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WO2022228424A1 - 一种抗egfr/vegf双功能融合蛋白及其用途 - Google Patents

一种抗egfr/vegf双功能融合蛋白及其用途 Download PDF

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WO2022228424A1
WO2022228424A1 PCT/CN2022/089244 CN2022089244W WO2022228424A1 WO 2022228424 A1 WO2022228424 A1 WO 2022228424A1 CN 2022089244 W CN2022089244 W CN 2022089244W WO 2022228424 A1 WO2022228424 A1 WO 2022228424A1
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egfr
fusion protein
vegf
seq
acid sequence
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PCT/CN2022/089244
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French (fr)
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邓岚
黄浩旻
朱祯平
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丹生医药技术(上海)有限公司
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Priority to CN202280030685.7A priority Critical patent/CN117242097A/zh
Publication of WO2022228424A1 publication Critical patent/WO2022228424A1/zh

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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39566—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against immunoglobulins, e.g. anti-idiotypic antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the technical field of fusion proteins, in particular, to an anti-EGFR/VEGF bifunctional fusion protein and uses thereof.
  • EGFR Epidermal growth factor
  • EGFR is a receptor for epidermal growth factor (EGF) and belongs to the ErbB receptor family.
  • EGFR is a transmembrane glycoprotein with a molecular weight of 170KDa, which belongs to receptor-type tyrosine kinase.
  • TGF ⁇ transforming growth factor ⁇
  • EGFR is composed of monomers. Converted into dimers and activated, thereby further activating downstream signaling pathways and regulating cell proliferation.
  • TGF ⁇ transforming growth factor ⁇
  • Abnormal EGFR function is related to tumor cell proliferation, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis.
  • the abnormality of its function is mainly manifested in two aspects: one is the excessive abnormal expression in tumor tissue, and the other is the persistent activation of EGFR mutants in tumor cells (without ligand stimulation or the formation of self-circulating stimulation pathways).
  • the expression rate of EGFR is about 25-77%. Relevant clinical data show that the amount of EGFR expression is closely related to the malignant degree of tumor and the prognosis of tumor patients.
  • VEGFs vascular endothelial growth factor
  • VEGFR2 vascular endothelial growth factor
  • VEGFR1-D2 competes with VEGF to block the binding of VEGFR2 and VEGF, thereby blocking the signaling pathway, inhibiting endothelial cell proliferation and angiogenesis, thereby inhibiting the rapid proliferation and metastasis of tumors.
  • the purpose of the present invention is to provide a novel anti-EGFR/VEGF bifunctional fusion protein, which can combine with EGFR and VEGF at the same time, thereby blocking the signaling pathways of EGFR and VEGF.
  • Another object of the present invention is to provide a nucleic acid molecule encoding the fusion protein; to provide an expression vector comprising the nucleic acid molecule; to provide a host cell comprising the expression vector; to provide a method for preparing the fusion protein; A pharmaceutical composition of the protein; the application of the fusion protein or the pharmaceutical composition in the preparation of a medicine for treating cancer is provided; and a method for the fusion protein or the pharmaceutical composition for the treatment of cancer is provided.
  • a first aspect of the present invention provides an anti-EGFR/VEGF bifunctional fusion protein, comprising: (a) an anti-EGFR antibody or an antigen-binding fragment thereof, and (b) the D2 domain of the extracellular region of VEGFR1.
  • the anti-EGFR antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 16 , the amino acid sequence of HCDR2 is shown in SEQ ID NO: 17, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 18; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 19, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 20 As shown, the amino acid sequence of LCDR3 is shown in SEQ ID NO:21.
  • the anti-EGFR antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the amino acid sequence of VH is shown in SEQ ID NO: 22, and the amino acid sequence of VL is as shown in SEQ ID NO: 22.
  • the sequence is shown in SEQ ID NO:24.
  • the heavy chain variable region VH comprises at least one deglycosylation mutation; preferably, the mutation is located within the framework region FR of the heavy chain variable region VH.
  • the mutation is located at position 85 of the VH of the heavy chain variable region, and the mutation is numbered according to the Kabat numbering system; preferably, the mutation is N85E.
  • amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO: 23
  • amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: 24.
  • the anti-EGFR antibody comprises a heavy chain constant region and a light chain constant region; more preferably, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3 or human IgG4, the The light chain constant region is selected from human kappa (Kappa) or human lambda (Lambda).
  • the anti-EGFR antibody or antigen-binding fragment thereof is selected from a full-length antibody, scFv, Fv, Fab or F(ab')2.
  • the anti-EGFR antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
  • the anti-EGFR antibody is a monoclonal antibody.
  • the anti-EGFR antibody comprises a heavy chain whose amino acid sequence is set forth in SEQ ID NO:2 or SEQ ID NO:3, and a light chain whose amino acid sequence is set forth in SEQ ID NO:7.
  • the D2 domain of the extracellular region of VEGFR1 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:8.
  • the anti-EGFR antibody or antigen-binding fragment thereof is linked directly by a peptide bond or by a peptide linker to the D2 domain of the extracellular region of VEGFRl.
  • the peptide linker comprises the amino acid sequence (GGGGS)n selected from 1, 2, 3, 4 or 5.
  • the anti-EGFR antibody is a full-length antibody, comprising 2 heavy chains and 2 light chains; the N-terminus or C-terminus of the D2 domain of the extracellular region of the VEGFR1 is linked by a peptide linker to N-terminus or C-terminus of the heavy or light chain of an anti-EGFR antibody.
  • the N-terminus of the D2 domain of the extracellular region of said VEGFR1 is linked to the C-terminus of the heavy chain of the anti-EGFR antibody via a peptide linker.
  • the fusion protein is selected from:
  • the fusion protein comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 5, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7; or,
  • the fusion protein comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 6, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7; or,
  • the fusion protein comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 9, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7; or,
  • the fusion protein comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 10, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7.
  • the fusion protein is selected from: 602-D2, 602dN-D2, 602-D2-M2 or 602dN-D2-M2.
  • the second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned anti-EGFR/VEGF bifunctional fusion protein.
  • the nucleic acid molecule comprises:
  • nucleic acid sequence shown in SEQ ID NO: 11 encoding the heavy chain of the fusion protein
  • nucleic acid sequence shown in SEQ ID NO: 13 encoding the light chain of the fusion protein
  • nucleic acid sequence shown in SEQ ID NO: 12 encoding the heavy chain of the fusion protein
  • nucleic acid sequence shown in SEQ ID NO: 13 encoding the light chain of the fusion protein
  • nucleic acid sequence shown in SEQ ID NO: 15 encoding the heavy chain of the fusion protein
  • nucleic acid sequence shown in SEQ ID NO: 13 encoding the light chain of the fusion protein.
  • a third aspect of the present invention provides an expression vector containing the above-mentioned nucleic acid molecule.
  • a fourth aspect of the present invention provides a host cell containing the above-mentioned nucleic acid molecule.
  • a fifth aspect of the present invention provides a method for preparing an anti-EGFR/VEGF bifunctional fusion protein, the preparation method comprising the following steps:
  • the above-mentioned host cells are cultured to express the anti-EGFR/VEGF bifunctional fusion protein;
  • step b Isolation and purification of the anti-EGFR/VEGF bifunctional fusion protein described in step a).
  • the sixth aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned anti-EGFR/VEGF bifunctional fusion protein and one or more pharmaceutically acceptable carriers, diluents or excipients .
  • the seventh aspect of the present invention provides the use of the above-mentioned anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition in the preparation of a medicament for the treatment of cancer, preferably, the cancer highly expresses or abnormally expresses EGFR and/or highly expresses or abnormal expression of VEGF.
  • the cancer is selected from the group consisting of: colorectal cancer, skin cancer, squamous cell carcinoma, breast cancer, glial cell carcinoma, kidney cancer, lung cancer, prostate cancer, pancreatic cancer, head and neck cancer (including but not limited to Not limited to throat cancer and nasopharyngeal cancer), or a combination thereof; more preferably, the cancer is selected from colon cancer, skin squamous cell carcinoma, breast cancer, throat cancer, or a combination thereof.
  • the treatment comprises administering to the subject an effective amount of an anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition administered by injection ; More preferably, the injection is selected from intravenous injection, intramuscular injection, or subcutaneous injection.
  • the treatment comprises administering to the subject an effective amount of an anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition, the effective amount being a dose of 0.5-30 mg/kg; more preferably , the administration interval of the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is selected from: a) once a week; b) twice a week; c) three times a week; further more preferably, the anti-EGFR/VEGF
  • the administration time of the bifunctional fusion protein or pharmaceutical composition is selected from: a) at least 1 week; b) at least 2 weeks; c) at least 3 weeks; d) at least 4 weeks.
  • the eighth aspect of the present invention provides an immunoconjugate, the immunoconjugate comprises:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • the ninth aspect of the present invention provides a method for treating cancer, comprising administering to a subject an effective amount of the above-mentioned anti-EGFR/VEGF bifunctional fusion protein, immunoconjugate or pharmaceutical composition, preferably, the Cancer overexpresses or aberrantly expresses EGFR and/or overexpresses or overexpresses VEGF.
  • the cancer is selected from the group consisting of: colorectal cancer, skin cancer, squamous cell carcinoma, breast cancer, glial cell carcinoma, kidney cancer, lung cancer, prostate cancer, pancreatic cancer, head and neck cancer (including but not limited to Not limited to throat cancer and nasopharyngeal cancer), or a combination thereof; more preferably, the cancer is selected from colon cancer, skin squamous cell carcinoma, breast cancer, throat cancer, or a combination thereof.
  • the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is administered by injection; more preferably, the injection is selected from intravenous injection, intramuscular injection, or subcutaneous injection.
  • the effective amount refers to a dose of 0.5-30 mg/kg; more preferably, the administration interval of the anti-EGFR/VEGF bifunctional fusion protein or pharmaceutical composition is selected from: a) weekly once; b) twice a week; c) three times a week; further more preferably, the administration time of the anti-EGFR/VEGF bifunctional fusion protein or the pharmaceutical composition is selected from: a) at least 1 week; b) at least 2 weeks ; c) at least 3 weeks; d) at least 4 weeks.
  • the fusion protein of the present invention can simultaneously bind to EGFR and VEGF with high affinity at the molecular level, and has good thermal stability; at the cellular level, it can effectively inhibit the proliferation of A431 cells, and its resistance to The ability to block the binding of VEGF to the receptor KDR is better than that of the anti-VEGF monoclonal antibody Bevacizumab; at the animal level, it played a significant tumor suppressor effect in the LIM1215 xenograft, SW48 xenograft, JIMT-1 xenograft, and FaDu xenograft models.
  • the fusion protein of the present invention has great clinical application prospects in the treatment of diseases related to the high expression or abnormal expression of EGFR and VEGF.
  • Figure 1 Schematic diagram of the anti-EGFR/VEGF bifunctional fusion protein structure.
  • Figure 2A HPLC detection result of anti-EGFR/VEGF bifunctional fusion protein 602-D2.
  • Figure 2B HPLC detection result of anti-EGFR/VEGF bifunctional fusion protein 602-D2-M2.
  • Figure 2C HPLC detection result of anti-EGFR/VEGF bifunctional fusion protein 602dN-D2.
  • Figure 2D HPLC detection result of anti-EGFR/VEGF bifunctional fusion protein 602dN-D2-M2.
  • Figure 3 ELISA test results of the affinity of the anti-EGFR/VEGF bifunctional fusion protein to EGFR.
  • Figure 4 ELISA test results of the affinity of the anti-EGFR/VEGF bifunctional fusion protein to VEGF.
  • Figure 5 ELISA detection result of the anti-EGFR/VEGF bifunctional fusion protein simultaneously binding to EGFR and VEGF.
  • Figure 6 FACS detection results of the anti-EGFR/VEGF bifunctional fusion protein binding to the target cell surface antigen EGFR.
  • Figure 7 Graph of the detection results of the inhibitory activity of anti-EGFR/VEGF bifunctional fusion protein on the proliferation of A431 cells.
  • Figure 8 Graph of the detection result of anti-EGFR/VEGF bifunctional fusion protein blocking the binding of VEGF to receptor KDR.
  • Figure 9 The result of anti-tumor effect of anti-EGFR/VEGF bifunctional fusion protein on LIM1215 xenograft model.
  • Figure 10 Results of anti-tumor effect of anti-EGFR/VEGF bifunctional fusion protein on SW48 xenograft model.
  • Figure 11 The result of anti-tumor effect of anti-EGFR/VEGF bifunctional fusion protein on JIMT-1 xenograft model.
  • Figure 12 The result of anti-tumor effect of anti-EGFR/VEGF bifunctional fusion protein on FaDu xenograft model.
  • the bifunctional fusion protein of the present invention can bind to EGFR and VEGF with high affinity, at the cellular level, it can effectively inhibit the proliferation of A431 cells, and the ability to block the binding of VEGF to the receptor KDR is better than that of the anti-VEGF monoclonal antibody Bevacizumab; On the level, it played a significant tumor suppressing effect in LIM1215 xenograft model, SW48 xenograft model, JIMT-1 xenograft model and FaDu xenograft model.
  • the antitumor effect of the bifunctional protein of the present invention in the SW48 xenograft tumor is better than the combination of Bevacizumab and the anti-EGFR antibody 602, showing a synergistic effect; in the JIMT-1 xenograft model, it is also superior.
