WO2021013064A1 - Humanized vegfr2 antibody and application thereof - Google Patents

Humanized vegfr2 antibody and application thereof Download PDF

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WO2021013064A1
WO2021013064A1 PCT/CN2020/102559 CN2020102559W WO2021013064A1 WO 2021013064 A1 WO2021013064 A1 WO 2021013064A1 CN 2020102559 W CN2020102559 W CN 2020102559W WO 2021013064 A1 WO2021013064 A1 WO 2021013064A1
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antibody
vegfr2
antigen
seq
binding fragment
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PCT/CN2020/102559
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French (fr)
Chinese (zh)
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谢良志
孙春昀
马娟
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神州细胞工程有限公司
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Priority to CN202080045177.7A priority Critical patent/CN114174335B/en
Publication of WO2021013064A1 publication Critical patent/WO2021013064A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the invention belongs to the field of tumor immunotherapy, and relates to a recombinant humanized anti-VEGFR2 antibody and its application.
  • Tumor blood vessels transport sufficient nutrients for the occurrence and development of tumors and provide escape channels for tumor cells.
  • Drugs that target angiogenesis can block the nutrient supply of tumors, thereby achieving the effect of "starving" the tumor.
  • tumor angiogenesis is comprehensively regulated by a variety of growth factors, receptors and downstream signaling pathways in the tumor microenvironment.
  • vascular endothelial growth factor vascular endothelial growth factor, VEGF
  • VEGF vascular endothelial growth factor
  • VEGFR vascular endothelial growth factor receptor
  • VEGFR2 (also known as FLK-1, or KDR), is the main member of the VEGFR family, is a type III receptor tyrosine kinase, and is mainly distributed on the surface of the endothelial cell membrane of blood vessels and lymphatic vessels.
  • the extracellular domain of VEGFR2 contains 7 immunoglobulin-like domains (ie D1-D7), including the ligand VEGF binding domain (D2-D3) and dimerization domain (D4-D7), and its intracellular domain contains Tyrosine kinase domain [2-4].
  • VEGF vascular endothelial growth factor
  • concentration of VEGF in normal human plasma is about 137 ⁇ 7.7 pg/mL [5]
  • concentration of VEGF in serum of patients with pancreatic cancer and ovarian cancer is significantly increased to 200- 400pg/mL
  • concentration of VEGF in tumor tissue or tumor cyst effusion is as high as 0.5-20ng/mL[5-7].
  • VEGFR2 When the high concentration of VEGF dimer in the tumor microenvironment binds to VEGFR2, it induces VEGFR2 receptors to form homodimers (primary), and can also form heterodimers with VEGFR1 or VEGFR3 (secondary), causing VEGFR2 intracellular Autophosphorylation of tyrosine residues in the region, activate downstream phospholipase C (PLC) and other signaling pathways, promote tumor vascular endothelial cell proliferation and survival, mediate tumor cell migration, change vascular permeability, and ultimately cause tumor angiogenesis[2 ].
  • PLC phospholipase C
  • VEGFR2 antibody drugs can block the binding of VEGFR2 and VEGF, inhibit angiogenesis, reduce vascular permeability, and restore the balance of the tumor microenvironment in a short period of time, thereby obtaining a good tumor suppressor effect.
  • CYRAMZA first anti-VEGFR2 tumor treatment drug ramucirumab
  • CYRAMZA tumor treatment drug ramucirumab
  • Avastin due to the important role of VEGF in normal physiological metabolism, although Avastin is well tolerated in many diseases, clinical studies have found that Avastin has serious side effects such as gastric perforation [13], and targeting VEGFR-2 can retain VEGF binding to VEGFR1 The function of homodimer retains part of the function of human normal tissue neovascularization. Therefore, it is expected that the systemic toxic side effects such as gastrointestinal perforation caused by targeting VEGFR-2 are lower and have better safety advantages. There is a need for drugs with both safety and efficacy in clinical practice.
  • ramucirumab is the only VEGFR2 monoclonal antibody drug approved by the FDA for the second-line treatment of advanced gastric cancer, colon cancer and non-small cell lung cancer, and it has not yet been approved for marketing in China.
