FIG. 2B ; with Huh7 as the target cell, the test results are shown in FIG. 2D ).
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。 Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims (31)

  1. 嵌合抗原受体，其包含抗原结合结构域，所述抗原结合结构域包含如下的互补决定区(CDRs)：A chimeric antigen receptor comprising an antigen binding domain comprising the following complementarity determining regions (CDRs):
    (a)SEQ ID NO:1所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:2所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(a) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:1; and/or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:2;
    或，or,
    (b)SEQ ID NO:3所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:4所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(b) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:3; and/or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:4;
    或，or,
    (c)SEQ ID NO:5所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:6所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(c) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:5; and/or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:6;
    或，or,
    (d)SEQ ID NO:7所示的重链可变区(VH)中含有的CDR-H1、CDR-H2以及CDR-H3；和/或，SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR-L1、CDR-L2以及CDR-L3；(d) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:7; and/or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:8;
    或，or,
    (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；(e) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and/or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and/or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and/or light chain variable region of any one of (a) to (d), and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution;
    优选地，所述CDR根据Kabat、IMGT、Chothia或AbM编号系统定义。Preferably, the CDRs are defined according to the Kabat, IMGT, Chothia or AbM numbering systems.
  2. 权利要求1所述的嵌合抗原受体，其中，所述抗原结合结构域包含：The chimeric antigen receptor of claim 1, wherein the antigen binding domain comprises:
    (1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义： (1) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:
    (1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：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；(1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 9 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 10 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 11 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 12 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 14 or a variant thereof;
    或，or,
    (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；(1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 24 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 25 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 26 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 27 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 29 or a variant thereof;
    或，or,
    (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，(1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 38 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 39 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 40 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 41 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 42 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 43 or a variant thereof; or,
    (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；(1d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 53 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 56 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 57 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 58 or a variant thereof;
    或，or,
    (2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(2) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:
    (2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：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； (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 15 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 16 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 17 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 18 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 19 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 14 or a variant thereof;
    或，or,
    (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；(2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 30 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 31 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 32 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 33 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 34 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 29 or a variant thereof;
    或，or,
    (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，(2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 44 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 45 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 46 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 47 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 48 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 43 or a variant thereof; or,
    (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；(2d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 59 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 62 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 63 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 58 or a variant thereof;
    或，or,
    (3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(3) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:
    (3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：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；(3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 20 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 12 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 14 or a variant thereof;
    或，or,
    (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；(3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 35 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 21 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 26 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 27 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 29 or a variant thereof. CDR-L3 of the body;
    或，or,
    (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，(3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 49 or a variant thereof; 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; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 41 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 42 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 43 or a variant thereof; or,
    (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；(3d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 64 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 56 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 57 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 58 or a variant thereof;
    或，or,
    (4)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：(4) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:
    (4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：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；(4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 22 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 12 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 14 or a variant thereof;
    或，or,
    (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；(4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 36 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 27 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 29 or a variant thereof;
    或，or,
    (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；(4c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 51 or a variant thereof; 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; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 51 or a variant thereof CDR-L1 of the present invention; CDR-L2 of SEQ ID NO: 42 or a variant thereof; CDR-L3 of SEQ ID NO: 43 or a variant thereof;
    或，or,
    (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；(4d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 66 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 67 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 55 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 56 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 57 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 58 or a variant thereof;
    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。Among them, the variant described in any one of (1a)-(1d), (2a)-(2d), (3a)-(3d), and (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; preferably, the substitutions are conservative substitutions.
  3. 权利要求1或2所述的嵌合抗原受体，其中，所述抗原结合结构域包含：The chimeric antigen receptor of claim 1 or 2, wherein the antigen binding domain comprises:
    (a)包含如SEQ ID NO:1所示的序列或其变体的VH和/或包含如SEQ ID NO:2所示的序列或其变体的VL；(a) a 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;
    或，or,
    (b)包含如SEQ ID NO:3所示的序列或其变体的VH和/或包含如SEQ ID NO:4所示的序列或其变体的VL；(b) a VH comprising the sequence shown in SEQ ID NO:3 or a variant thereof and/or a VL comprising the sequence shown in SEQ ID NO:4 or a variant thereof;
    或，or,
    (c)包含如SEQ ID NO:5所示的序列或其变体的VH和/或包含如SEQ ID NO:6所示的序列或其变体的VL；(c) a 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;
    或or
    (d)包含如SEQ ID NO:7所示的序列或其变体的VH和/或包含如SEQ ID NO:8所示的序列或其变体的VL；(d) a 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;
    其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。 Wherein, 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; preferably, the substitutions are conservative substitutions.
  4. 权利要求1-3任一项所述的嵌合抗原受体，其中，所述抗原结合结构域选自全长抗体、Fab片段、Fab'片段、Fab'-SH、F(ab)'2片段、F(ab)'3片段、Fv片段、单链抗体(例如scFv、di-scFv或(scFv)2)、微型抗体、二硫键稳定的Fv蛋白(dsFv)、单结构域抗体(sdAb，纳米抗体)、双抗体(diabody)、双特异性抗体和多特异性抗体。The chimeric antigen receptor of any one of claims 1 to 3, wherein the antigen binding domain is selected from a full-length antibody, a Fab fragment, a Fab' fragment, a Fab'-SH, a F(ab)' 2 fragment, a F(ab)' 3 fragment, a Fv fragment, a single-chain antibody (e.g., scFv, di-scFv or (scFv) 2 ), a minibody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb, nanobody), a diabody, a bispecific antibody and a multispecific antibody.