  • the combination of Bevacizumab and anti-EGFR antibody 602 also showed a synergistic effect.
  • the bifunctional fusion protein of the present invention also has good thermal stability. Therefore, the bifunctional fusion protein of the present invention has an excellent clinical application prospect in the treatment of diseases related to the high expression or abnormal expression of EGFR and VEGF. The present invention has been completed on this basis.
  • fusion protein refers to a new polypeptide sequence obtained by fusion of two or more identical or different polypeptide sequences.
  • fusion refers to direct attachment by peptide bonds or via one or more linking peptides (peptide linkers).
  • linker peptide refers to a short peptide that can link two polypeptide sequences, typically a peptide of 2-30 amino acids in length.
  • it is a flexible peptide linker, such as the amino acid sequence (GGGGS)n, said n being selected from 1, 2, 3, 4 or 5.
  • antibody refers to a full-length antibody
  • antigen-binding fragment refers to a fragment derived from an antibody and capable of binding antigenic epitopes, including but not limited to scFv, Fv, Fab or F(ab')2 .
  • full-length antibody refers to a heterotetraglycan protein of about 150,000 Daltons with the same structural characteristics, comprising a variable region (V) and a constant region (C), which are composed of two identical heavy Chain (H) and two identical light chains (L). At one end of each heavy chain is a heavy chain variable region (VH) followed by a heavy chain constant region consisting of three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region (VL) at one end and a light chain constant region at the other end.
  • the light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain constant region is The chain variable region is paired with the heavy chain variable region.
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC), etc.
  • Heavy chain constant regions include IgGl, IgG2, IgG3, IgG4 subtypes; light chain constant regions include kappa (Kappa) or lambda (Lambda).
  • the heavy and light chains of the antibody are covalently linked together by a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are interpolypeptide disulfide formed between the hinge regions. The bonds are covalently linked together.
  • variable means that certain portions of the variable regions of antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the heavy and light chains. The more conserved part of the variable region is called the frame region (FR).
  • CDRs complementarity-determining regions
  • FR frame region
  • the variable regions of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the CDRs of the heavy chain variable region (VH) and light chain variable region (VL) are called HCDRs and LCDRs, respectively.
  • humanized antibody means that the complementarity determining regions (CDRs) of the antibody are derived from non-human species (such as rodents), and the remaining parts of the antibody molecule (including the framework region FR and the constant region C) are derived from human of antibodies. Among them, the FR residues of the framework regions can be altered to maintain binding affinity.
  • CDRs complementarity determining regions
  • FR framework region
  • the light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • a light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L), and the heavy chain variable domain can thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H )-(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is substantially identical to a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to that of a naturally occurring human antibody .
  • chimeric antibody refers to an antibody in which the variable region (V) is derived from non-human species (eg, rodents) and the constant region (C) is derived from human.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, and the individual antibodies contained in the population are identical except for a few possible natural mutations. Monoclonal antibodies are directed against a single determinant on an antigen and are highly specific for a single antigenic site. Monoclonal antibodies can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the terms “anti” and “binding” refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which it is directed.
  • the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10-7 M, eg, less than about 10-8 M, 10-9 M, 10-10 M, 10-11 M, or less.
  • KD refers to the equilibrium dissociation constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • surface plasmon resonance Surface Plasmon Resonance, abbreviated SPR
  • SPR Surface Plasmon Resonance
  • affinity (energy) binding (ability)
  • binding affinity (energy) binding affinity
  • D2 domain of the extracellular region of VEGFR1 includes the native sequence and variants thereof (containing at least 1 amino acid mutation), as long as the mutation does not alter the biological activity of the D2 domain.
  • expression vector refers to a conventional expression vector in the art containing appropriate regulatory sequences, such as promoter, terminator, enhancer, etc.
  • the expression vector may be a virus or a plasmid.
  • the expression vector preferably includes: pcDNA3.4, pDR1, pDHFR or pTT5.
  • the term "host cell” refers to various conventional host cells in the art, as long as the vector can stably replicate itself and the nucleic acid molecule carried by it can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells preferably include: COS, CHO, NSO, sf9, sf21, DH5 ⁇ , BL21(DE3), TG1, BL21(DE3) or 293F cells .
  • the term "effective amount” refers to the amount or dose of the active ingredient that produces the desired effect in the treated individual after the fusion protein or pharmaceutical composition of the present invention is administered to a patient, and the desired effect includes the improvement of the individual's condition.
  • the term "subject” includes, but is not limited to, mammals, such as humans, non-human primates, rats, mice, and the like.
  • TIW refers to administration three times a week
  • mpk refers to mg/kg (milligrams per kilogram).
  • SEM scanning electron microscope
  • mAU mAbsorbance Unit
  • the bifunctional fusion protein of the present invention is a bifunctional fusion protein of the present invention.
  • the bifunctional fusion protein of the present invention is a bifunctional fusion protein targeting both EGFR and VEGF, comprising: (a) an anti-EGFR antibody or an antigen-binding fragment thereof, and (b) the D2 domain of the extracellular region of VEGFR1.
  • bifunctional fusion protein As used herein, “bifunctional fusion protein”, “diabody fusion protein of the invention”, “bifunctional fusion protein targeting EGFR and VEGF”, “anti-EGFR/VEGF bifunctional fusion protein” are used interchangeably, All refer to the bifunctional fusion protein comprising the anti-EGFR antibody part and the D2 domain part of VEGFR1 according to the first aspect of the present invention.
  • the anti-EGFR antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the heavy chain complementarity determining regions HCDR1-3, and
  • the light chain variable region comprises the light chain complementarity determining region LCDR1-3; wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 16, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 17, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 17 ID NO: 18; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 19, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 20, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 21.
  • variable region of the heavy chain and the variable region of the light chain of the anti-EGFR antibody further include the framework regions of the heavy chain and the light chain.
  • the anti-EGFR antibody part in the bifunctional fusion protein of the present invention also includes its mutants, preferably, the Mutations include 1, 2, 3, 4, or 5 deglycosylation mutations.
  • the mutation is located within the framework region FRs of the heavy chain variable region; preferably, the mutation is N85E (numbering according to the Kabat numbering system).
  • the anti-EGFR antibody comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 3, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7.
  • D2 domain of the extracellular region of VEGFRl As used herein, "D2 domain of the extracellular region of VEGFRl", “VEGFRl-D2” or “D2 domain of VEGFRl” are used interchangeably to refer to the D2 domain of the extracellular region of human VEGFRl (wild-type sequence As shown in SEQ ID NO.1), it competes to bind VEGF, blocks the binding of VEGFR2 and VEGF, thereby blocking the signaling pathway, inhibiting endothelial cell proliferation and angiogenesis, thereby inhibiting the rapid proliferation and metastasis of tumors.
  • the "D2 domain of VEGFR1" also includes a protein variant of the D2 domain of VEGFR1, preferably a mutant obtained by amino acid substitution, mutation, or deletion with respect to the D2 domain of wild-type VEGFR1.
  • the D2 domain protein variant of VEGFR1 is truncated by 2 amino acids at the C-terminus relative to the sequence of the wild type (SEQ ID NO. 1). The two amino acids are easy to fall off during the fermentation process, and the removal does not affect their binding activity.
  • the sequence of the D2 domain protein variant of VEGFR1 is shown in SEQ ID NO.8.
  • the bifunctional fusion protein includes a mutant obtained by performing amino acid substitution, mutation or deletion, etc. on the anti-EGFR antibody and/or the domain D2 domain of VEGFR1 in the fusion protein.
  • the N-terminus or C-terminus of the D2 domain of the extracellular region of VEGFR1 is linked to the N-terminus of the heavy chain or the light chain of the anti-EGFR antibody through a peptide linker terminal or C-terminal.
  • the N-terminus of the D2 domain of the extracellular region of VEGFR1 is linked to the C-terminus of the heavy chain of the anti-EGFR antibody through a peptide linker.
  • sequences of the heavy and light chains of the bifunctional fusion protein are selected from the group consisting of:
  • the bifunctional fusion protein comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 5, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7; or,
  • the bifunctional fusion protein comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 6, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7; or,
  • the bifunctional fusion protein comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 9, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7; or,
  • the bifunctional fusion protein comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 10, and a light chain whose amino acid sequence is shown in SEQ ID NO: 7.
  • bifunctional fusion protein of the present invention can also modify the bifunctional fusion protein of the present invention through techniques well known in the art, such as adding, deleting and/or replacing one or several amino acid residues, so as to further increase the affinity or structure of the bifunctional fusion protein of the present invention stability, and obtain modified results by conventional assay methods.
  • the bifunctional fusion protein of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the bifunctional fusion protein of the present invention, there are at most 10, preferably at most 8, more preferably at most 5 Each, optimally up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the bifunctional fusion proteins of the present invention may be used alone, or may be combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, or a combination of any of the above.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • expression vector refers to a vector carrying an expression cassette for expressing a specific target protein or other substances, such as plasmid, viral vector (such as adenovirus, retrovirus), phage, yeast plasmid or other vectors.
  • conventional expression vectors in the art that include appropriate regulatory sequences, such as promoters, terminators, enhancers, etc. include but are not limited to: viral vectors (such as adenoviruses, retroviruses), plasmids, phages , yeast plasmids or other vectors.
  • the expression vector preferably includes pDR1, pcDNA3.4(+), pDHFR or pTT5.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the term "host cell” refers to various conventional host cells in the field, as long as the vector can stably replicate itself and the polynucleotide molecule carried by it can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells preferably include: COS, CHO, NSO, sf9, sf21, DH5 ⁇ , BL21(DE3), TG1, BL21(DE3), 293F or 293E cells.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned bifunctional fusion protein or an active fragment thereof, or an immunoconjugate thereof, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 4-8, preferably about pH 5-7, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.
  • routes including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.
  • the term "pharmaceutical composition” means that the bifunctional fusion protein of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to stably exert the curative effect, and these preparations can ensure the bifunctional disclosed in the present invention.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned bifunctional fusion protein (or its conjugate) of the present invention and A pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the bifunctional fusion proteins of the present invention can also be used with other therapeutic agents.
  • a safe and effective amount of the bifunctional fusion protein or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the dosage form of the pharmaceutical composition includes parenteral administration dosage form or parenteral administration dosage form.
  • the parenteral dosage forms include intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavitary injection.
  • the pharmaceutical composition of the present invention can be directly used to bind VEGF protein molecules or EGFR, and thus can be used to treat tumors.
  • other therapeutic agents may also be used concomitantly.
  • the term "effective amount” refers to an amount or dose of a pharmaceutical composition of the present invention that, after administration to a subject, produces a desired effect, including amelioration of the individual's condition, in the treated individual.
  • subject includes, but is not limited to, mammals such as humans, non-human primates, rats, mice, and the like.
  • immunoconjugates of bifunctional fusion proteins of the present invention include:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • the radionuclide includes:
  • a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.
  • the coupling moiety is a drug or a toxin; the drug is a cytotoxic drug.
  • the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
  • the bifunctional fusion protein of the present invention or the composition thereof can be used to prepare a drug for treating tumors or a drug for inhibiting the proliferation, angiogenesis, tumor invasion, metastasis and apoptosis of tumor cells, or for preparing a drug for blocking the binding of VEGFR2 to VEGF or inhibiting the interior of tumors Angiogenesis drugs.
  • the tumor is a tumor or cancer overexpressing EGFR and/or VEGF2; preferably, the indication includes colorectal cancer, skin cancer, squamous cell carcinoma, breast cancer, glial cell carcinoma, kidney cancer, Lung cancer, prostate cancer, pancreatic cancer, head and neck cancer (including but not limited to throat cancer and nasopharyngeal cancer), or a combination thereof.
  • the indication includes colorectal cancer, skin cancer, squamous cell carcinoma, breast cancer, glial cell carcinoma, kidney cancer, Lung cancer, prostate cancer, pancreatic cancer, head and neck cancer (including but not limited to throat cancer and nasopharyngeal cancer), or a combination thereof.
  • the positive control anti-EGFR monoclonal antibody 602 used in the following examples is a human-mouse chimeric monoclonal prepared after cell culture and production according to the amino acid sequence of Erbitux (Cetuximab, IMC-C225), expressed in CHO cell expression system Antibody.
  • the positive control anti-VEGF monoclonal antibody Bevacizumab (601) is a humanized monoclonal antibody prepared according to the sequence and method disclosed in US Patent US6884879B1.
  • the positive control Fc-D2 is a fusion protein constructed by linking the D2 domain of VEGFR1 at the C-terminus of Fc.
  • the EGFR-ECD-Fc protein used in the following examples is obtained by linking the extracellular domain (Met 1-Ser 645) of the human EGFR protein (NP_005219) with an FC tag and expressing it by transient transfection of 293F cells.
  • EGFR-ECD-Fc-biotin was obtained by biotinylation of EGFR-ECD-Fc protein using biotinylation reagent (thermofisher, Cat. #20217).
  • the VEGF referred to in the following examples is VEGF-A165.
  • the HRP referred to in the following examples is horseradish peroxidase.
  • fusion proteins or antibodies referred to in the following examples are numbered or defined according to the Kabat system.
  • A431 cells human skin squamous cell carcinoma cell line, purchased from ATCC.
  • KDR cells purchased from promega, Cat. #GA1082.