  • the antibody disclosed in the present invention can specifically bind to tumor cells and block the effects of VEGF-A, VEGF-C and VEGF-D at the same time, thus having a better effect of inhibiting angiogenesis.
  • the antibody can also activate the antibody-dependent cytotoxicity (ADCC) reaction of NK cells, inhibit tumor growth and metastasis, and can be used for clinical treatment of melanoma.
  • ADCC antibody-dependent cytotoxicity
  • the present invention provides an isolated anti-VEGFR2 antibody or antigen-binding fragment thereof, which comprises a heavy chain CDR1 domain having the amino acid sequence shown in SEQ ID NO: 13, and having a heavy chain CDR1 domain as shown in SEQ ID NO: 14/50.
  • the heavy chain CDR2 domain of the amino acid sequence and the heavy chain CDR3 domain of the heavy chain CDR3 domain with the amino acid sequence shown in SEQ ID NO: 15, and the light chain CDR1 domain with the amino acid sequence of SEQ ID NO: 10 are light chain variable regions.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 8 or at least 90%, 92%, 95%, 98%, or at least 90% to SEQ ID NO: 8
  • the heavy chain variable region of the amino acid sequence with 99% sequence identity and the amino acid sequence shown in SEQ ID NO: 9 or at least 90%, 92%, 95%, 98% or 99% with SEQ ID NO: 9
  • the light chain variable region of the amino acid sequence of sequence identity is shown in SEQ ID NO: 8 or at least 90%, 92%, 95%, 98% or 99%.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 22 or at least 85%, 90%, 95% or 99% sequence with SEQ ID NO: 22
  • the antibody further comprises a light chain constant region and a heavy chain constant region.
  • the light chain constant region is the amino acid sequence of the kappa light chain constant region of SEQ ID NO: 25 or the amino acid sequence of the kappa light chain constant region.
  • ID NO: 25 has an amino acid sequence with at least 90%, 92%, 95%, 98% or 99% sequence identity
  • the heavy chain constant region is an IgG1 heavy chain with the amino acid sequence of SEQ ID NO: 24
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof is an IgG antibody, preferably an IgG1 antibody.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof is a monoclonal antibody.
  • the binding affinity K D of the anti-VEGFR2 antibody or antigen-binding fragment thereof to recombinant VEGFR2 is 1-100 pM, preferably 5-50 pM, more preferably 10.6 pM.
  • the antigen binding fragment is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecule or single-domain antibody;
  • the antibody molecule is preferably scFv, di-scFv, tri-scFv, diabody or scFab.
  • the binding epitope of the anti-VEGFR2 antibody or antigen-binding fragment thereof is Y137/K142, R164/Y165, D257, S311/G312 of VEGFR2.
  • the present invention provides an anti-VEGFR2 antibody or antigen-binding fragment thereof, which binds to the same epitope on the antigen as the anti-VEGFR2 antibody or antigen-binding fragment thereof of the present invention.
  • the present invention provides a molecular epitope of VEGFR2, which is Y137/K142, R164/Y165, D257, S311/G312 of VEGFR2.
  • the present invention provides an antibody-drug conjugate comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-9 and another therapeutic agent, preferably the anti-VEGFR2 antibody
  • another therapeutic agent preferably the anti-VEGFR2 antibody
  • the VEGFR2 antibody or antigen-binding fragment thereof and the other therapeutic agent are connected by a linker.
  • the present invention provides a nucleic acid encoding the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention.
  • the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO: 4 and/or a nucleotide sequence as shown in SEQ ID NO: 5, or comprises a nucleotide sequence as shown in SEQ ID NO: 30 The nucleotide sequence of and/or the nucleotide sequence shown in SEQ ID NO: 31.
  • the invention provides an expression vector comprising the nucleic acid according to the invention.
  • the present invention provides a host cell comprising the nucleic acid or expression vector according to the present invention.
  • the present invention provides a method for producing the anti-VEGFR2 antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell under conditions suitable for antibody expression, and recovering the expression from the culture medium.