  5. 权利要求1-4任一项所述的嵌合抗原受体，其中，所述抗原结合结构域是单链抗体，例如scFv、di-scFv或(scFv)2；The chimeric antigen receptor of any one of claims 1 to 4, wherein the antigen binding domain is a single chain antibody, such as scFv, di-scFv or (scFv)2;
    优选地，所述单链抗体从其N端至C端依次包括：Preferably, the single-chain antibody comprises, from its N-terminus to its C-terminus:
    (1)包含如SEQ ID NO:1所示的序列或其变体的VH-连接子-包含如SEQ ID NO:2所示的序列或其变体的VL；或，包含如SEQ ID NO:2所示的序列或其变体的VL-连接子-包含如SEQ ID NO:1所示的序列或其变体的VH；(1) a VH-linker comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof; or, a 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;
    或or
    (2)包含如SEQ ID NO:3所示的序列或其变体的VH-连接子-包含如SEQ ID NO:4所示的序列或其变体的VL；或，包含如SEQ ID NO:4所示的序列或其变体的VL-连接子-包含如SEQ ID NO:3所示的序列或其变体的VH；(2) a VH-linker comprising a sequence as shown in SEQ ID NO: 3 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 4 or a variant thereof; or, a 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;
    或or
    (3)包含如SEQ ID NO:5所示的序列或其变体的VH-连接子-包含如SEQ ID NO:6所示的序列或其变体的VL；或，包含如SEQ ID NO:6所示的序列或其变体的VL-连接子-包含如SEQ ID NO:5所示的序列或其变体的VH；(3) a VH-linker comprising a sequence as shown in SEQ ID NO: 5 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 6 or a variant thereof; or, a 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;
    或or
    (4)包含如SEQ ID NO:7所示的序列或其变体的VH-连接子-包含如SEQ ID NO:8所示的序列或其变体的VL；或，包含如SEQ ID NO:8所示的序列或其变体的VL-连接子-包含如SEQ ID NO:7所示的序列或其变体的VH；(4) a VH-linker comprising a sequence as shown in SEQ ID NO: 7 or a variant thereof-a VL comprising a sequence as shown in SEQ ID NO: 8 or a variant thereof; or, a 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;
    其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；wherein 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 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; preferably, the substitutions are conservative substitutions;
    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。Preferably, the linker is a polypeptide; preferably, the linker comprises one or several (e.g., 1, 2 or 3) sequences as shown in (G m S) 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; more preferably, the linker has the sequence of SEQ ID NO: 76.
  6. 权利要求1-5任一项所述的嵌合抗原受体，其中，所述抗原结合结构域是单链抗体，所述单链抗体包含SEQ ID NO:68,69,70,71任一项所示的序列或其变体，所述变体与SEQ ID NO:68,69,70,71相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个，或10个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。The chimeric antigen receptor of any one of claims 1-5, wherein the antigen binding domain is a single-chain antibody, and the single-chain antibody comprises the sequence shown in any one of SEQ ID NO:68,69,70,71 or a variant thereof, and the variant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:68,69,70,71, or has 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); preferably, the substitutions are conservative substitutions.
  7. 权利要求1-6任一项所述的嵌合抗原受体，其进一步包含跨膜结构域，所述跨膜结构域选自下列蛋白的跨膜区：T细胞受体的α、β或ζ链、CD28、CD45、CD3ε、CD3ζ、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD152、CD154和PD-1；The chimeric antigen receptor of any one of claims 1 to 6, further comprising a transmembrane domain, wherein the transmembrane domain is selected from the transmembrane region of the following proteins: α, β or ζ chain of T cell receptor, CD28, CD45, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1;
    优选地，所述跨膜结构域选自下列蛋白的跨膜区：CD8、CD28、CD4、PD-1、CD152和CD154；Preferably, the transmembrane domain is selected from the transmembrane regions of the following proteins: CD8, CD28, CD4, PD-1, CD152 and CD154;
    优选地，所述跨膜结构域包含SEQ ID NO:77所示的CD8跨膜区。Preferably, the transmembrane domain comprises the CD8 transmembrane region shown in SEQ ID NO:77.
  8. 权利要求1-7任一项所述的嵌合抗原受体，其进一步包含间隔结构域，所述间隔结构域位于抗原结合结构域与跨膜结构域之间，所述间隔结构域选自铰链结构域和/或免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区；The chimeric antigen receptor of any one of claims 1 to 7, further comprising a spacer domain, wherein the spacer domain is located between the antigen binding domain and the transmembrane domain, and the spacer domain is selected from the hinge domain and/or the CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4);
    优选地，所述铰链结构域包含CD8、IgG4、PD-1、CD152或CD154的铰链区；更优选地，所述铰链结构域包含SEQ ID NO:78所示的CD8铰链区。Preferably, the hinge domain comprises the hinge region of CD8, IgG4, PD-1, CD152 or CD154; more preferably, the hinge domain comprises the CD8 hinge region shown in SEQ ID NO:78.