  • LIM1215 human colorectal cancer cell line, purchased from Guangzhou Genio Biotechnology Co., Ltd.
  • SW48 Human colon cancer cell line, purchased from ATCC.
  • SD rats purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • BALB/C nude mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • thermofisher purchased from thermofisher, Cat. No. A14697.
  • Recombinant VEGF-A165 protein purchased from acrobiosystems, Cat. #VE5-H4210.
  • HRP-labeled goat anti-human Fab antibody purchased from abcam, Cat. #ab87422.
  • Goat anti-human IgG-FITC purchased from sigma, Cat. #F9512.
  • Goat anti-human FC antibody purchased from Abcam, Cat. #ab97225.
  • Protein A chip purchased from GE HealthCare, Cat. #29127558.
  • EDC purchased from Fortebio, Cat. #18-1033.
  • s-NHS purchased from Fortebio, Cat. #18-1067.
  • AR2G probe purchased from Fortebio, Cat. #18-5092.
  • Sodium acetate purchased from Fortebio, Cat. #18-1070.
  • Ethanolamine Available from Fortebio, Cat. #18-1071.
  • 1*Kinetic Buffer purchased from Fortebio, Cat. #18-1072.
  • DMEM medium purchased from Gibco, Cat. #11995.
  • Bio-Glo purchased from promega, Cat. #G7940.
  • TMB purchased from BD Company, item number 555214.
  • HRP-tagged Streptavidin (streptavidin): purchased from BD Pharmingen, Cat. No. 554066.
  • HRP-labeled mouse anti-human Fab antibody purchased from GenScript, Cat. #A01855.
  • EGFR-his purchased from SINO, Cat. #10001-H08H.
  • thermofisher purchased from thermofisher, Cat. #21184.
  • nucleic acid sequences used in the following examples were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. or Jinweizhi Biotechnology Co., Ltd.
  • the anti-EGFR monoclonal antibody and the D2 domain of VEGFR1 are connected in series to construct an anti-EGFR/VEGF bifunctional fusion protein.
  • the schematic diagram of the structure is shown in FIG. 1 .
  • the N-terminus of the D2 domain (SEQ ID NO:1) of VEGFR1 and the C-terminus of the heavy chain (SEQ ID NO:2) of the anti-EGFR monoclonal antibody 602 are linked by a peptide linker L (SEQ ID NO:4),
  • the heavy chain (SEQ ID NO: 5) of fusion protein 602-D2 was obtained, and the light chain sequence of fusion protein 602-D2 was SEQ ID NO: 7.
  • the N-terminus of the D2 domain (SEQ ID NO: 1) of VEGFR1 and the C-terminus of the heavy chain (SEQ ID NO: 3) of the anti-EGFR monoclonal antibody 602dN are linked by a peptide linker L (SEQ ID NO: 4),
  • the heavy chain (SEQ ID NO: 6) of the fusion protein 602dN-D2 was obtained, and the light chain sequence of the fusion protein 602dN-D2 was SEQ ID NO: 7.
  • the N-terminus of the D2 domain (SEQ ID NO:8) of VEGFR1 and the C-terminus of the heavy chain (SEQ ID NO:2) of the anti-EGFR monoclonal antibody 602 are linked by a peptide linker L (SEQ ID NO:4),
  • the heavy chain (SEQ ID NO: 9) of fusion protein 602-D2-M2 was obtained, and the light chain sequence of fusion protein 602-D2-M2 was SEQ ID NO: 7.
  • the N-terminus of the D2 domain (SEQ ID NO:8) of VEGFR1 and the C-terminus of the heavy chain (SEQ ID NO:3) of the anti-EGFR monoclonal antibody 602dN are linked by a peptide linker L (SEQ ID NO:4),
  • the heavy chain (SEQ ID NO: 10) of the fusion protein 602dN-D2-M2 was obtained, and the light chain sequence of the fusion protein 602dN-D2-M2 was SEQ ID NO: 7.
  • the D2 domain (SEQ ID NO: 8) of VEGFR1 in the fusion proteins 602-D2-M2 and 602dN-D2-M2 is relative to the D2 domain (SEQ ID NO: 8) of VEGFR1 in the fusion proteins 602-D2 and 602dN-D2 : 1) Truncating 2 amino acids at the C-terminus, the two amino acids are easy to fall off during the fermentation process, and the removal does not affect the efficacy.
  • the heavy chain (SEQ ID NO: 3) of the anti-EGFR monoclonal antibody 602dN was mutated to glutamic acid at position 85 relative to the heavy chain (SEQ ID NO: 2) of the anti-EGFR monoclonal antibody 602 ( N85E), reducing the charge heterogeneity caused by glycosylation, facilitating the development of subsequent production and preparation processes, and deglycosylation has no obvious effect on the activity of the bifunctional fusion protein.
  • the fusion protein 602-D2 heavy chain nucleic acid sequence is SEQ ID NO: 11, and the light chain nucleic acid sequence is SEQ ID NO: 13.
  • the fusion protein 602dN-D2 heavy chain nucleic acid sequence is SEQ ID NO: 12, and the light chain nucleic acid sequence is SEQ ID NO: 13.
  • the fusion protein 602-D2-M2 heavy chain nucleic acid sequence is SEQ ID NO: 14, and the light chain nucleic acid sequence is SEQ ID NO: 13.
  • the fusion protein 602dN-D2-M2 heavy chain nucleic acid sequence is SEQ ID NO: 15, and the light chain nucleic acid sequence is SEQ ID NO: 13.
  • the DNA fragments of the heavy chain and light chain of the above four anti-EGFR/VEGF bifunctional fusion proteins were subcloned into pcDNA3.4 vector, respectively, and the recombinant plasmids were extracted and co-transfected into CHO cells and/or 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, vacuum filtered through a microporous membrane, and then loaded onto a HiTrap MabSelect SuRe column. The 100 mM citric acid (pH 3.5) eluent was used to elute the protein in one step, and the target sample was recovered and the medium was dialyzed and exchanged. to PBS.
  • the purified protein was detected by HPLC, and the detection results in Figures 2A-2D showed that the molecular states of the four fusion proteins were uniform, and the monomer purity reached more than 97%.
  • Example 3 Determination of the affinity of anti-EGFR/VEGF bifunctional fusion protein to antigen by enzyme-linked immunosorbent assay (ELISA)
  • the recombinant EGFR-ECD-Fc protein was diluted with coating solution to 1 ⁇ g/ml, added to the ELISA plate at 50 ⁇ l/well, and kept at 4°C overnight.
  • the plate was washed three times with PBST, 200 ⁇ l/well of blocking solution was added, and the plate was placed at 37° C. for 1 hour, and then the plate was washed once with PBST for use.
  • the anti-EGFR/VEGF bifunctional fusion protein was diluted to 60 nM with diluent, and 4-fold dilution was performed to form 8 concentration gradients.
  • the plate was washed three times with PBST, added with HRP-labeled mouse anti-human Fab antibody, and placed at 37°C for 30 minutes.
  • the recombinant VEGF-A165 protein was coated with 100 ng/well on the ELISA plate, overnight at 4°C.
  • the plate was washed three times with PBST, 200 ⁇ l/well of blocking solution was added, and the plate was placed at 37° C. for 1 hour, and then the plate was washed once with PBST for use.
  • the anti-EGFR/VEGF bifunctional fusion protein was diluted to 100 nM with diluent, and 4-fold dilution was performed to form 8 concentration gradients.
  • the plate was washed 3 times with PBST, added with HRP-labeled goat anti-human Fab antibody, and placed at 37°C for 30 minutes.
  • the experimental results are shown in Figure 4.
  • the anti-EGFR/VEGF bifunctional fusion proteins 602dN-D2, 602-D2-M2, 602dN-D2-M2 and the positive control anti-VEGF monoclonal antibody Bevacizumab (601) have EC50s of 0.497 for binding to VEGF, respectively.
  • nM, 0.603nM, 0.422nM and 0.747nM indicating that the fusion proteins 602dN-D2, 602-D2-M2, and 602dN-D2-M2 can specifically bind to VEGF, and their affinity for VEGF is similar to that of the anti-VEGF monoclonal antibody Bevacizumab (601 ) are comparable.
  • Steric hindrance may affect the ability of the anti-EGFR/VEGF bifunctional fusion protein to bind both antigens simultaneously.
  • the recombinant VEGF-A165 protein was plated at 20ng/well, overnight at 4°C. The plate was washed three times with PBST, 200 ⁇ l/well of blocking solution was added, and the plate was placed at 37° C. for 1 hour, and then the plate was washed once with PBST for use.
  • the anti-EGFR/VEGF bifunctional fusion protein was diluted to 200 nM with diluent, and 2-fold dilution was performed to form 12 concentration gradients.
  • the plate was washed three times with PBST, EGFR-ECD-Fc-biotin was added at 150 ng/well, and the plate was placed at 37°C for 1 hour. After washing the plate three times with PBST, HRP-labeled Streptavidin (streptavidin) was added, and the plate was placed at 37° C. for 30 minutes.
  • HRP-labeled Streptavidin streptavidin
  • the anti-EGFR/VEGF bifunctional fusion proteins 602dN-D2 and 602dN-D2-M2 can both specifically bind to EGFR and VEGF at the same time, with EC50 of 4.599nM and 4.518nM, respectively.
  • Example 4 Determination of the binding affinity of anti-EGFR/VEGF bifunctional fusion protein to the target cell surface antigen EGFR by FACS
  • A431 cells expressing EGFR on the cell surface were used as target cells.
  • the target cells were seeded in 96-well plates at 2 ⁇ 10 5 /well, washed three times with PBS containing 0.5% BSA, centrifuged at 300g for 5 minutes each time, and the supernatant was discarded.
  • Anti-EGFR/VEGF bifunctional fusion protein and control antibody were diluted to 50nM, 3-fold dilution to form 10 gradients as primary antibody, and added to 96-well plate, cells were suspended and incubated at 4°C for 1 hour.
  • the experimental results are shown in Figure 6.
  • the anti-EGFR/VEGF bifunctional fusion proteins 602dN-D2, 602dN-D2-M2 and the positive control anti-EGFR monoclonal antibody 602 can specifically bind to the EGFR expressed on the cell surface with EC50 of 1.784nM, respectively. , 1.840nM and 1.831nM, the affinity is similar.
  • the negative control negative control is an isotype control that does not bind to the EGFR antigen.
  • the Biacore 8K molecular interaction analyzer was used to measure the kinetic parameters of the binding and dissociation between the anti-EGFR/VEGF bifunctional fusion protein and the antigen EGFR using the capture method.
  • the fusion protein was diluted to 3 ⁇ g/ml with 1*HBS working solution, and the protein A
  • the chip captures the fusion protein; the antigen EGFR-his is diluted with 1 ⁇ HBS working solution, and 6 concentration gradients with a maximum concentration of 25nM are set to bind to the fusion protein and dissociate in the HBS working solution.
  • the experimental results are shown in Table 2.
  • the anti-EGFR/VEGF bifunctional fusion proteins 602dN-D2-M2, 602-D2-M2, and 602-D2 have comparable affinity and dissociation constants for EGFR-his to the positive control anti-EGFR monoclonal antibody 602. .
  • KD is the affinity constant
  • kon is the association rate constant
  • kdis is the dissociation rate constant
  • the kinetic parameters of the binding and dissociation of the anti-EGFR/VEGF bifunctional fusion protein and the antigen VEGF-A165 were determined by the octet molecular interaction analyzer using the capture method.
  • the AR2G probe was activated with 20 mM EDC and 10 mM s-NHS, and the antigen VEGF was activated.
  • -A165 was diluted to 5 ⁇ g/ml with 10 mM Sodium Acetate (sodium acetate, pH 6.0), bound to the activated AR2G probe, and the probe was blocked with 1 M Ethanolamine (ethanolamine, pH 8.5) solution. Dilute the fusion protein with 1*Kinetic Buffer working solution, set 5 concentration gradients with a maximum concentration of 12.5nM, and dissociate in 1*Kinetic Buffer working solution.
  • the experimental results are shown in Table 3.
  • the anti-EGFR/VEGF bifunctional fusion protein 602dN-D2-M2 has a slightly better affinity for VEGF165 than the positive control anti-VEGF monoclonal antibody Bevacizumab.
  • KD is the affinity constant
  • kon is the association rate constant
  • kdis is the dissociation rate constant
  • Example 7 Inhibitory activity of anti-EGFR/VEGF bifunctional fusion protein on A431 cell proliferation
  • the log phase growing A431 cells in the adherent culture were taken, and the growth medium RPMI 1640 (10% FBS, 1% penicillin-streptomycin) was discarded. After washing once with DPBS, trypsinization was performed, and the trypsin was neutralized with growth medium. Trypan blue cells were counted and centrifuged at 300g for 5min. Resuspend in RPMI 1640 (1% FBS, 1% penicillin-streptomycin) medium, count and plate, adjust the density to 5000 cells/well, 100 ⁇ l/well, place at 37°C, 5% CO 2 , incubate overnight for about 24 hours .
  • RPMI 1640 1% FBS, 1% penicillin-streptomycin
  • the experimental results are shown in Figure 7.
  • the anti-EGFR/VEGF bifunctional fusion proteins 602-D2-M2, 602dN-D2-M2 and the positive control 602 mAb can effectively inhibit the proliferation of A431 cells with IC50 of 2.942nM and 3.976nM, respectively. and 3.556nM, indicating that the anti-EGFR/VEGF bifunctional fusion protein has a similar inhibitory activity on the proliferation of A431 cells as the positive control anti-EGFR monoclonal antibody 602.