  • a method for producing the anti-VEGFR2 antibody or antigen-binding fragment thereof of the present invention which comprises culturing the host cell under conditions suitable for antibody expression, and recovering the expression from the culture medium.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the nucleic acid or expression vector according to the present invention, and a pharmaceutical composition Acceptable carrier.
  • the present invention relates to an anti-VEGFR2 antibody or antigen-binding fragment thereof or antibody-drug conjugate or pharmaceutical composition for the treatment of melanoma.
  • the present invention provides a method for treating melanoma, comprising administering to a subject in need a therapeutically effective amount of the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug according to the present invention A conjugate or a pharmaceutical composition according to the present invention, thereby treating the melanoma.
  • the present invention relates to an anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or an antibody-drug conjugate according to the present invention or a pharmaceutical composition according to the present invention in the preparation of a medicament for the treatment of melanoma use.
  • the present invention provides a pharmaceutical combination comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the pharmaceutical composition according to the present invention and one or A variety of additional therapeutic agents.
  • the present invention provides a kit comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the pharmaceutical composition according to the present invention, preferably, The kit further includes a device for administration.
  • Figure 1 shows the binding of the anti-VEGFR2 chimeric antibody to the recombinant protein VEGFR2-His detected by ELISA.
  • Figure 2 shows the binding of anti-VEGFR2 chimeric antibody on 293FT-VEGFR2 cell line detected by FACS.
  • Figure 3 shows that the anti-VEGFR2 chimeric antibody detected by ELISA blocks the binding of recombinant human VEGFR2-His to human VEGF165 recombinant protein.
  • Figure 4 shows that the anti-VEGFR2 chimeric antibody blocks the proliferation effect of VEGF165 on HUVEC cells.
  • Figure 5 shows the detection of the binding of VEGFR2-HK19 to recombinant human VEGFR2-His by ELSIA.
  • Figure 6 shows the binding of VEGFR2-HK19 on the 293FT-VEGFR2 cell line detected by FACS.
  • Figure 7 shows the species cross-binding of VEGFR2-HK19 detected by ELISA.
  • Figure 8 shows that VEGFR2-HK19 blocked the binding of VEGF165 to VEGFR2 detected by ELISA.
  • Figure 9 shows that VEGFR2-HK19 blocked the binding of VEGF165 to 293FT-VEGFR2 detected by FACS.
  • Figure 10 shows that VEGFR2-HK19 blocked the binding of VEGFR2-Fc recombinant protein to VEGF-C detected by ELISA.
  • Figure 11 shows that VEGFR2-HK19 blocked the binding of VEGFR2-Fc recombinant protein to VEGF-D detected by ELISA.
  • FIG. 12 shows that VEGFR2-HK19 blocks the proliferation effect of VEGF165 on HUVEC cells.
  • FIG. 13 shows that VEGFR2-HK19 blocks the proliferation effect of VEGF-C on HUVEC cells.
  • FIG. 14 shows that VEGFR2-HK19 blocks the proliferation effect of VEGF-C+VEGF-D on HUVEC cells.
  • Figure 15 shows that VEGFR2-HK19 blocks the proliferation effect of VEGF-A+VEGF-C+VEGF-D on HUVEC cells.
  • Figure 16 shows the ADCC effect of VEGFR2-HK19 detected by the CD16A recombinant reporter gene.
  • Figure 17 shows the docking of VEGFR2-HK19 homology modeling with the crystal structure of VEGFR2.
  • Figure 18 shows the epitopes that VEGFR2-HK19 and VEGF-A bind to VEGFR2.
  • Figure 19 shows the effect of VEGFR2-HK19 on the body weight of B16-F1 melanoma transplanted KDR humanized mice.
  • Figure 20 shows the effect of VEGFR2-HK19 on the tumor volume of B16-F1 melanoma transplanted KDR humanized mice.
  • Figure 21 shows the drug concentration-time curve of a single administration of VEGFR2-HK19 antibody to CD-1 mice.
  • Various aspects of the present invention relate to isolated anti-VEGFR2 antibodies or antigen-binding fragments thereof, antibody-drug conjugates comprising the antibodies or antigen-binding fragments thereof, nucleic acids and expression vectors encoding the antibodies or antigen-binding fragments thereof, and containing the nucleic acids Or a host cell expressing a vector, a method for producing the anti-VEGFR2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising the anti-VEGFR2 antibody or an antigen-binding fragment thereof, and a method for treating melanoma using the anti-VEGFR2 antibody or the antigen-binding fragment thereof .