  9. 权利要求1-8任一项所述的嵌合抗原受体，其进一步包含胞内信号传导结构域，所述胞内信号传导结构域包含初级信号传导结构域和/或共刺激信号传导结构域；The chimeric antigen receptor of any one of claims 1 to 8, further comprising an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary signaling domain and/or a co-stimulatory signaling domain;
    优选地，所述胞内信号传导结构域从N端到C端依次包含共刺激信号传导结构域和初级信号传导结构域；Preferably, the intracellular signaling domain comprises a co-stimulatory signaling domain and a primary signaling domain in sequence from the N-terminus to the C-terminus;
    优选地，所述胞内信号传导结构域包含初级信号传导结构域以及至少一个共刺激信 号传导结构域；Preferably, the intracellular signaling domain comprises a primary signaling domain and at least one co-stimulatory signaling domain. signal transduction domain;
    优选地，所述初级信号传导结构域包含免疫受体酪氨酸活化基序(ITAM)；Preferably, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM);
    优选地，所述初级信号传导结构域包含选自以下蛋白的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CDS、CD22、CD79a、CD79b或CD66d；更优选地，所述初级信号传导结构域包含SEQ ID NO:113所示的CD3ζ胞内信号传导结构域；Preferably, the primary signaling domain comprises an intracellular signaling domain selected from the following proteins: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b or CD66d; More preferably, the primary signaling domain comprises the CD3ζ intracellular signaling domain shown in SEQ ID NO: 113;
    优选地，所述共刺激信号传导结构域包含选自下列蛋白的胞内信号传导结构域：CARD11、CD2、CD7、CD27、CD28、CD30、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)或DAP10；Preferably, the co-stimulatory signaling domain comprises an intracellular signaling domain selected from the following proteins: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10;
    优选地，所述共刺激信号传导结构域选自CD28的胞内信号传导结构域或CD137(4-1BB)的胞内信号传导结构域或二者片段的组合；更优选地，所述共刺激信号传导结构域包含SEQ ID NO:80所示的CD137(4-1BB)胞内信号传导结构域；Preferably, 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 thereof; more preferably, the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) shown in SEQ ID NO: 80;
    更优选地，所述胞内信号传导结构域序列包含SEQ ID NO:81所示的序列。More preferably, the intracellular signaling domain sequence comprises the sequence shown in SEQ ID NO:81.
  10. 权利要求1-9任一项所述的嵌合抗原受体，其中，所述嵌合抗原受体进一步在其N端包含信号肽；The chimeric antigen receptor according to any one of claims 1 to 9, wherein the chimeric antigen receptor further comprises a signal peptide at its N-terminus;
    优选地，所述信号肽包含重链信号肽(例如IgG1的重链信号肽)、粒细胞-巨噬细胞集落刺激因子受体2(GM-CSFR2)信号肽、IL2信号肽、或CD8α信号肽；更优选地，所述信号肽包含SEQ ID NO:82所示的序列。Preferably, 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 CD8α signal peptide; more preferably, the signal peptide comprises the sequence shown in SEQ ID NO:82.
  11. 权利要求1-10任一项所述的嵌合抗原受体，其中，所述嵌合抗原受体从其N端至C端依次包含信号肽、抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域；The chimeric antigen receptor according to any one of claims 1 to 10, wherein the chimeric antigen receptor comprises, from its N-terminus to its C-terminus, a signal peptide, an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain;
    优选地，所述信号肽包含IgG1的重链信号肽或CD8α信号肽(例如，序列如SEQ ID NO:82所示的信号肽)；Preferably, the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (for example, a signal peptide having a sequence as shown in SEQ ID NO: 82);
    优选地，所述抗原结合结构域如权利要求1-6任一项中所定义(例如，包含SEQ ID NO:68,69,70,71任一项所示的序列)；Preferably, the antigen binding domain is as defined in any one of claims 1-6 (for example, comprising a sequence as shown in any one of SEQ ID NO: 68, 69, 70, 71);
    优选地，所述间隔结构域包含CD8(例如CD8α)的铰链区(例如，序列如SEQ ID NO:78所示的铰链区)；Preferably, the spacer domain comprises a hinge region of CD8 (e.g., CD8α) (e.g., a hinge region whose sequence is shown in SEQ ID NO: 78);
    优选地，所述跨膜结构域包含CD8(例如CD8α)的跨膜区(例如，序列如SEQ ID  NO:77所示的跨膜区)；Preferably, the transmembrane domain comprises a transmembrane region of CD8 (eg, CD8α) (eg, a sequence such as SEQ ID NO: transmembrane region shown in 77);
    优选地，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域(例如，如SEQ ID NO:79所示序列)，所述共刺激信号传导结构域包含CD137(4-1BB)的胞内信号传导结构域(例如，如SEQ ID NO:80所示序列)；更优选地，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO:81所示序列；Preferably, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., a sequence as shown in SEQ ID NO: 79), and the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., a sequence as shown in SEQ ID NO: 80); more preferably, the intracellular signaling domain of the chimeric antigen receptor has a sequence as shown in SEQ ID NO: 81;
    优选地，所述嵌合抗原受体包含SEQ ID NO:85、86、87、88任一项所示的序列或其变体，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换为保守置换。Preferably, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NO:85, 86, 87, 88 or a variant thereof, and the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.
  12. 权利要求1-11任一项所述的嵌合抗原受体，其中，所述抗原结合结构域包含权利要求1-6任一项中所定义的抗原结合结构域作为第一抗原结合结构域，并且进一步包含不结合GPC3的第二抗原结合结构域；更优选地，所述第二抗原结合结构域结合的抗原选自：PD-1、PD-L1、CTLA-4、CD3、ASGPR1、CD19、MSLN、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2；The chimeric antigen receptor of any one of claims 1 to 11, wherein the antigen binding domain comprises the antigen binding domain defined in any one of claims 1 to 6 as a first antigen binding domain, and further comprises a second antigen binding domain that does not bind to GPC3; more preferably, the antigen bound by the second antigen binding domain is selected from: PD-1, PD-L1, CTLA-4, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2;
    优选地，所述抗原结合结构域是单链抗体，例如scFv、di-scFv或(scFv)2；Preferably, the antigen binding domain is a single chain antibody, such as scFv, di-scFv or (scFv) 2 ;
    优选地，所述抗原结合结构域中的VH和VL通过连接子连接；优选地，所述连接子包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为1或2；更优选地，所述连接子具有SEQ ID NO：76的序列；Preferably, the VH and VL in the antigen binding domain are connected by a linker; preferably, the linker comprises one or several (e.g., 1, 2 or 3) sequences as shown in (G m S) 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; more preferably, the linker has the sequence of SEQ ID NO: 76;
    优选地，所述第一抗原结合结构域和第二抗原结合结构域通过自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)连接；Preferably, the first antigen binding domain and the second antigen binding domain are connected by a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof);
    优选地，所述自裂解肽是P2A；例如，所述自裂解肽的氨基酸序列如SEQ ID NO:83所示；Preferably, the self-cleaving peptide is P2A; for example, the amino acid sequence of the self-cleaving peptide is shown in SEQ ID NO:83;
    优选地，所述第二抗原结合结构域在其N端进一步包含信号肽；优选地，所述信号肽不同于所述GPC3特异性嵌合抗原受体所包含的信号肽；优选地，所述第二抗原结合结构域N端的信号肽是IL2信号肽(例如，氨基酸序列如SEQ ID NO:84所示)。 Preferably, the second antigen binding domain further comprises a signal peptide at its N-terminus; preferably, the signal peptide is different from the signal peptide contained in the GPC3-specific chimeric antigen receptor; preferably, the signal peptide at the N-terminus of the second antigen binding domain is an IL2 signal peptide (e.g., the amino acid sequence is shown in SEQ ID NO: 84).