  • Example 8 Cell experiment of anti-EGFR/VEGF bifunctional fusion protein blocking the binding of VEGF to receptor KDR
  • VEGF-A165 was diluted to 30 ng/ml in DMEM medium containing 10% FBS, and fusion proteins were fold-diluted in VEGF-containing medium, 3-fold dilution, 10 gradients.
  • the diluted fusion protein was added to 25 ⁇ l of each well of the cell well (the final VEGF concentration was 10 ng/ml, and the initial concentration of fusion protein was 50 nM). After incubation at 37°C for 6 hours, 75 ⁇ l of the detection reagent Bio-Glo was added to each well. After 10 min incubation at room temperature, the luminescence (fluorescence intensity) was read with a SpectraMax i3x. All data are double wells, and the obtained signal values are averaged and then fitted by the 4-parameter method, and the data is analyzed by GraphPad Prism7 software.
  • LIM1215 cells cultured in vitro (the surface of LIM1215 cells expresses EGFR, and anti-EGFR antibodies can effectively inhibit the proliferation of LIM1215 cells) were collected, and the concentration of the cell suspension was adjusted to 2 ⁇ 10 7 /ml and mixed with an equal volume of Matrigel. Under sterile conditions, 100 ⁇ l of the cell suspension was inoculated subcutaneously on the right flank of nude mice. The diameter of the transplanted tumor was measured with a vernier caliper, and the animals were randomly divided into groups after the average tumor volume grew to about 200 mm 3 .
  • the doses of the test sample 602dN-D2-M2 were set to 5.65mg/kg and 28.25mg/kg, the doses of the control drug 602 were set to 5mg/kg and 25mg/kg, and the doses of the control drug Bevacizumab were set At 5 mg/kg and 25 mg/kg, the blank control group (CONTROL) was given the same volume of normal saline. It was administered by intraperitoneal injection 3 times a week for 3 consecutive weeks. During the whole experiment, the diameter of the transplanted tumor was measured twice a week, and the mice were weighed at the same time.
  • Relative tumor volume (RTV) was calculated from the measured results.
  • SW48 cells cultured in vitro (the surface of SW48 cells expresses EGFR, and anti-EGFR antibodies can effectively inhibit the proliferation of SW48 cells) were collected, and the concentration of the cell suspension was adjusted to 6 ⁇ 10 7 /ml, and mixed with an equal volume of Matrigel. Under sterile conditions, 100 ⁇ l of the cell suspension was inoculated subcutaneously on the right flank of nude mice. The diameter of the transplanted tumor was measured with a vernier caliper, and the animals were randomly divided into groups after the average tumor volume grew to 200 mm 3 .
  • test sample 602dN-D2-M2 The dose of test sample 602dN-D2-M2 was set to 11.3mg/kg, the dose of control drug 602 and Bevacizumab were both set to 10mg/kg, and the combination group was given 602 and Bevacizumab (10mg/kg and 10mg/kg) at the same time. /kg), the blank control group (CONTROL) was given the same volume of normal saline. It was administered by intraperitoneal injection 3 times a week for 3 consecutive weeks. During the whole experiment, the diameter of the transplanted tumor was measured twice a week, and the mice were weighed at the same time.
  • the thermal stability of 602dN-D2-M2 samples in PBS buffer system was detected by DSC (Differential scanning calorimetry, differential scanning calorimetry).
  • the samples were exchanged into PBS buffer, and the concentration of the samples was controlled at 1 mg/ml, and detected by MicroCal*Vp-Capillary DSC (Malvern).
  • the samples and blank buffer were filtered through a 0.22 ⁇ m filter. 400 ⁇ l of sample or blank buffer was added to each well of the sample plate (6 groups of blank buffer pairs were set), and ddH 2 O was added to the last three pairs of well plates for cleaning. After the sample plate is added, put on the plastic soft cover.
  • the scanning temperature started at 25°C and ended at 100°C, and the scan rate was 150°C/h.
  • Example 13 In vivo antitumor effect of anti-EGFR/VEGF bifunctional fusion protein on human breast cancer mouse xenograft model
  • a subcutaneous xenograft model of human breast cancer JIMT-1 was established in CB-17 SCID mice to evaluate the in vivo antitumor activity of fusion protein 602dN-D2-M2.
  • the specific implementation steps are as follows: collecting human breast cancer JIMT-1 cells cultured in vitro, adjusting the cell suspension concentration to 1.6 ⁇ 10 8 /ml, and mixing with Matrigel in an equal ratio of 1:1.
  • the right flank of CB-17 SCID mice was shaved, and under sterile conditions, 100 ⁇ l of the mixed suspension of cells was inoculated subcutaneously on the right flank of the mouse.
  • mice When the subcutaneous tumor grows to a volume of about 150 mm3 , the mice are randomly divided into 5 groups according to the tumor volume, with 8 mice in each group, including: blank control group, injected with normal saline only, as control; anti-EGFR control antibody 602 group , dose 10mg/kg; anti-VEGF positive control antibody Avastin (ie Bevacizumab) group, dose 10mg/kg; 602+Avastin combination group, dose 10mg/kg+10mg/kg and 602dN-D2-M2 group, dose 11.3mg/kg kg. Dosing 3 times a week for a total of 4 weeks. Tumor volume was measured twice a week. Finally, the measured growth curves of each group of tumors over time are shown in Figure 11 .
  • anti-EGFR antibody 602 can significantly inhibit tumor growth, with a tumor inhibition rate of 66.2%; anti-VEGF antibody Avastin can also inhibit tumor growth, with a tumor inhibition rate of 22.3%, which is weaker than that of anti-EGFR.
  • Monoclonal antibody; anti-EGFR monoclonal antibody 602 and anti-VEGF monoclonal antibody Avastin combined therapy can significantly inhibit tumor growth, with a tumor inhibition rate of 80.7%.
  • the fusion protein 602dN-D2-M2 can also significantly inhibit tumor growth, and its tumor inhibition rate is 88.7%, which is significantly better than that of either monoclonal antibody alone and higher than the combination of two monoclonal antibodies.
  • Example 14 In vivo antitumor effect of anti-EGFR/VEGF bifunctional fusion protein on human throat cancer mouse xenograft model
  • the human throat cancer FaDu subcutaneous xenograft model was established in BALB/C-nude nude mice to evaluate the in vivo antitumor activity of fusion protein 602dN-D2-M2.
  • the specific implementation steps are as follows: collect FaDu cells of human throat cancer cultured in vitro, adjust the cell suspension concentration to 1 ⁇ 10 8 /ml, and mix with Matrigel in an equal ratio of 1:1. Under sterile conditions, 100 ⁇ l of the cell suspension was inoculated subcutaneously on the right flank of nude mice.
  • mice When the subcutaneous tumor grows to a volume of about 150mm3 , the mice are randomly divided into 5 mice in each group according to the tumor volume, including: blank control group, only injected with PBS, as control; Fc-D2 positive control group, dose of 2.5mg /kg; anti-VEGF positive control antibody Avastin (ie Bevacizumab) group, dose 5mg/kg and 602dN-D2-M2 group, dose 5.65mg/kg. 2 times a week for a total of 7 doses. Tumor volume was measured twice a week. Finally, the measured growth curves of each group of tumors over time are shown in FIG. 12 .