  • antibody means an immunoglobulin molecule, and refers to any form of antibody that exhibits the desired biological activity. Including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (such as bispecific antibodies), and even antibody fragments.
  • the full-length antibody structure preferably contains 4 polypeptide chains, usually 2 heavy (H) chains and 2 light (L) chains connected to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, its structure also includes other derivative forms.
  • the heavy chain variable region and light chain variable region can be further subdivided into more conservative regions (called framework regions (FR)) and hypervariable regions interspersed (called complementarity determining regions (CDR)).
  • framework regions FR
  • CDR complementarity determining regions
  • CDR complementarity determining region
  • CDR1, CDR2, and CDR3 refers to the amino acid residues of the variable region of an antibody, the presence of which is necessary for antigen binding.
  • Each variable region usually has 3 CDR regions identified as CDR1, CDR2, and CDR3.
  • Each complementarity determining region can contain amino acid residues from the "complementarity determining region” defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. 1991 )) and/or those residues from the "hypervariable loop" (Chothia and Lesk; J Mol Biol 196:901-917 (1987)).
  • framework or "FR” residues are those variable region residues other than the CDR residues as defined herein.
  • Each heavy chain variable region and light chain variable region usually contains 3 CDRs and up to 4 FRs.
  • the CDRs and FRs are arranged in the following order from the amino terminal to the carboxy terminal, for example: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • CDR complementarity determining region
  • FR framework region
  • constant region refers to such amino acid sequences on the light chain and heavy chain of an antibody that do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as antibody-dependent cytotoxicity.
  • the heavy chain of an antibody can be divided into five categories: ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ .
  • ⁇ When it forms a complete antibody with the light chain, it can be divided into five categories: IgA , IgD, IgE, IgG and IgM, several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the light chain of an antibody can be classified into ⁇ and ⁇ .
  • an "antigen-binding fragment of an antibody” includes a portion of a complete antibody molecule that retains at least some of the binding specificity of the parent antibody, and usually includes at least a portion of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody.
  • antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecules (e.g., scFv, di-scFv or tri-scFv , Diabody or scFab), single domain antibody.
  • antibody fragment refers to an incomplete antibody molecule that retains at least some biological properties of the parent antibody, and examples thereof include, but are not limited to, Fc fragments in addition to those mentioned in the above-mentioned "antigen-binding fragments".
  • antibody-drug conjugate refers to a binding protein chemically linked to one or more chemical drugs, such as an antibody or antigen-binding fragment thereof, which may optionally be a therapeutic agent or a cytotoxic agent (such as a One or more cytokines or chemotherapy drugs).
  • the ADC includes an antibody, cytotoxic or therapeutic drug, and a linker that enables the drug to be linked or conjugated to the antibody.
  • ADCs usually have any value of 1 to 8 drugs conjugated to antibodies, including 2, 4, 6, or 8 drug-loading substances.
  • Non-limiting examples of drugs that can be included in the ADC are mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemotherapy protection Agents, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors and radiosensitizers.
  • chimeric antibody refers to an antibody in which a part of the heavy chain and/or light chain is derived from a specific source or species, and the remaining part is derived from a different source or species.
  • the “chimeric antibody” may also be a functional fragment as defined above.
  • Humanized antibodies are a subset of “chimeric antibodies.”
  • humanized antibody or “humanized antigen-binding fragment” is defined herein as an antibody or antibody fragment: (i) derived from a non-human source (for example, a transgenic mouse carrying a heterologous immune system) And based on human germline sequence; or (ii) the variable region is of non-human origin and the constant region is a chimeric antibody of human origin; or (iii) CDR grafted, wherein the CDR of the variable region is derived from a non-human source, and the variable One or more framework regions of the region are of human origin, and the constant region (if any) is of human origin.
  • the purpose of "humanization” is to eliminate the immunogenicity of non-human source antibodies in the human body, while retaining the greatest possible affinity.