  13. 分离的核酸分子，其包含编码权利要求1-12任一项所述的嵌合抗原受体的核苷酸序列。An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 1 to 12.
  14. 权利要求13所述的分离的核酸分子，其包含选自下列核苷酸序列：(1)SEQ ID NO:72、73、74、75任一项所示的序列或其简并变体；(2)与(1)中任一项所示序列相比基本上相同的序列(例如，与(1)中任一项所示的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)中任一项所示的序列相比具有一个或更多个核苷酸取代的序列)。The isolated nucleic acid molecule of claim 13, comprising a nucleotide sequence selected from the following: (1) a sequence shown in any one of SEQ ID NOs: 72, 73, 74, 75, or a degenerate variant thereof; (2) a sequence substantially identical to the sequence shown in any one of (1) (for example, 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 compared to the sequence shown in any one of (1) or a sequence having one or more nucleotide substitutions compared to the sequence shown in any one of (1)).
  15. 核酸构建体，其包含编码GPC3特异性嵌合抗原受体的核酸序列；其中，所述GPC3特异性嵌合抗原受体包含靶向GPC3的抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域，所述靶向GPC3的抗原结合结构域包含权利要求1-6任一项中定义的抗原结合结构域。A nucleic acid construct comprising a nucleic acid sequence encoding a GPC3-specific chimeric antigen receptor; wherein the GPC3-specific chimeric antigen receptor comprises an antigen binding domain targeting GPC3, a spacer domain, a transmembrane domain, and an intracellular signaling domain, and the antigen binding domain targeting GPC3 comprises the antigen binding domain defined in any one of claims 1 to 6.
  16. 权利要求15所述的核酸构建体，其具备以下特征中的一项或多项：The nucleic acid construct of claim 15, which has one or more of the following characteristics:
    (1)所述跨膜结构域如权利要求7中定义；(1) The transmembrane domain is as defined in claim 7;
    (2)所述间隔结构域如权利要求8中定义；(2) The spacer domain is defined in claim 8;
    (3)所述胞内信号传导结构域如权利要求9中定义；(3) The intracellular signaling domain is as defined in claim 9;
    (4)所述核酸序列所编码的GPC3特异性嵌合抗原受体进一步在其N端包含信号肽，所述信号肽如权利要求10中定义。(4) The GPC3-specific chimeric antigen receptor encoded by the nucleic acid sequence further comprises a signal peptide at its N-terminus, wherein the signal peptide is as defined in claim 10.
  17. 权利要求15-16任一项所述的核酸构建体，其中，所述核酸构建体从其5’端至3’端依次包含：编码所述信号肽的核苷酸序列、编码所述靶向GPC3的抗原结合结构域的核苷酸序列、编码所述间隔结构域的核苷酸序列、编码所述跨膜结构域的核苷酸序列、编码所述胞内信号传导结构域的核苷酸序列；The nucleic acid construct according to any one of claims 15 to 16, wherein the nucleic acid construct comprises, from its 5' end to its 3' end, in order: a nucleotide sequence encoding the signal peptide, a nucleotide sequence encoding the antigen binding domain targeting GPC3, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, and a nucleotide sequence encoding the intracellular signaling domain;
    优选地，所述信号肽包含IgG1的重链信号肽或CD8α信号肽(例如，序列如SEQ ID NO:82所示的信号肽)； Preferably, the signal peptide comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (eg, a signal peptide having a sequence as shown in SEQ ID NO: 82);
    优选地，所述靶向GPC3的抗原结合结构域选自权利要求1-6任一项中所定义的抗原结合结构域(例如，包含SEQ ID NO:68,69,70,71任一项所示的序列)；Preferably, the antigen binding domain targeting GPC3 is selected from the antigen binding domain defined in any one of claims 1 to 6 (for example, comprising a sequence shown in any one of SEQ ID NO: 68, 69, 70, 71);
    优选地，所述间隔结构域包含CD8(例如CD8α)的铰链区(例如，序列如SEQ ID NO:78所示的铰链区)；Preferably, the spacer domain comprises a hinge region of CD8 (e.g., CD8α) (e.g., a hinge region whose sequence is shown in SEQ ID NO: 78);
    优选地，所述跨膜结构域包含CD8(例如CD8α)的跨膜区(例如，序列如SEQ ID NO:77所示的跨膜区)；Preferably, the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8α) (e.g., a transmembrane region whose sequence is shown in SEQ ID NO: 77);
    优选地，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域(例如，如SEQ ID NO:79所示序列)，所述共刺激信号传导结构域包含CD137(4-1BB)的胞内信号传导结构域(例如，如SEQ ID NO:80所示序列)；更优选地，所述嵌合抗原受体的胞内信号传导结构域具有SEQ ID NO:81所示序列。Preferably, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO:79), and the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO:80); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:81.