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Abstract

本发明涉及融合蛋白技术领域，具体地，涉及一种抗EGFR/VEGF双功能融合蛋白及其用途。所述抗EGFR/VEGF双功能融合蛋白包含：(a)抗EGFR抗体或其抗原结合片段，和(b)VEGFR1的胞外区的D2结构域。本发明的融合蛋白在治疗与EGFR和VEGF高表达或异常表达相关的疾病中具有很大临床应用前景。

Description

一种抗EGFR/VEGF双功能融合蛋白及其用途 技术领域
本发明涉及融合蛋白技术领域，具体地，涉及一种抗EGFR/VEGF双功能融合蛋白及其用途。
背景技术
EGFR(Epidermal Growth Factor Receptor)是表皮生长因子(EGF)的受体，属于ErbB受体家族。EGFR是一种分子量为170KDa的跨膜糖蛋白，属于受体型酪氨酸激酶，在相关配体如EGF和转化生长因子-α(transforming growth factor α,TGFα)的作用下，EGFR由单体转化为二聚体而被激活，从而进一步活化下游信号传导通路，调控细胞的增殖。大量研究表明，在大多数肿瘤如胶质细胞癌、肾癌、肺癌、前列腺癌、胰腺癌、乳腺癌等组织中存在EGFR的高表达或者异常表达。EGFR功能的异常与肿瘤细胞的增殖、血管生成、肿瘤侵袭、转移以及细胞凋亡的抑制有关。其功能的异常主要表现为两个方面：一是在肿瘤组织中的过度异常表达，二是EGFR突变体在肿瘤细胞中的持续性激活(不需要配体刺激或形成自循环刺激通路)。在结肠癌病人组织中，EGFR的表达率约为25-77％。相关临床数据表明，EGFR表达量的多少与肿瘤的恶性程度以及肿瘤患者的预后密切相关。
肿瘤的生长有两个阶段，从无血管的缓慢生长期到有血管的快速增殖期。如果肿瘤内部没有血管的生成，则原发肿瘤生长缓慢，转移无法实现。因此抑制肿瘤血管生成被认为是当前具有前途的肿瘤治疗方法之一。血管内皮生长因子(VEGFs)家族中，VEGF-A165(以下简称VEGF)是最丰富活跃的亚型。VEGF通过与II型受体VEGFR2结合，激活信号通路发生一系列级联反应，促进新生血管形成并维持其完整性。但I型受体VEGFR1与VEGF结合的能力远大于VEGFR2，发生作用部位主要是VEGFR1的胞外区D2结构域。VEGFR1-D2通过竞争结合VEGF，阻断VEGFR2与VEGF结合，从而阻断信号通路，抑制内皮细胞增殖与血管生成，从而抑制肿瘤的快速增殖与转移。
目前，仍有患者对抗EGFR抗体、抗VEGF抗体或VEGF-Trap的治疗无反应或反应较差，尚需要开发一种可同时靶向EGFR和VEGF的双功能融合蛋白， 以满足临床需求。
发明内容
本发明的目的在于提供一种新的抗EGFR/VEGF双功能融合蛋白，该融合蛋白能同时与EGFR及VEGF结合，从而阻断EGFR和VEGF信号通路。本发明的目的还在于提供编码所述融合蛋白的核酸分子；提供包含所述核酸分子的表达载体；提供包含所述表达载体的宿主细胞；提供所述融合蛋白的制备方法；提供包含所述融合蛋白的药物组合物；提供所述融合蛋白或所述药物组合物在制备治疗癌症的药物中的应用；提供所述融合蛋白或所述药物组合物用于治疗癌症的方法。
为了达到上述目的，本发明提供了以下技术方案：
本发明的第一个方面提供了一种抗EGFR/VEGF双功能融合蛋白，包含：(a)抗EGFR抗体或其抗原结合片段，和(b)VEGFR1的胞外区的D2结构域。
在一个优选的实施方案中，所述抗EGFR抗体或其抗原结合片段包含重链互补决定区HCDR1-3和轻链互补决定区LCDR1-3，其中HCDR1的氨基酸序列如SEQ ID NO：16所示，HCDR2的氨基酸序列如SEQ ID NO：17所示，HCDR3的氨基酸序列如SEQ ID NO：18所示；LCDR1的氨基酸序列如SEQ ID NO：19所示，LCDR2的氨基酸序列如SEQ ID NO：20所示，LCDR3的氨基酸序列如SEQ ID NO：21所示。
在一个优选的实施方案中，所述抗EGFR抗体或其抗原结合片段包含重链可变区VH和轻链可变区VL，其中VH的氨基酸序列如SEQ ID NO：22所示，VL的氨基酸序列如SEQ ID NO：24所示。
在一个优选的实施方案中，所述重链可变区VH包含至少一个去糖基化突变；优选的，所述突变位于重链可变区VH的框架区FR内。
在一个优选的实施方案中，所述突变位于重链可变区VH的第85位，所述突变根据Kabat编号系统编号；优选的，所述突变是N85E。
在一个优选的实施方案中，所述重链可变区VH的氨基酸序列如SEQ ID NO：23所示，轻链可变区VL的氨基酸序列如SEQ ID NO：24所示。
在一个优选的实施方案中，所述抗EGFR抗体包含重链恒定区和轻链恒定区；更优选的，所述重链恒定区选自人IgG1、人IgG2、人IgG3或人IgG4，所述轻 链恒定区选自人κ(Kappa)或人λ(Lambda)。
在一个优选的实施方案中，所述抗EGFR抗体或其抗原结合片段选自全长抗体、scFv、Fv、Fab或F(ab')2。
在一个优选的实施方案中，所述抗EGFR抗体或其抗原结合片段为嵌合抗体或其抗原结合片段，或人源化抗体或其抗原结合片段。
在一个优选的实施方案中，所述抗EGFR抗体为单克隆抗体。
在一个优选的实施方案中，所述抗EGFR抗体包含氨基酸序列如SEQ ID NO：2或SEQ ID NO：3所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链。
在一个优选的实施方案中，所述VEGFR1的胞外区的D2结构域包含如SEQ ID NO：1或SEQ ID NO：8所示的氨基酸序列。
在一个优选的实施方案中，所述抗EGFR抗体或其抗原结合片段与VEGFR1的胞外区的D2结构域通过肽键直接连接或通过肽接头连接。
在一个优选的实施方案中，所述肽接头包含氨基酸序列(GGGGS)n，所述n选自1、2、3、4或5。
在一个优选的实施方案中，所述抗EGFR抗体为全长抗体，包含2条重链和2条轻链；所述VEGFR1的胞外区D2结构域的N末端或C末端通过肽接头连接至抗EGFR抗体的重链或轻链的N末端或C末端。
在一个优选的实施方案中，所述VEGFR1的胞外区的D2结构域的N末端通过肽接头连接至抗EGFR抗体的重链的C末端。
在一个优选的实施方案中，所述融合蛋白选自：
a)所述融合蛋白包含氨基酸序列如SEQ ID NO：5所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
b)所述融合蛋白包含氨基酸序列如SEQ ID NO：6所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
c)所述融合蛋白包含氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
d)所述融合蛋白包含氨基酸序列如SEQ ID NO：10所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链。
在一个优选的实施方案中，所述融合蛋白选自：602-D2、602dN-D2、602-D2-M2或602dN-D2-M2。
本发明的第二个方面提供了一种核酸分子，所述核酸分子编码上述的抗EGFR/VEGF双功能融合蛋白。
在一个优选的实施方案中，所述核酸分子包含：
a)编码融合蛋白重链的如SEQ ID NO：11所示的核酸序列，和编码融合蛋白轻链的如SEQ ID NO：13所示的核酸序列；或，
b)编码融合蛋白重链的如SEQ ID NO：12所示的核酸序列，和编码融合蛋白轻链的如SEQ ID NO：13所示的核酸序列；或，
c)编码融合蛋白重链的如SEQ ID NO：14所示的核酸序列，和编码融合蛋白轻链的如SEQ ID NO：13所示的核酸序列；或，
d)编码融合蛋白重链的如SEQ ID NO：15所示的核酸序列，和编码融合蛋白轻链的如SEQ ID NO：13所示的核酸序列。
本发明的第三个方面提供了一种表达载体，所述表达载体含有上述的核酸分子。
本发明的第四个方面提供了一种宿主细胞，所述宿主细胞含有上述的核酸分子。
本发明的第五个方面提供了一种抗EGFR/VEGF双功能融合蛋白的制备方法，所述制备方法包括以下步骤：
a)在表达条件下，培养上述宿主细胞，从而表达抗EGFR/VEGF双功能融合蛋白；
b)分离并纯化步骤a)所述的抗EGFR/VEGF双功能融合蛋白。
本发明的第六个方面提供了一种药物组合物，所述药物组合物包含上述的抗EGFR/VEGF双功能融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明的第七个方面提供了上述的抗EGFR/VEGF双功能融合蛋白或药物组合物在制备治疗癌症的药物中的用途，优选的，所述癌症高表达或异常表达EGFR和/或高表达或异常表达VEGF。
在一个优选的实施方案中，所述癌症选自：结直肠癌、皮肤癌、鳞状细胞癌、乳腺癌、胶质细胞癌、肾癌、肺癌、前列腺癌、胰腺癌、头颈癌(包括但不限于咽喉癌和鼻咽癌)、或其组合；更优选的，所述癌症选自结肠癌、皮肤鳞状细胞癌、乳腺癌、咽喉癌、或其组合。
在一个优选的实施方案中，所述治疗包括向受试者施用有效量的抗EGFR/VEGF双功能融合蛋白或药物组合物，所述抗EGFR/VEGF双功能融合蛋白或药物组合物通过注射施用；更优选的，所述注射选自静脉注射、肌肉注射、或皮下注射。
在一个优选的实施方案中，所述治疗包括向受试者施用有效量的抗EGFR/VEGF双功能融合蛋白或药物组合物，所述有效量是指0.5-30mg/kg的剂量；更优选的，所述抗EGFR/VEGF双功能融合蛋白或药物组合物的施用间隔选自：a)每周一次；b)每周二次；c)每周三次；进一步更优选的，所述抗EGFR/VEGF双功能融合蛋白或药物组合物的施用时间选自：a)至少1周；b)至少2周；c)至少3周；d)至少4周。
本发明的第八个方面提供了一种免疫偶联物，所述免疫偶联物包括：
(a)如上述的抗EGFR/VEGF双功能融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
本发明的第九个方面提供了一种治疗癌症的方法，包括向受试者施用有效量的上述的抗EGFR/VEGF双功能融合蛋白、免疫偶联物或药物组合物，优选的，所述癌症高表达或异常表达EGFR和/或高表达或异常表达VEGF。
在一个优选的实施方案中，所述癌症选自：结直肠癌、皮肤癌、鳞状细胞癌、乳腺癌、胶质细胞癌、肾癌、肺癌、前列腺癌、胰腺癌、头颈癌(包括但不限于咽喉癌和鼻咽癌)、或其组合；更优选的，所述癌症选自结肠癌、皮肤鳞状细胞癌、乳腺癌、咽喉癌、或其组合。
在一个优选的实施方案中，所述抗EGFR/VEGF双功能融合蛋白或药物组合物通过注射施用；更优选的，所述注射选自静脉注射、肌肉注射、或皮下注射。
在一个优选的实施方案中，所述有效量是指0.5-30mg/kg的剂量；更优选的，所述抗EGFR/VEGF双功能融合蛋白或药物组合物的施用间隔选自：a)每周一次；b)每周二次；c)每周三次；进一步更优选的，所述抗EGFR/VEGF双功能融合蛋白或药物组合物的施用时间选自：a)至少1周；b)至少2周；c)至少3周；d)至少4周。
本发明的积极效果具体体现在：本发明的融合蛋白在分子水平可同时与EGFR和VEGF高亲和力结合，且具有良好的热稳定性；在细胞水平，其能够有 效地抑制A431细胞增殖，其阻断VEGF与受体KDR结合的能力优于抗VEGF单抗Bevacizumab；在动物水平，其在LIM1215移植瘤、SW48移植瘤、JIMT-1移植瘤、FaDu移植瘤模型中，均起到了显著的抑瘤作用，并且其在LIM1215移植瘤、SW48移植瘤移植瘤、JIMT-1移植瘤中的抑瘤效果均优于对照抗EGFR单抗602以及抗VEGF单抗Bevacizumab，出人意料地，还发现其在SW48和JIMT-1移植瘤中的抑瘤效果优于Bevacizumab和602的联用，展现出了协同效应。本发明的融合蛋白在治疗与EGFR和VEGF高表达或异常表达相关的疾病中具有很大临床应用前景。
附图说明
图1：抗EGFR/VEGF双功能融合蛋白结构示意图。
图2A：抗EGFR/VEGF双功能融合蛋白602-D2的HPLC检测结果图。
图2B：抗EGFR/VEGF双功能融合蛋白602-D2-M2的HPLC检测结果图。
图2C：抗EGFR/VEGF双功能融合蛋白602dN-D2的HPLC检测结果图。
图2D：抗EGFR/VEGF双功能融合蛋白602dN-D2-M2的HPLC检测结果图。
图3：抗EGFR/VEGF双功能融合蛋白与EGFR的亲和力的ELISA检测结果图。
图4：抗EGFR/VEGF双功能融合蛋白与VEGF的亲和力的ELISA检测结果图。
图5：抗EGFR/VEGF双功能融合蛋白同时结合EGFR和VEGF的ELISA检测结果图。
图6：抗EGFR/VEGF双功能融合蛋白结合靶细胞表面抗原EGFR的FACS检测结果图。
图7：抗EGFR/VEGF双功能融合蛋白对A431细胞增殖的抑制活性检测结果图。
图8：抗EGFR/VEGF双功能融合蛋白阻断VEGF与受体KDR结合检测结果图。
图9：抗EGFR/VEGF双功能融合蛋白在LIM1215移植瘤模型上的抗肿瘤作用结果图。
图10：抗EGFR/VEGF双功能融合蛋白在SW48移植瘤模型上的抗肿瘤作 用结果图。
图11：抗EGFR/VEGF双功能融合蛋白在JIMT-1移植瘤模型上的抗肿瘤作用结果图。
图12：抗EGFR/VEGF双功能融合蛋白在FaDu移植瘤模型上的抗肿瘤作用结果图。
具体实施方式
本发明人通过广泛而深入的研究，构建了一种包含抗EGFR抗体或其抗原结合片段和VEGFR1的胞外区的D2结构域的双功能融合蛋白。本发明的双功能融合蛋白能与EGFR和VEGF高亲和力结合，在细胞水平，其能够有效地抑制A431细胞增殖，并且阻断VEGF与受体KDR结合的能力优于抗VEGF单抗Bevacizumab；在动物水平上，其在LIM1215移植瘤、SW48移植瘤模型、JIMT-1移植瘤模型以及FaDu移植瘤模型中，均起到了显著的抑瘤作用。
令人惊讶地是，本发明的双功能蛋白在SW48移植瘤中的抑瘤效果优于Bevacizumab和抗EGFR抗体602的联用，展现出了协同效应；在JIMT-1移植瘤模型中，也优于Bevacizumab和抗EGFR抗体602的联用，同样地展现出了协同效应。本发明的双功能融合蛋白还具有良好的热稳定性。因此，本发明的双功能融合蛋白在治疗与EGFR和VEGF高表达或异常表达相关的疾病中具有优异的临床应用前景。在此基础上完成了本发明。
本发明中，术语“融合蛋白”是指由两个或多个相同或不同的多肽序列融合得到的新的多肽序列。术语“融合”是指由肽键直接连接或借助一个或多个连接肽(肽接头)连接。术语“连接肽(肽接头)”是指可以连接两个多肽序列的短肽，一般是指长度为2-30个氨基酸的肽。优选地，为柔性肽接头，如氨基酸序列(GGGGS)n，所述n选自1、2、3、4或5。
本发明中，术语“抗体”是指全长抗体，术语“抗原结合片段”是指来源于抗体且能结合抗原表位的片段，包括但不限于scFv、Fv、Fab或F(ab')2。
本发明中，术语“全长抗体”是指有相同结构特征的约150000道尔顿的异四聚糖蛋白，包含可变区(V)和恒定区(C)，其由两条相同的重链(H)和两条相同的轻链(L)组成。每条重链的一端有重链可变区(VH)，其后是重链恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端 有轻链可变区(VL)，另一端有轻链恒定区，轻链恒定区包括一个结构域CL；轻链恒定区与重链恒定区的CH1结构域配对，轻链可变区与重链可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于重链可变区和轻链可变区中称为互补决定区(complementarity-determining region，CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(frame region，FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。重链可变区(VH)和轻链可变区(VL)的CDR分别称为HCDR和LCDR。