  18. 权利要求15-17任一项所述的核酸构建体，其中，所述核酸构建体包含选自下列核苷酸序列：(1)SEQ ID NO:72、73、74、75任一项所示的序列或其简并变体；(2)与(1)中任一项所示序列相比基本上相同的序列(例如，与(1)中任一项所示的序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，或，与(1)中任一项所示的序列相比具有一个或更多个核苷酸取代的序列)。The nucleic acid construct of any one of claims 15 to 17, wherein the nucleic acid construct comprises a nucleotide sequence selected from the following: (1) a sequence shown in any one of SEQ ID NOs: 72, 73, 74, 75, or a degenerate variant thereof; (2) a sequence substantially identical to the sequence shown in any one of (1) (e.g., 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 compared to the sequence shown in any one of (1), or a sequence having one or more nucleotide substitutions compared to the sequence shown in any one of (1)).
  19. 权利要求15-18任一项所述的核酸构建体，其包含：(1)编码GPC3特异性嵌合抗原受体的第一核酸序列；和The nucleic acid construct of any one of claims 15 to 18, comprising: (1) a first nucleic acid sequence encoding a GPC3-specific chimeric antigen receptor; and
    (2)编码另外的生物活性分子的第二核酸序列；(2) a second nucleic acid sequence encoding another biologically active molecule;
    优选地，所述GPC3特异性嵌合抗原受体包含靶向GPC3的抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域，所述靶向GPC3的抗原结合结构域包含权利要求1-7任一项中定义的第一抗原结合结构域；Preferably, the GPC3-specific chimeric antigen receptor comprises an antigen binding domain targeting GPC3, a spacer domain, a transmembrane domain, and an intracellular signaling domain, and the antigen binding domain targeting GPC3 comprises the first antigen binding domain defined in any one of claims 1 to 7;
    优选地，所述第二核酸序列所编码的另外的生物活性分子选自下列组分的一种或一种以上：免疫检查点抑制剂(例如，抗PD-1、PD-L1、CTLA-4或LAG3抗体或其抗原结合片段、细胞因子(例如，IL-15、IL-7、IL-12、IL-18或IL-21)、T细胞表面抗原(例如，CD3等)或膜嵌合型多肽(例如，mIL-15、mIL-7、mIL-12、mIL-18或mIL-21)；优 选地，所述第一核酸序列和第二核酸序列通过编码自裂解肽(例如P2A，E2A，F2A，T2A或其任意组合)的核苷酸序列连接；Preferably, the additional biologically active molecule encoded by the second nucleic acid sequence is selected from one or more of the following components: immune checkpoint inhibitors (e.g., anti-PD-1, PD-L1, CTLA-4 or LAG3 antibodies or antigen-binding fragments thereof, cytokines (e.g., IL-15, IL-7, IL-12, IL-18 or IL-21), T cell surface antigens (e.g., CD3, etc.) or membrane chimeric polypeptides (e.g., mIL-15, mIL-7, mIL-12, mIL-18 or mIL-21); preferably Optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof);
    优选地，所述自裂解肽是P2A；例如，所述自裂解肽的氨基酸序列如SEQ ID NO:83所示；Preferably, the self-cleaving peptide is P2A; for example, the amino acid sequence of the self-cleaving peptide is shown in SEQ ID NO:83;
    优选地，所述第二核苷酸序列所编码的另外的生物活性分子在其N端进一步包含信号肽；优选地，所述信号肽不同于所述第一核酸序列所编码的GPC3特异性嵌合抗原受体所包含的信号肽；优选地，所述另外的生物活性分子N端的信号肽是IL2信号肽(例如，所述IL2信号肽的氨基酸序列如SEQ ID NO:84所示)。Preferably, the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide at its N-terminus; preferably, the signal peptide is different from the signal peptide contained in the GPC3-specific chimeric antigen receptor encoded by the first nucleic acid sequence; preferably, the signal peptide at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (for example, the amino acid sequence of the IL2 signal peptide is shown in SEQ ID NO:84).
  20. 载体，其包含权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体；A vector comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19;
    优选地，所述载体选自DNA载体，RNA载体，质粒，转座子载体，CRISPR/Cas9载体，或病毒载体；Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, or a viral vector;
    优选的，所述载体是表达载体；Preferably, the vector is an expression vector;
    优选地，所述载体是游离型载体；Preferably, the vector is an episomal vector;
    优选地，所述载体是病毒载体；更优选地，所述病毒载体是慢病毒载体，腺病毒载体或逆转录病毒载体。Preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.
  21. 宿主细胞，其包含权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体，或权利要求20所述的载体。A host cell comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19, or the vector of claim 20.