本发明中，术语“人源化抗体”是指抗体互补决定区(CDR)来源于非人物种(例如啮齿动物)，抗体分子中残余的部分(包括框架区FR和恒定区C)来源于人的抗体。其中，框架区FR残基可被改变以维持结合亲和性。
如本文所用，术语“框架区”(FR)指免疫球蛋白可变区内超变区之外的氨基酸组成和排列顺序变化相对较小的部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)，且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语“人框架区”是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
本发明中，术语“嵌合抗体”是指可变区(V)来源于非人物种(例如啮齿动物)，恒定区(C)来源于人的抗体。
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体中获得的抗体，除少数可能存在的天然突变外，该群体中包含的单个抗体是相同的。单克隆抗体针对抗原上的单个决定簇，对单个抗原位点高特异。单克隆抗体可以通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。
本发明中，术语“抗”和“结合”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。通常，抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M、10 -10M、10 -11M或更小的平衡解离常数(KD)结合该抗原。术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，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。例如，使用表面等离子体共振术(Surface Plasmon Resonance，缩写SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。
本发明中，术语“亲和(能)力”、“结合(能)力”或“结合亲和(能)力”可以互换使用，是指两个分子之间的结合相互作用的强度。
本发明中，术语“VEGFR1的胞外区的D2结构域”包括天然序列及其变体(包含至少1个氨基酸突变)，只要所述突变不改变D2结构域的生物学活性。
本发明中，术语“表达载体”是指包含适当的调控序列，例如启动子、终止子、增强子等的本领域的常规表达载体，所述表达载体可以是病毒或质粒。所述表达载体较佳地包括：pcDNA3.4、pDR1、pDHFR或pTT5。
本发明中，术语“宿主细胞”为本领域常规的各种宿主细胞，只要能使载体稳定地自行复制，且所携带的核酸分子可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述宿主细胞较佳地包括：COS、CHO、NS0、sf9、sf21、DH5α、BL21(DE3)、TG1、BL21(DE3)或293F细胞。
本发明中，术语“有效量”是指本发明的融合蛋白或药物组合物施用患者后， 在治疗的个体中产生预期效果的活性成分的量或剂量，该预期效果包括个体病症的改善。
本发明中，术语“受试者”包括但不限于哺乳动物，例如人、非人灵长类动物、大鼠和小鼠等。
本发明中，术语“测定”或“检测”可以互换使用。
本发明中，术语“TIW”是指一周给药三次，术语“mpk”是指mg/kg(毫克/千克)。
本发明中，术语“SEM”是指扫描电镜。
本发明中，术语“mAU(m Absorbance Unit)”是指毫吸光度单位。
本发明的双功能融合蛋白
本发明的双功能融合蛋白是一种同时靶向EGFR和VEGF的双功能融合蛋白，包含：(a)抗EGFR抗体或其抗原结合片段，和(b)VEGFR1的胞外区的D2结构域。
如本文所用，“双功能融合蛋白”、“本发明的双功能抗体融合蛋白”、“靶向EGFR和VEGF的双功能融合蛋白”、“抗EGFR/VEGF双功能融合蛋白”可以互换使用，皆指本发明第一方面所述的包含抗EGFR抗体部分以及VEGFR1的D2结构域部分的双功能融合蛋白。
在本发明的具体实施例中，所述抗EGFR抗体或其抗原结合片段包括重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1-3，和所述轻链可变区包含轻链互补决定区LCDR1-3；其中HCDR1的氨基酸序列如SEQ ID NO：16所示，HCDR2的氨基酸序列如SEQ ID NO：17所示，HCDR3的氨基酸序列如SEQ ID NO：18所示；LCDR1的氨基酸序列如SEQ ID NO：19所示，LCDR2的氨基酸序列如SEQ ID NO：20所示，LCDR3的氨基酸序列如SEQ ID NO：21所示。
在另一优选例中，所述抗EGFR抗体的重链可变区和轻链可变区还包括重链、轻链的框架区。进一步地，为了减少氨基酸糖基化导致的电荷异质性对双功能融合蛋白生产制备的影响，本发明的双功能融合蛋白中的抗EGFR抗体部分还包括其突变体，较佳地，所述突变包括1、2、3、4、或5个去糖基化突变。优选地，所述突变位于重链可变区的框架区FR内；优选地，所述突变是N85E(根据Kabat 编号系统编号)。
较佳地，所述抗EGFR抗体包含氨基酸序列如SEQ ID NO：2或SEQ ID NO：3所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链。
如本文所用，“VEGFR1的胞外区的D2结构域”、“VEGFR1-D2”或“VEGFR1的D2结构域”可以互换使用，指人的VEGFR1的胞外区的D2结构域(野生型序列如SEQ ID NO.1所示)，其通过竞争结合VEGF，阻断VEGFR2与VEGF结合，从而阻断信号通路，抑制内皮细胞增殖与血管生成，从而抑制肿瘤的快速增殖与转移。“VEGFR1的D2结构域”还包括VEGFR1的D2结构域蛋白变体，优选为相对野生型的VEGFR1的D2结构域进行氨基酸的替换、突变或删除等而获得的突变体。较佳地，所述VEGFR1的D2结构域蛋白变体相对于野生型(SEQ ID NO.1)的序列在C末端截断2个氨基酸。该两个氨基酸在发酵过程中容易脱落，去除并不影响其结合活性。较佳地，所述VEGFR1的D2结构域蛋白变体的序列如SEQ ID NO.8所示。
在另一优选例中，所述双功能融合蛋白包括对融合蛋白中的抗EGFR抗体和/或VEGFR1的结构域D2结构域进行氨基酸的替换、突变或删除等而获得的突变体。
在另一优选例中，所述双功能融合蛋白中，所述VEGFR1的胞外区的D2结构域的N末端或C末端通过肽接头连接至所述抗EGFR抗体的重链或轻链的N末端或C末端。较佳地，所述VEGFR1的胞外区的D2结构域的N末端通过肽接头连接至所述抗EGFR抗体的重链的C末端。
在具体实施例中，所述双功能融合蛋白的重链和轻链的序列选自下组：
a)所述双功能融合蛋白包含氨基酸序列如SEQ ID NO：5所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
b)所述双功能融合蛋白包含氨基酸序列如SEQ ID NO：6所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
c)所述双功能融合蛋白包含氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
d)所述双功能融合蛋白包含氨基酸序列如SEQ ID NO：10所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链。
本领域技术人员也可以通过本领域熟知的技术对本发明双功能融合蛋白进行修饰，例如添加、缺失和/或取代一个或几个氨基酸残基，从而进一步增加本 发明双功能融合蛋白的亲和力或结构稳定性，并通过常规的测定方法获得修饰后的结果。
在本发明中，本发明双功能融合蛋白还包括其保守性变异体，指与本发明双功能融合蛋白的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明的双功能融合蛋白可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、或任何以上这些物质的组合结合或偶联。
编码核酸和表达载体
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明中，术语“表达载体”是指携带表达盒用于表达特定目的蛋白或其他 物质的载体，如质粒、病毒载体(如腺病毒、逆转录病毒)、噬菌体、酵母质粒或其他载体。例如包含适当的调控序列，例如启动子、终止子、增强子等的本领域的常规表达载体，所述表达载体包括但并不限于：病毒载体(如腺病毒、逆转录病毒)、质粒、噬菌体、酵母质粒或其他载体。所述表达载体较佳地包括pDR1、pcDNA3.4(+)、pDHFR或pTT5。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
本发明中，术语“宿主细胞”为本领域常规的各种宿主细胞，只要能使载体稳定地自行复制，且所携带的多核苷酸分子可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述宿主细胞较佳地包括：COS、CHO、NS0、sf9、sf21、DH5α、BL21(DE3)、TG1、BL21(DE3)、293F或293E细胞。
药物组合物和应用
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的双功能融合蛋白或其活性片段、或其免疫偶联物，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为4-8，较佳地pH约为5-7，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射(如腹膜内)、颅内注射、或腔内注射。
本发明中，术语“药物组合物”是指本发明的双功能融合蛋白可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双功能融合蛋白的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的双功能融合蛋白(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、 甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的双功能融合蛋白还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的双功能融合蛋白或其免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
在另一优选例中，所述药物组合物的剂型包括胃肠给药剂型或胃肠外给药剂型。所述的胃肠外给药剂型包括静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射、颅内注射、或腔内注射。
本发明的药物组合物可直接用于结合VEGF蛋白分子或EGFR，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
如本文所用，术语“有效量”是指本发明的药物组合物施用受试者后，在治疗的个体中产生预期效果的量或剂量，该预期效果包括个体病症的改善。术语“受试者”包括但不限于哺乳动物，例如人、非人灵长类动物、大鼠和小鼠等。
如本文所用，本发明的双功能融合蛋白的免疫偶联物包括：
(a)如本发明第一方面所述的双功能融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、 Xe-133Yb-169、Yb-177、或其组合。
在另一优选例中，所述偶联部分为药物或毒素；所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱、或其组合。
本发明的双功能融合蛋白或其组合物可用于制备治疗肿瘤的药物或抑制肿瘤细胞的增殖、血管生成、肿瘤侵袭、转移以及细胞凋亡的药物或制备阻断VEGFR2与VEGF结合或抑制肿瘤内部血管生成的药物。优选地，所述肿瘤是过表达EGFR和/或VEGF2的肿瘤或癌症；优选地，所述适应症包括结直肠癌、皮肤癌、鳞状细胞癌、乳腺癌、胶质细胞癌、肾癌、肺癌、前列腺癌、胰腺癌、头颈癌(包括但不限于咽喉癌和鼻咽癌)、或其组合。
以下实验例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法或本领域常规方法的详细描述，如核酸分子的制备方法、用于构建载体和质粒的方法、将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法、宿主细胞的培养方法等，这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。除非另外说明，否则百分比和份数按重量计算。
本发明中涉及的序列信息总结在表1中。
表1序列表
Figure PCTCN2022089244-appb-000001
Figure PCTCN2022089244-appb-000002
Figure PCTCN2022089244-appb-000003
Figure PCTCN2022089244-appb-000004
Figure PCTCN2022089244-appb-000005
Figure PCTCN2022089244-appb-000006
以下实施例中使用的阳性对照抗EGFR单抗602为按照Erbitux(Cetuximab,IMC-C225)的氨基酸序列、用CHO细胞表达系统进行表达、经细胞培养与生产后制备获得的人鼠嵌合单克隆抗体。阳性对照抗VEGF单抗Bevacizumab(601)为按照美国专利US6884879B1中披露的序列与方法制备获得的人源化单克隆抗体。阳性对照Fc-D2为通过在Fc的C端连接VEGFR1的D2结构域而构建的融合蛋白。
以下实施例中使用的EGFR-ECD-Fc蛋白为将人源EGFR蛋白(NP_005219)的胞外结构域(Met 1-Ser 645)连接FC标签，通过瞬时转染293F细胞进行表达获得。EGFR-ECD-Fc-biotin为采用生物素化试剂(thermofisher，Cat.#20217)将EGFR-ECD-Fc蛋白进行生物素化获得。以下实施例中涉及的VEGF为VEGF-A165。
以下实施例中涉及的HRP为辣根过氧化物酶。
以下实施例中涉及的融合蛋白或抗体根据Kabat系统编号或定义。
以下实施例中使用的实验材料以及实验试剂的来源具体说明如下。若无特殊说明，均可从商业途径购得。
实验材料：
A431细胞：人皮肤鳞癌细胞株，购自ATCC。
KDR细胞：购自promega，Cat.#GA1082。
LIM1215：人结直肠癌细胞株，购自广州吉妮欧生物科技有限公司。
SW48：人结肠癌细胞株，购自ATCC。
SD大鼠：购自浙江维通利华实验动物技术有限公司。
BALB/C裸小鼠：购自北京维通利华实验动物科技有限公司。
实验试剂：
pcDNA3.4载体：购自thermofisher，货号A14697。
重组VEGF-A165蛋白：购自acrobiosystems，Cat.#VE5-H4210。
HRP标记的羊抗人Fab抗体：购自abcam，Cat.#ab87422。
羊抗人IgG-FITC：购自sigma，Cat.#F9512。
山羊抗人FC抗体：购自Abcam，Cat.#ab97225。
1*HBS工作液：购自GE HealthCare，Cat.#BR100669。
Protein A芯片：购自GE HealthCare，Cat.#29127558。
EDC：购自Fortebio，Cat.#18-1033。
s-NHS：购自Fortebio，Cat.#18-1067。
AR2G探针：购自Fortebio，Cat.#18-5092。
乙酸钠：购自Fortebio，Cat.#18-1070。
乙醇胺：购自Fortebio，Cat.#18-1071。
1*Kinetic Buffer：购自Fortebio，Cat.#18-1072。
Figure PCTCN2022089244-appb-000007
solution：购自Sigma，Cat.#A6964。