  22. 制备表达嵌合抗原受体细胞的方法，其包括：(1)提供宿主细胞；(2)将权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体、或包含它们的载体引入步骤(1)所述的宿主细胞，获得能够共表达所述嵌合抗原受体以及任选的另外的生物活性分子的宿主细胞；A method for preparing a cell expressing a chimeric antigen receptor, comprising: (1) providing a host cell; (2) introducing the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19, or a vector comprising them into the host cell of step (1), to obtain a host cell capable of co-expressing the chimeric antigen receptor and optionally another biologically active molecule;
    优选地，所述宿主细胞选自免疫细胞，例如T淋巴细胞、NK细胞、单核细胞、树突状细胞、巨噬细胞及其任意组合；Preferably, the host cell is selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages and any combination thereof;
    优选地，所述免疫细胞选自T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及这些细胞的任意组合；Preferably, the immune cells are selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination of these cells;
    优选地，在步骤(1)中，所述宿主细胞提供自患者或者健康供体，并且经过预处理； 所述预处理包括免疫细胞的分选、激活和/或增殖；优选地，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞；Preferably, in step (1), the host cells are provided from a patient or a healthy donor and are pretreated; The pretreatment includes sorting, activating and/or proliferating immune cells; preferably, 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;
    优选地，在步骤(2)中，将核酸分子或载体通过病毒感染引入宿主细胞；Preferably, in step (2), the nucleic acid molecule or vector is introduced into the host cell by viral infection;
    优选地，在步骤(2)中将核酸分子或载体通过非病毒载体转染的方式引入宿主细胞，如通过转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法、电穿孔方法、磷酸钙转染、DEAE-葡聚糖介导的转染或显微注射等方法；Preferably, in step (2), the nucleic acid molecule or vector is introduced into the host cell by non-viral vector transfection, such as a transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran-mediated transfection or microinjection;
    优选地，在步骤(2)之后，所述方法还包括：扩增步骤(2)获得的宿主细胞。Preferably, after step (2), the method further comprises: amplifying the host cells obtained in step (2).
  23. 经改造的免疫细胞，其包含权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体，或包含它们的载体；A modified immune cell comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19, or a vector comprising the same;
    优选地，所述经改造的免疫细胞表达权利要求1-12任一项所述的嵌合抗原受体。Preferably, the engineered immune cell expresses the chimeric antigen receptor according to any one of claims 1-12.
  24. 权利要求23所述的经改造的免疫细胞，其中，所述免疫细胞来源于T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合；优选地，所述免疫细胞得自患者；可选地，所述免疫细胞得自健康供体；优选地，所述免疫细胞来源于T淋巴细胞或NK细胞。The modified immune cells of claim 23, wherein the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are obtained from patients; alternatively, the immune cells are obtained from healthy donors; preferably, the immune cells are derived from T lymphocytes or NK cells.
  25. 权利要求23或24所述的经改造的免疫细胞，其中，所述免疫细胞还表达不特异性针对GPC3的CAR；优选地，所述不特异性针对GPC3的CAR具有针对选自下列靶点的特异性：PD-1、PD-L1、CTLA-4、CD3、ASGPR1、CD19、MSLN、PSMA、MUC1、EGFR、HER2、CD276、GD2、BCMA、CD33或Claudin18.2。The modified immune cell of claim 23 or 24, wherein the immune cell also expresses a CAR that is not specific to GPC3; preferably, the CAR that is not specific to GPC3 has specificity for a target selected from the following: PD-1, PD-L1, CTLA-4, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.2.
  26. 权利要求23-25任一项所述的经改造的免疫细胞，其中，所述经改造的免疫细胞的免疫排斥有关的基因(例如，TRAC、TRBC、B2M、HLA-A、HLA-B或HLA-C)和免疫共抑制通路或信号分子的基因(例如，PD-1、CTLA-4或LAG-3)中的一种或两种靶基因的转录或表达被抑制；优选地，所述靶基因的转录或表达被抑制采用的方法选自基因敲除(例如，CRISPR、CRISPR/Cas9)、同源重组、干扰RNA。The modified immune cell according to any one of claims 23 to 25, wherein the transcription or expression of one or two target genes of the immune rejection-related genes (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B or HLA-C) and the genes of the immune co-inhibitory pathway or signaling molecules (e.g., PD-1, CTLA-4 or LAG-3) of the modified immune cell is inhibited; preferably, the transcription or expression of the target gene is inhibited by a method selected from gene knockout (e.g., CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.
  27. 制备经改造的免疫细胞的方法，其包括：(1)提供来自患者或者健康供体的免 疫细胞；(2)将权利要求13或14所述的分离的核酸分子或包含其的载体引入步骤(1)所述的免疫细胞，以获得能够表达嵌合抗原受体的免疫细胞；或者，将权利要求15-19任一项所述的核酸构建体或包含其的载体引入步骤(1)所述的免疫细胞，以获得能够共表达嵌合抗原受体和另外的生物活性分子的免疫细胞；A method for preparing modified immune cells, comprising: (1) providing immune cells from a patient or a healthy donor; (2) introducing the isolated nucleic acid molecule of claim 13 or 14 or a vector comprising the same into the immune cell of step (1) to obtain an immune cell capable of expressing a chimeric antigen receptor; or, introducing the nucleic acid construct of any one of claims 15 to 19 or a vector comprising the same into the immune cell of step (1) to obtain an immune cell capable of co-expressing a chimeric antigen receptor and another biologically active molecule;
    优选地，在步骤(1)中，所述免疫细胞经预处理，所述预处理包括免疫细胞的分选、激活和/或增殖；更优选地，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞；Preferably, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of the immune cells; more preferably, 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;
    优选地，在步骤(2)中将所述核酸分子或载体通过病毒感染引入免疫细胞；Preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by viral infection;
    优选地，在步骤(2)中将所述核酸分子或载体通过非病毒载体转染的方式引入免疫细胞，如通过磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法或电穿孔方法；Preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method;
    优选地，在步骤(2)之后还包括扩增步骤(2)获得的免疫细胞的步骤。Preferably, after step (2), the method further includes a step of amplifying the immune cells obtained in step (2).
  28. 免疫细胞组合物，包括权利要求23-26任一项所述的经改造的免疫细胞；可选地，所述组合物还包括未改造和/或未成功改造的免疫细胞；优选地，所述经改造的免疫细胞数占所述免疫细胞组合物细胞总数的10％-100％，更优选40％-80％。An immune cell composition comprising the modified immune cells described in any one of claims 23-26; optionally, the composition also includes unmodified and/or unsuccessfully modified immune cells; preferably, the number of the modified immune cells accounts for 10%-100%, more preferably 40%-80% of the total number of cells in the immune cell composition.
  29. 试剂盒，所述试剂盒包括权利要求1-12任一项所述的嵌合抗原受体，或权利要求15-19任一项所述的核酸构建体；A kit comprising the chimeric antigen receptor according to any one of claims 1 to 12, or the nucleic acid construct according to any one of claims 15 to 19;
    优选地，所述试剂盒包括权利要求13或14所述的分离的核酸分子或包含所述分离的核酸分子的载体；所述试剂盒用于制备权利要求1-12任一项所述的嵌合抗原受体；Preferably, the kit comprises the isolated nucleic acid molecule of claim 13 or 14 or a vector comprising the isolated nucleic acid molecule; the kit is used to prepare the chimeric antigen receptor of any one of claims 1 to 12;
    优选地，所述试剂盒包括权利要求13或14所述的分离的核酸分子或包含所述分离的核酸分子的载体；所述试剂盒用于制备权利要求23-26所述的经改造的免疫细胞或包含所述经改造的免疫细胞的免疫细胞组合物；Preferably, the kit comprises the isolated nucleic acid molecule of claim 13 or 14 or a vector comprising the isolated nucleic acid molecule; the kit is used to prepare the modified immune cell of claims 23-26 or an immune cell composition comprising the modified immune cell;
    优选地，所述试剂盒包括权利要求15-19任一项所述的核酸构建体或包含所述核酸构建体的载体；所述试剂盒用于制备权利要求23-26所述的经改造的免疫细胞或包含所述经改造的免疫细胞的免疫细胞组合物。Preferably, the kit comprises the nucleic acid construct of any one of claims 15 to 19 or a vector comprising the nucleic acid construct; the kit is used to prepare the modified immune cells of claims 23 to 26 or an immune cell composition comprising the modified immune cells.
  30. 药物组合物，其含有权利要求1-12任一项所述的嵌合抗原受体，或权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体，或权利要求 20所述的载体，或权利要求21所述的宿主细胞，或权利要求23-26任一项所述的经改造的免疫细胞，或权利要求28所述的免疫细胞组合物，以及药学上可接受的载体和/或赋形剂；A pharmaceutical composition comprising a chimeric antigen receptor according to any one of claims 1 to 12, or an isolated nucleic acid molecule according to claim 13 or 14, or a nucleic acid construct according to any one of claims 15 to 19, or The vector of claim 20, or the host cell of claim 21, or the modified immune cell of any one of claims 23 to 26, or the immune cell composition of claim 28, and a pharmaceutically acceptable carrier and/or excipient;
    优选地，所述药物组合物还包含另外的药学活性剂，例如具有抗肿瘤活性的药物；优选地，所述另外的药学活性剂包括anti-PD1抗体、anti-PD-L1抗体、anti-CTLA-4抗体、anti-CD3抗体、anti-ASGPR1抗体、索拉菲尼或其衍生物、瑞格菲尼或其衍生物、培美曲塞、顺铂、紫杉醇、吉西他滨、卡培他滨或FOLFIRINOX；Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as a drug having anti-tumor activity; preferably, the other pharmaceutically active agent comprises 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;
    可选地，所述药物组合物包含的嵌合抗原受体、或分离的核酸分子、或载体、或宿主细胞、或核酸构建体、或经改造的免疫细胞、或免疫细胞组合物，可与所述另外的药学活性剂同时、分开或相继施用。Optionally, the chimeric antigen receptor, or isolated nucleic acid molecule, or vector, or host cell, or nucleic acid construct, or modified immune cell, or immune cell composition contained in the pharmaceutical composition can be administered simultaneously, separately or sequentially with the additional pharmaceutically active agent.
  31. 权利要求1-12任一项所述的嵌合抗原受体，或权利要求13或14所述的分离的核酸分子，或权利要求15-19任一项所述的核酸构建体，或权利要求20所述的载体，或权利要求21所述的宿主细胞，或权利要求23-26任一项所述的经改造的免疫细胞，或权利要求28所述的免疫细胞组合物，或权利要求30所述的药物组合物，在制备用于预防和/或治疗与GPC3的表达相关的疾病的药物中的用途；Use of the chimeric antigen receptor according to any one of claims 1 to 12, or the isolated nucleic acid molecule according to claim 13 or 14, or the nucleic acid construct according to any one of claims 15 to 19, or the vector according to claim 20, or the host cell according to claim 21, or the modified immune cell according to any one of claims 23 to 26, or the immune cell composition according to claim 28, or the pharmaceutical composition according to claim 30 in the preparation of a medicament for preventing and/or treating a disease associated with the expression of GPC3;
    优选地，所述与GPC3的表达相关的疾病选自增生性疾病，例如肿瘤，或是与GPC3的表达相关的非肿瘤相关的适应症；Preferably, the disease associated with the expression of GPC3 is selected from a proliferative disease, such as a tumor, or a non-tumor-related indication associated with the expression of GPC3;
    优选地，所述肿瘤是GPC3阳性肿瘤；Preferably, the tumor is a GPC3-positive tumor;
    优选地，所述肿瘤选自实体瘤；优选地，所述实体瘤选自肝癌、肝细胞癌、胰腺癌、肺癌、结肠癌、乳腺癌、前列腺癌、卵巢癌、卵巢透明细胞癌、黑色素瘤、非小细胞肺癌、小细胞肺癌、鳞状细胞癌、肾细胞癌、直结肠癌、胃癌、神经胶质瘤中的一种或其组合；Preferably, the tumor is selected from solid tumors; preferably, the solid tumor is selected from one or a combination of liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, and glioma;
    优选地，所述肿瘤选自血液肿瘤；优选地，所述血液肿瘤选自白血病、淋巴瘤。 Preferably, the tumor is selected from blood tumors; preferably, the blood tumor is selected from leukemia and lymphoma.