DMEM培养基：购自Gibco，Cat.#11995。
Bio-Glo：购自promega，Cat.#G7940。
TMB：购自BD公司，货号555214。
HRP标记的Streptavidin(链霉亲和素)：购自BD Pharmingen，货号554066。
HRP标记的鼠抗人Fab抗体：购自金斯瑞，Cat.#A01855。
EGFR-his：购自SINO，Cat.#10001-H08H。
protein A：购自thermofisher，Cat.#21184。
以下实施例中使用的核酸序列，由生工生物工程(上海)股份有限公司或金唯智生物科技有限公司合成。
实施例1.抗EGFR/VEGF双功能融合蛋白构建
本发明采用了抗EGFR单克隆抗体和VEGFR1的D2结构域串联的方式，构建了抗EGFR/VEGF双功能融合蛋白，结构示意图如图1所示。
融合蛋白602-D2
将VEGFR1的D2结构域(SEQ ID NO：1)的N末端和抗EGFR单克隆抗体602的重链(SEQ ID NO：2)的C末端通过肽接头L(SEQ ID NO：4)连接起来，得到融合蛋白602-D2的重链(SEQ ID NO：5)，融合蛋白602-D2的轻链序列为SEQ ID NO：7。
融合蛋白602dN-D2
将VEGFR1的D2结构域(SEQ ID NO：1)的N末端和抗EGFR单克隆抗体602dN的重链(SEQ ID NO：3)的C末端通过肽接头L(SEQ ID NO：4)连接起来，得到融合蛋白602dN-D2的重链(SEQ ID NO：6)，融合蛋白602dN-D2的轻链序列为SEQ ID NO：7。
融合蛋白602-D2-M2
将VEGFR1的D2结构域(SEQ ID NO：8)的N末端和抗EGFR单克隆抗体602的重链(SEQ ID NO：2)的C末端通过肽接头L(SEQ ID NO：4)连接起来，得到融合蛋白602-D2-M2的重链(SEQ ID NO：9)，融合蛋白602-D2-M2的轻链序列为SEQ ID NO：7。
融合蛋白602dN-D2-M2
将VEGFR1的D2结构域(SEQ ID NO：8)的N末端和抗EGFR单克隆抗体602dN的重链(SEQ ID NO：3)的C末端通过肽接头L(SEQ ID NO：4)连接起来，得到融合蛋白602dN-D2-M2的重链(SEQ ID NO：10)，融合蛋白602dN-D2-M2的轻链序列为SEQ ID NO：7。
其中，融合蛋白602-D2-M2和602dN-D2-M2中的VEGFR1的D2结构域(SEQ ID NO：8)相对于融合蛋白602-D2和602dN-D2的VEGFR1的D2结构域(SEQ ID NO：1)在C末端截断2个氨基酸，该两个氨基酸在发酵过程中容易脱落，且去除并不影响药效。
其中，抗EGFR单克隆抗体602dN的重链(SEQ ID NO：3)相对于抗EGFR单克隆抗体602的重链(SEQ ID NO：2)，将其85位天冬酰胺突变为谷氨酸(N85E)，减少了由于糖基化导致的电荷异质性，方便后续生产制备工艺的开发，并且去糖基化对双功能融合蛋白的活性没有明显的影响。
实施例2.抗EGFR/VEGF双功能融合蛋白的表达与纯化
融合蛋白602-D2重链核酸序列为SEQ ID NO：11，轻链核酸序列为SEQ ID NO：13。
融合蛋白602dN-D2重链核酸序列为SEQ ID NO：12，轻链核酸序列为SEQ ID NO：13。
融合蛋白602-D2-M2重链核酸序列为SEQ ID NO：14，轻链核酸序列为SEQ ID NO：13。
融合蛋白602dN-D2-M2重链核酸序列为SEQ ID NO：15，轻链核酸序列为SEQ ID NO：13。
将以上4种抗EGFR/VEGF双功能融合蛋白的重链和轻链的DNA片段分别亚克隆到pcDNA3.4载体中，提取重组质粒共转染CHO细胞和/或293F细胞。细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后上样至HiTrap MabSelect SuRe柱，100mM柠檬酸(pH 3.5)洗脱液一步洗脱蛋白，回收目标样品并透析换液至PBS。
将纯化后的蛋白用HPLC检测，图2A-图2D检测结果表明4种融合蛋白分子状态均一，单体纯度达到97％以上。
实施例3.酶联免疫吸附法(ELISA)测定抗EGFR/VEGF双功能融合蛋白对抗原的亲和力
3.1 ELISA检测抗EGFR/VEGF双功能融合蛋白与EGFR的亲和力
将重组EGFR-ECD-Fc蛋白用包被液稀释至1μg/ml，以50μl/孔加入酶标板， 4℃过夜。PBST洗板3次，加入200μl/孔封闭液，37℃放置1小时后PBST洗板1次待用。用稀释液稀释抗EGFR/VEGF双功能融合蛋白至60nM，4倍比稀释形成8个浓度梯度，依次加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板3次，加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism7软件进行数据分析，作图并计算EC50。
实验结果如图3所示，4种抗EGFR/VEGF双功能融合蛋白602-D2、602-D2-M2、602dN-D2、602dN-D2-M2以及阳性对照抗EGFR单抗602与EGFR-ECD结合的EC50分别为0.633nM、0.749nM、0.738nM、0.633nM以及0.656nM，表明融合蛋白602-D2、602-D2-M2、602dN-D2、602dN-D2-M2均能够特异性地结合EGFR，其与EGFR的亲和力与抗EGFR单抗602相当。
3.2 ELISA检测抗EGFR/VEGF双功能融合蛋白与VEGF的亲和力
将重组VEGF-A165蛋白以100ng/孔包被酶标板，4℃过夜。PBST洗板3次，加入200μl/孔封闭液，37℃放置1小时后PBST洗板1次待用。用稀释液稀释抗EGFR/VEGF双功能融合蛋白至100nM，4倍比稀释形成8个浓度梯度，依次加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板3次，加入HRP标记的羊抗人Fab抗体，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism7软件进行数据分析，作图并计算EC50。
实验结果如图4所示，抗EGFR/VEGF双功能融合蛋白602dN-D2、602-D2-M2、602dN-D2-M2以及阳性对照抗VEGF单抗Bevacizumab(601)与VEGF结合的EC50分别为0.497nM、0.603nM、0.422nM以及0.747nM，表明融合蛋白602dN-D2、602-D2-M2、602dN-D2-M2均能够特异性地结合VEGF，其与VEGF的亲和力与抗VEGF单抗Bevacizumab(601)的亲和力相当。
3.3 ELISA检测抗EGFR/VEGF双功能融合蛋白同时结合EGFR和VEGF的能力
空间位阻可能会影响抗EGFR/VEGF双功能融合蛋白同时结合两种抗原的能力。为了检测抗EGFR/VEGF双功能融合蛋白同时结合EGFR和VEGF的能力，将重组VEGF-A165蛋白以20ng/孔包板，4℃过夜。PBST洗板3次，加入200μl/孔封闭液，37℃放置1小时后PBST洗板1次待用。用稀释液稀释抗EGFR/VEGF双功能融合蛋白至200nM，2倍比稀释形成12个浓度梯度，依次加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板3次，按150ng/孔加入EGFR-ECD-Fc-biotin，37℃放置1小时。PBST洗板3次后加入HRP标记的Streptavidin(链霉亲和素)，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism7软件进行数据分析，作图并计算EC50。
实验结果如图5所示，抗EGFR/VEGF双功能融合蛋白602dN-D2、602dN-D2-M2均能同时特异性地结合EGFR和VEGF，EC50分别为4.599nM及4.518nM。
实施例4.FACS法测定抗EGFR/VEGF双功能融合蛋白对靶细胞表面抗原EGFR的结合亲和力
本实验以细胞表面表达EGFR的A431细胞作为靶细胞，将靶细胞按照2×10 5/孔接种于96孔板，用含有0.5％BSA的PBS洗涤三次，每次300g离心5分钟，弃上清。将抗EGFR/VEGF双功能融合蛋白及对照抗体稀释至50nM，3倍比稀释形成10个梯度作为一抗加入96孔板，将细胞悬浮后于4℃孵育1小时。用含有0.5％BSA的PBS洗涤细胞两次，以去除未结合的抗体，再将细胞与100μl的1g/ml羊抗人IgG-FITC于4℃孵育30分钟。300g离心5分钟，用含有0.5％BSA的PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μl PBS中，通过Beckman Coμlter CytoFLEX流式细胞仪测定融合蛋白对A431细胞表面EGFR的结合亲和力。所得数据通过GraphPad Prism7软件拟合分析。
实验结果如图6所示，抗EGFR/VEGF双功能融合蛋白602dN-D2、602dN-D2-M2和阳性对照抗EGFR单抗602均可特异性地结合细胞表面表达的EGFR，EC50分别为1.784nM、1.840nM以及1.831nM，亲和力相当。其中，阴性对照(negative control)是与EGFR抗原不结合的同型对照。
实施例5.抗EGFR/VEGF双功能融合蛋白对抗原EGFR的亲和解离常数KD的测定
利用Biacore 8K分子相互作用分析仪，使用捕获法测定抗EGFR/VEGF双功能融合蛋白和抗原EGFR结合解离的动力学参数，用1*HBS工作液将融合蛋白稀释至3μg/ml，用Protein A芯片捕获融合蛋白；将抗原EGFR-his用1×HBS工作液稀释，设最高浓度为25nM的6个浓度梯度与融合蛋白结合，于HBS工作液中解离。
实验结果如表2所示，抗EGFR/VEGF双功能融合蛋白602dN-D2-M2、602-D2-M2、602-D2与阳性对照抗EGFR单抗602，对于EGFR-his的亲和解离常数相当。
表2亲和解离常数
Figure PCTCN2022089244-appb-000008
注：KD为亲和力常数；kon为结合速率常数；kdis为解离速率常数。
实施例6.抗EGFR/VEGF双功能融合蛋白对抗原VEGF的亲和解离常数KD的测定
利用octet分子相互作用分析仪，使用捕获法测定抗EGFR/VEGF双功能融合蛋白和抗原VEGF-A165结合解离的动力学参数，用20mM EDC和10mM s-NHS将AR2G探针活化，将抗原VEGF-A165用10mM Sodium Acetate(乙酸钠，pH6.0)稀释至5μg/ml，结合在活化过的AR2G探针上，再用1M Ethanolamine(乙醇胺，pH8.5)溶液封闭探针。将融合蛋白用1*Kinetic Buffer工作液稀释，设最高浓度为12.5nM的5个浓度梯度，于1*Kinetic Buffer工作液中解离。
实验结果如表3所示，抗EGFR/VEGF双功能融合蛋白602dN-D2-M2对于VEGF165的亲和力略优于阳性对照抗VEGF单抗Bevacizumab。
表3亲和解离常数
Figure PCTCN2022089244-appb-000009
注：KD为亲和力常数；kon为结合速率常数；kdis为解离速率常数。
实施例7.抗EGFR/VEGF双功能融合蛋白对A431细胞增殖的抑制活性
取贴壁培养的对数期生长的A431细胞，弃掉生长培养基RPMI 1640(10％FBS、1％青链霉素)。用DPBS洗一次后，用胰酶消化，再用生长培养基中和胰酶。台盼蓝细胞计数，300g离心5min。用RPMI 1640(1％FBS、1％青链霉素)培养基重悬后计数铺板，调整密度为5000个/孔，100μl/孔，置于37℃，5％CO 2，孵育过夜约24小时。次日准备RPMI 1640纯培养基稀释融合蛋白至100nM作为起始浓度，3倍梯度稀释10个梯度。后加入细胞孔，每孔90μl，37℃温箱继续培养72小时后，加入80μl/孔检测试剂Cell-Titer Glo(提前30分钟放25℃水浴锅解冻平衡温度)。室温孵育10分钟后，用spectramax i3读取luminescence(荧光强度)。
实验结果如图7所示，抗EGFR/VEGF双功能融合蛋白602-D2-M2、602dN-D2-M2以及阳性对照602单抗均能够有效地抑制A431细胞增殖，IC50分别为2.942nM、3.976nM以及3.556nM，表明抗EGFR/VEGF双功能融合蛋白对A431细胞增殖的抑制活性与阳性对照抗EGFR单抗602相当。
实施例8.抗EGFR/VEGF双功能融合蛋白阻断VEGF与受体KDR结合的细胞实验
取贴壁培养的对数期生长的密度约在80％-90％的KDR细胞，弃掉生长培养基。用DPBS洗一次后，用
Figure PCTCN2022089244-appb-000010
solution消化，中和胰酶后，200g离心5分钟，用含10％FBS的DMEM培养基将细胞重悬后，台盼蓝细胞计数，调整细胞密度以40000个/孔铺板，50μl/孔。用含10％FBS的DMEM培养基稀释VEGF-A165至30ng/ml，用含VEGF的培养基倍比稀释融合蛋白，3倍稀释，10个梯度。将稀释好的融合蛋白加入细胞孔每孔25μl(最终VEGF浓度为10ng/ml，融合蛋白起始浓度为50nM)，37℃孵育6小时后，每孔加入75μl检测 试剂Bio-Glo。室温孵育10分钟后，用SpectraMax i3x读取luminescence(荧光强度)。所有数据均为双复孔，所得信号值取平均值后用4-parameter法拟合，用GraphPad Prism7软件进行数据分析。
实验结果如图8所示，抗EGFR/VEGF双功能融合蛋白与阳性对照抗VEGF单抗Bevacizumab均能够有效地阻断VEGF与其受体KDR之间的相互作用，并且融合蛋白602dN-D2、602dN-D2-M2的阻断能力均优于阳性对照抗VEGF单抗Bevacizumab，602dN-D2、602dN-D2-M2和Bevacizumab的IC 50分别为0.147nM、0.120nM以及0.448nM。
实施例9.抗EGFR/VEGF双功能融合蛋白的药代动力学研究
实验用SD大鼠4只，雌性，体重200g左右，12/12小时光/暗调节，温度20-26℃，湿度40-70％，自由进食饮水。每只大鼠通过尾静脉注射融合蛋白，剂量为10mg/kg，注射体积为5ml/kg。分别在给药后的1h、4h、24h、72h、96h、168h、336h、504h，于大鼠眼底静脉取血，每次200μl左右。收集的血样在室温下置放半小时至凝集，然后4℃下8000rpm，离心10分钟后取血清。血清立即放置-80℃储存。用ELISA检测血清中融合蛋白浓度。
1)用EGFR-ECD-Fc包被ELISA板4℃过夜，包被量为100ng/孔；包被完毕后用PBST洗板3次，然后用PBS+2％BSA于37℃封闭2小时；然后加入500倍稀释的大鼠血清孵育1小时；PBST洗板3次后加入HRP标记的山羊抗人FC抗体，37℃放置30分钟；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl 2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
2)包被protein A，检测EGFR。用protein A包被ELISA板4℃过夜，包被量为100ng/孔；包被完毕后用PBST洗板3次，然后用PBS+2％BSA于37℃封闭2小时；然后加入500倍稀释的大鼠血清孵育1小时；PBST洗板3次洗板后加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
用Phoenix软件计算抗EGFR/VEGF双功能融合蛋白药物在大鼠体内的半衰 期，药代动力学参数见表4，PK分析结果表明，融合蛋白602dN-D2-M2在大鼠体内的半衰期约为138小时。
表4 602dN-D2-M2在大鼠体内药代动力学参数
Figure PCTCN2022089244-appb-000011
实施例10.抗EGFR/VEGF双功能融合蛋白在LIM1215移植瘤模型上的抗肿瘤作用