PCT/CN2023/132244 2022-11-28 2023-11-17 Chimeric antigen receptor specifically binding to gpc3 and use thereof WO2024114404A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211499852.0 2022-11-28
CN202211499852 2022-11-28

Publications (1)

Publication Number Publication Date
WO2024114404A1 true WO2024114404A1 (en) 2024-06-06

Family

ID=91322969

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/132244 WO2024114404A1 (en) 2022-11-28 2023-11-17 Chimeric antigen receptor specifically binding to gpc3 and use thereof

Country Status (1)

Country Link
WO (1) WO2024114404A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103168104A (en) * 2010-07-22 2013-06-19 加利福尼亚大学董事会 Anti-tumor antigen antibodies and methods of use
CN109021108A (en) * 2018-08-27 2018-12-18 南京医科大学 The full humanized antibody of resisting GPC 3, its Chimeric antigen receptor cell and application
US20220056408A1 (en) * 2018-12-13 2022-02-24 Carsgen Therapeutics Co., Ltd. Immune effector cell targeting gpc3 and application thereof
WO2022166876A1 (en) * 2021-02-03 2022-08-11 江苏先声药业有限公司 Monoclonal antibody for specifically recognizing glypican-3, and application thereof
WO2022171100A1 (en) * 2021-02-10 2022-08-18 江苏先声药业有限公司 Humanized gpc3 antibody and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103168104A (en) * 2010-07-22 2013-06-19 加利福尼亚大学董事会 Anti-tumor antigen antibodies and methods of use
CN109021108A (en) * 2018-08-27 2018-12-18 南京医科大学 The full humanized antibody of resisting GPC 3, its Chimeric antigen receptor cell and application
US20220056408A1 (en) * 2018-12-13 2022-02-24 Carsgen Therapeutics Co., Ltd. Immune effector cell targeting gpc3 and application thereof
WO2022166876A1 (en) * 2021-02-03 2022-08-11 江苏先声药业有限公司 Monoclonal antibody for specifically recognizing glypican-3, and application thereof
WO2022171100A1 (en) * 2021-02-10 2022-08-18 江苏先声药业有限公司 Humanized gpc3 antibody and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU QIAN-YONG; MARK GILBERT; LI YI-PING: "Recent Progress in the Research and Development of CAR-T Products", CHINESE JOURNAL OF NEW DRUGS, GAI-KAN BIANJIBU, BEIJING, CN, vol. 30, no. 19, 31 December 2021 (2021-12-31), CN , pages 1759 - 1767, XP009555269, ISSN: 1003-3734 *
WU, X.Q. ET AL.: "Combined Antitumor Effects of Sorafenib and GPC3-CAR T Cells in Mouse Models of Hepatocellular Carcinoma", MOLECULAR THERAPY, vol. 27, no. 8, 31 August 2019 (2019-08-31), XP093016416, DOI: 10.1016/j.ymthe.2019.04.020 *

Similar Documents

Publication Publication Date Title
JP7280828B2 (en) Antibodies targeting BCMA and uses thereof
JP7082620B2 (en) Anti-PD1 monoclonal antibody, its pharmaceutical composition and its use
JP7510246B2 (en) Anti-glypican 3 antibodies and uses thereof
CN110950953B (en) Monoclonal antibody against B7-H3 and application thereof in cell therapy
JP2022130393A (en) Anti-ctla4-anti-pd-1 bifunctional antibodies and pharmaceutical compositions and uses thereof
CN108373504B (en) CD 24-specific antibodies and anti-CD 24-CAR-T cells
WO2022078286A1 (en) Chimeric antigen receptor which specifically binds to msln, and application thereof
US20220331363A1 (en) Bcma-targeted antibody and chimeric antigen receptor
JP2022515487A (en) Claudin 18.2 coupling part and its utilization
CN113754780A (en) Chimeric antigen receptor targeting CLDN18.2, compositions and uses thereof
US20230203178A1 (en) Chimeric antigen receptor car or car construct targeting bcma and cd19 and application thereof
CN111454358A (en) Chimeric antigen receptor and application thereof
CN109796532B (en) Binding unit targeting fibroblast activation protein alpha and application thereof
KR20240046224A (en) Bispecific antibodies and their uses
EP3730519B1 (en) Antibody or antigen-binding fragment thereof that specifically recognizes b cell malignancies, chimeric antigen receptor comprising same, and uses thereof
CN115232209B (en) Antibodies targeting GPRC5D and uses thereof
CN115403671A (en) Claudin18.2-targeted nano antibody and application thereof
CN109970859B (en) Glyphican-3 specific antibody and CAR-T cell specific to the same
WO2024114404A1 (en) Chimeric antigen receptor specifically binding to gpc3 and use thereof
CN116063527A (en) Antibodies targeting mesothelin and uses thereof
WO2023246578A1 (en) Chimeric antigen receptor specifically binding to gpc3 and use thereof
WO2024114410A1 (en) Gpc3-targeting antibody and use thereof
TW202204419A (en) Chimeric antigen receptors
WO2023246574A1 (en) Gpc3-targeting antibody and use thereof
WO2023131063A1 (en) Chimeric antigen receptors specifically binding to msln and use thereof

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: 23896556

Country of ref document: EP

Kind code of ref document: A1