收集体外培养的LIM1215细胞(LIM1215细胞表面表达EGFR，抗EGFR抗体能有效抑制LIM1215细胞的增殖)，将细胞悬液浓度调整为2×10 7/ml，与等体积的基质胶混合。在无菌条件下，接种100μl细胞悬液于裸小鼠右侧肋部皮下。移植瘤用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至200mm 3左右后将动物随机分组给药。
受试样品602dN-D2-M2的给药剂量设置为5.65mg/kg和28.25mg/kg，对照药品602的给药剂量设置为5mg/kg和25mg/kg，对照药品Bevacizumab的给药剂量设置为5mg/kg和25mg/kg，空白对照组(CONTROL)给以相同体积的生理盐水。每周腹腔注射给药3次，连续给药3周。整个实验过程中，每周2次测量移植瘤直径，同时称小鼠体重。肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×a×b 2。其中a、b分别表示长、宽。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)。
实验结果如图9所示，在LIM1215移植瘤模型上，602dN-D2-M2在两个剂量的抑瘤作用均显著优于对照抗EGFR单抗602以及抗VEGF单抗Bevacizumab，表明在LIM1215移植瘤模型上，602dN-D2-M2通过阻断EGFR及VEGF从而抑制肿瘤生长。
实施例11.抗EGFR/VEGF双功能融合蛋白在SW48移植瘤模型上的抗肿瘤作用
收集体外培养的SW48细胞(SW48细胞表面表达EGFR，抗EGFR抗体能 有效抑制SW48细胞的增殖)，将细胞悬液浓度调整为6×10 7/ml，与等体积的基质胶混合。在无菌条件下，接种100μl细胞悬液于裸小鼠右侧肋部皮下。移植瘤用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至200mm 3后将动物随机分组给药。
受试样品602dN-D2-M2的给药剂量设置为11.3mg/kg，对照药品602和Bevacizumab的给药剂量均设置为10mg/kg，联用组同时给予602和Bevacizumab(10mg/kg和10mg/kg)，空白对照组(CONTROL)给以相同体积的生理盐水。每周腹腔注射给药3次，连续给药3周。整个实验过程中，每周2次测量移植瘤直径，同时称小鼠体重。肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×a×b 2。其中a、b分别表示长、宽。
实验结果如图10所示，在SW48移植瘤模型上，对照抗VEGF单抗Bevacizumab和抗EGFR单抗602均能显著抑制肿瘤生长。在等摩尔剂量下，602dN-D2-M2的抑瘤效果优于Bevacizumab或602，以及Bevacizumab和602的联用，展现出协同作用。
实施例12.抗EGFR/VEGF双功能融合蛋白的物理稳定性
利用DSC(Differential scanning calorimetry,差示扫描量热法)检测602dN-D2-M2样品在PBS缓冲体系下的热稳定性。将样品置换到PBS缓冲液中，控制样品浓度在1mg/ml，利用MicroCal*Vp-Capillary DSC(Malvern)进行检测。检测前，将样品及空白缓冲液用0.22μm滤膜过滤。样品板每个孔加入400μl样品或空白缓冲液(设置6组空白缓冲对)，最后三对孔板加入ddH 2O，以备清洗用。样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到100℃结束，扫描速率150℃/h。
具体结果如表5所示，样品602dN-D2-M2蛋白表现出良好的热稳定性。
表5 602dN-D2-M2热稳定性检测数据
Figure PCTCN2022089244-appb-000012
实施例13.抗EGFR/VEGF双功能融合蛋白在人乳腺癌小鼠移植瘤模型上的体内抗肿瘤作用
在CB-17 SCID小鼠体内建立人乳腺癌JIMT-1皮下移植瘤模型，用以评价融合蛋白602dN-D2-M2的体内抗肿瘤活性。具体实施步骤如下：收集体外培养的人乳腺癌JIMT-1细胞，将细胞悬液浓度调整为1.6×10 8/ml，与基质胶以1：1等比例混合。CB-17 SCID小鼠右侧肋部剃毛，在无菌条件下，接种100μl细胞混合悬液于小鼠右侧肋部皮下。待皮下瘤生长至体积约150mm 3时，按照肿瘤体积将小鼠随机分为5组，每组8只小鼠，包括：空白对照组，仅注射生理盐水，作为对照；抗EGFR对照抗体602组，剂量10mg/kg；抗VEGF阳性对照抗体Avastin(即Bevacizumab)组，剂量10mg/kg；602+Avastin联用组，剂量10mg/kg+10mg/kg和602dN-D2-M2组，剂量11.3mg/kg。每周给药3次，共给药4周。每周测定肿瘤体积2次。最终，测定的各组肿瘤随时间的生长曲线如图11所示。
实验结果显示，在此移植瘤模型上，抗EGFR抗体602能够显著抑制肿瘤生长，抑瘤率为66.2％；抗VEGF抗体Avastin，也可以抑制肿瘤生长，抑瘤率为22.3％，弱于抗EGFR单抗；抗EGFR单抗602和抗VEGF单抗Avastin二者联合治疗，能够显著抑制肿瘤生长，抑瘤率为80.7％。融合蛋白602dN-D2-M2也能够显著抑制肿瘤生长，其抑瘤率为88.7％，显著优于任一单抗单独治疗，且高于两种单抗联用。
实施例14.抗EGFR/VEGF双功能融合蛋白在人咽喉癌小鼠移植瘤模型上的体内抗肿瘤作用
在BALB/C-nude裸小鼠体内建立人咽喉癌FaDu皮下移植瘤模型，用以评价融合蛋白602dN-D2-M2的体内抗肿瘤活性。具体实施步骤如下：收集体外培养的人咽喉癌FaDu细胞，将细胞悬液浓度调整为1×10 8/ml，与基质胶以1：1等比例混合。在无菌条件下，接种100μl细胞混合悬液于裸小鼠右侧肋部皮下。待皮下瘤生长至体积约150mm 3时，按照肿瘤体积将小鼠随机分为每组5只小鼠，包括：空白对照组，仅注射PBS，作为对照；Fc-D2阳性对照组，剂量2.5mg/kg；抗VEGF阳性对照抗体Avastin(即Bevacizumab)组，剂量5mg/kg和602dN-D2-M2组，剂量5.65mg/kg。每周给药2次，共给药7次。每周测定肿瘤体积2次。最终，测定的各组肿瘤随时间的生长曲线如图12所示。
实验结果显示，在此移植瘤模型上，抗VEGF抗体Avastin和融合蛋白Fc-D2均可以抑制肿瘤生长，抑瘤率分别为64.3％和67.2％；融合蛋白602dN-D2-M2能够显著抑制肿瘤生长，抑瘤率为89.0％，显著优于Avastin或Fc-D2的单独治疗。

Claims (29)

  1. 一种抗EGFR/VEGF双功能融合蛋白，其特征在于，包含：(a)抗EGFR抗体或其抗原结合片段，和(b)VEGFR1的胞外区的D2结构域。
  2. 如权利要求1所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述抗EGFR抗体或其抗原结合片段包含重链互补决定区HCDR1-3和轻链互补决定区LCDR1-3，其中HCDR1的氨基酸序列如SEQ ID NO：16所示，HCDR2的氨基酸序列如SEQ ID NO：17所示，HCDR3的氨基酸序列如SEQ ID NO：18所示；LCDR1的氨基酸序列如SEQ ID NO：19所示，LCDR2的氨基酸序列如SEQ ID NO：20所示，LCDR3的氨基酸序列如SEQ ID NO：21所示。
  3. 如权利要求1或2所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述抗EGFR抗体或其抗原结合片段包含重链可变区VH和轻链可变区VL，其中VH的氨基酸序列如SEQ ID NO：22所示，VL的氨基酸序列如SEQ ID NO：24所示。
  4. 如权利要求1或2所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述重链可变区VH包含至少一个去糖基化突变；优选地，所述突变位于框架区FR内。
  5. 如权利要求4所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述突变位于重链可变区VH的第85位，所述突变根据Kabat编号系统编号；优选地，所述突变是N85E。
  6. 如权利要求5所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述重链可变区VH的氨基酸序列如SEQ ID NO：23所示，轻链可变区VL的氨基酸序列如SEQ ID NO：24所示。
  7. 如权利要求1所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述抗EGFR抗体包含重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、人IgG2、人IgG3或人IgG4，所述轻链恒定区选自人κ(Kappa)或人λ(Lambda)。
  8. 如权利要求1所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述抗EGFR抗体或其抗原结合片段为嵌合抗体或其抗原结合片段，或人源化抗体或其抗原结合片段。
  9. 如权利要求1所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述抗EGFR抗体或其抗原结合片段选自全长抗体、scFv、Fv、Fab或F(ab')2。
  10. 如权利要求1所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述抗EGFR抗体包含氨基酸序列如SEQ ID NO：2或SEQ ID NO：3所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链。
  11. 如权利要求1所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述VEGFR1的胞外区的D2结构域包含如SEQ ID NO：1或SEQ ID NO：8所示的氨基酸序列。
  12. 如权利要求1所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述抗EGFR抗体或其抗原结合片段与所述VEGFR1的胞外区的D2结构域通过肽键直接连接或通过肽接头连接。
  13. 如权利要求12所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述肽接头包含氨基酸序列(GGGGS)n，所述n选自1、2、3、4或5。
  14. 如权利要求1-13任一项所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述抗EGFR抗体为全长抗体，包含2条重链和2条轻链，所述VEGFR1的胞外区的D2结构域的N末端或C末端通过肽接头连接至所述抗EGFR抗体的重链或轻链的N末端或C末端。
  15. 如权利要求14所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述VEGFR1的胞外区的D2结构域的N末端通过肽接头连接至所述抗EGFR抗体的重链的C末端。
  16. 如权利要求15所述的抗EGFR/VEGF双功能融合蛋白，其特征在于，所述融合蛋白选自：
    a)所述融合蛋白包含氨基酸序列如SEQ ID NO：5所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
    b)所述融合蛋白包含氨基酸序列如SEQ ID NO：6所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
    c)所述融合蛋白包含氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链；或，
    d)所述融合蛋白包含氨基酸序列如SEQ ID NO：10所示的重链，和氨基酸序列如SEQ ID NO：7所示的轻链。
  17. 一种核酸分子，其特征在于，所述核酸分子编码如权利要求1-16任一项所述的抗EGFR/VEGF双功能融合蛋白。
  18. 如权利要求17所述的核酸分子，其特征在于，所述核酸分子包含：
    a)编码融合蛋白重链的如SEQ ID NO：11所示的核酸序列，和编码融合蛋白轻链的如SEQ ID NO：13所示的核酸序列；或，
    b)编码融合蛋白重链的如SEQ ID NO：12所示的核酸序列，和编码融合蛋白轻链的如SEQ ID NO：13所示的核酸序列；或，
    c)编码融合蛋白重链的如SEQ ID NO：14所示的核酸序列，和编码融合蛋白轻链的如SEQ ID NO：13所示的核酸序列；或，
    d)编码融合蛋白重链的如SEQ ID NO：15所示的核酸序列，和编码融合蛋白轻链的如SEQ ID NO：13所示的核酸序列。
  19. 一种表达载体，其特征在于，所述表达载体含有如权利要求17或18所述的核酸分子。
  20. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求19所述的表达载体。
  21. 一种如权利要求1-16任一项所述的抗EGFR/VEGF双功能融合蛋白的制备方法，其特征在于，所述制备方法包括以下步骤：
    a)在表达条件下，培养如权利要求20所述的宿主细胞，从而表达抗EGFR/VEGF双功能融合蛋白；
    b)分离并纯化步骤a)所述的抗EGFR/VEGF双功能融合蛋白。
  22. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-16任一项所述的抗EGFR/VEGF双功能融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。
  23. 如权利要求1-16任一项所述的抗EGFR/VEGF双功能融合蛋白、或如权利要求22所述的药物组合物在制备治疗癌症的药物中的用途。
  24. 如权利要求23所述的用途，其特征在于，所述癌症高表达或异常表达EGFR和/或高表达或异常表达VEGF。
  25. 如权利要求24所述的用途，其特征在于，所述癌症选自：结直肠癌、皮肤癌、鳞状细胞癌、乳腺癌、胶质细胞癌、肾癌、肺癌、前列腺癌、胰腺癌、头颈 癌、或其组合；优选地，所述癌症选自结肠癌、皮肤鳞状细胞癌、乳腺癌、咽喉癌、或其组合。
  26. 如权利要求23所述的用途，其特征在于，所述治疗包括向受试者施用有效量的抗EGFR/VEGF双功能融合蛋白或药物组合物，所述抗EGFR/VEGF双功能融合蛋白或药物组合物通过注射施用；优选地，所述注射选自静脉注射、肌肉注射、或皮下注射。
  27. 如权利要求23所述的用途，其特征在于，所述治疗包括向受试者施用有效量的抗EGFR/VEGF双功能融合蛋白或药物组合物，所述有效量是指0.5-30mg/kg的剂量；优选地，所述药物组合物的施用间隔选自：a)每周一次；b)每周二次；c)每周三次；更优选地，所述药物组合物的施用时间选自：a)至少1周；b)至少2周；c)至少3周；d)至少4周。
  28. 一种免疫偶联物，其特征在于，所述免疫偶联物包括：
    (a)如权利要求1-16任一项所述的抗EGFR/VEGF双功能融合蛋白；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  29. 一种治疗高表达或异常表达EGFR和/或高表达或异常表达VEGF癌症的方法，其特征在于，所述方法包括向有需要的受试者施用如权利要求1-16任一项所述的双功能融合蛋白、如权利要求22所述的药物组合物、或如权利要求28所述的免疫偶联物。
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CN109563166A (zh) * 2016-04-28 2019-04-02 拜奥穆尼克斯制药 靶向egfr和her2的双特异性抗体
CN110835375A (zh) * 2018-08-16 2020-02-25 上海洛启生物医药技术有限公司 一种抗pd-1/egfr双特异性抗体及其用途
CN112552410A (zh) * 2019-09-26 2021-03-26 三生国健药业（上海）股份有限公司 一种抗体融合蛋白及其制法和在抗肿瘤中的应用
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