  • a “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, that is, the population comprising a single antibody is identical except for possible mutations (such as natural mutations) that may be present in very small amounts. Therefore, the term “monoclonal” indicates the nature of the antibody, that is, it is not a mixture of unrelated antibodies. In contrast to polyclonal antibody preparations which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibody preparations is that they are generally not contaminated by other antibodies. The term “monoclonal” should not be understood as requiring the production of the antibody by any specific method.
  • the antibody "specifically binds" to an antigen of interest, such as a tumor-associated polypeptide antigen target (herein, VEGFR2), that is, binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent to target a target expressing the antigen Cells or tissues, and have no significant cross-reactivity with other proteins or with proteins other than homologs and variants (such as mutant forms, splice variants, or truncated forms of proteolysis) of the antigen target mentioned above No significant cross reaction.
  • VEGFR2 tumor-associated polypeptide antigen target
  • binding affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise stated, "binding affinity” as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • KD refers to the equilibrium dissociation constant of the antibody-antigen interaction.
  • kon refers to the rate constant at which an antibody binds to an antigen.
  • the term “koff” refers to the rate constant at which the antibody dissociates from the antibody/antigen complex.
  • KD association rate constant k on "and “dissociation rate constant k off” are usually used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen), that is, how tightly a ligand binds to a specific protein. degree. Binding affinity is affected by interactions between non-covalent molecules, such as hydrogen bonds, electrostatic interactions, hydrophobicity and van der Waals forces between two molecules. In addition, the binding affinity between the ligand and its target molecule may be affected by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including the ELISA described herein.
  • epitope includes any protein determinant capable of specifically binding to an antibody or T cell receptor.
  • Epitope determinants usually consist of chemically active surface groups of molecules (for example, amino acids or sugar side chains, or combinations thereof), and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • isolated antibody is an antibody that has been identified and isolated from a component of the cell that expresses it. Isolated antibodies include antibodies in situ in recombinant cells where at least one component of the antibody's natural environment is absent. However, usually, the isolated antibody is prepared through at least one purification step.
  • sequence identity between two polypeptide or nucleic acid sequences means the number of identical residues between the sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller of the compared molecules.
  • sequences being compared are aligned in a way that produces the largest match between the sequences, and the gaps in the alignment (if any) are resolved by a specific algorithm.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410) .
  • the above program can be publicly obtained from the International Center for Biotechnology Information (NCBI) and other sources.
  • NCBI International Center for Biotechnology Information
  • Smith Waterman algorithm can also be used to determine identity.
  • Fc receptor refers to a receptor that binds to the Fc region of an antibody.
  • Human FcR of natural sequence is preferred, and receptors ( ⁇ receptors) that bind to IgG antibodies are preferred, which include Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII subtypes, and variants of these receptors.
  • Other FcRs are included in the term “FcR”.
  • the term also includes the neonatal receptor (FcRn) which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunology 117:587 (1976) and Kim et al., J. Immunology 24:249 (1994)).
  • FcRn neonatal Fc receptor
  • the neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG antibodies in the body. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its clearance in serum and increasing its half-life. Therefore, the in vitro FcRn binding properties/characteristics of IgG indicate its in vivo pharmacokinetic properties in the blood circulation.
  • effector functions refers to those biological activities attributable to the Fc region of an antibody, which differ by antibody isotype.
  • antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complexes Mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors), and B cell activation.
  • effector cells refers to leukocytes that express one or more FcRs and perform effector functions.
  • the effector cell at least expresses FcyRIII and performs ADCC effector function.
  • human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells
  • neutrophils effector cells can be isolated from natural sources, for example, blood. Effector cells are usually lymphocytes associated with the effector stage and function to produce cytokines (helper T cells), kill cells infected by pathogens (cytotoxic T cells) or secrete antibodies (differentiated B cells) .
  • Immune cells include cells that have hematopoietic origin and play a role in immune responses. Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as NK cells, neutrophils, and macrophages
  • the secreted Ig on the Fc ⁇ receptor enables these cytotoxic effector cells to specifically bind to the target cell carrying the antigen, and then kill the target cell using, for example, a cytotoxin.
  • an in vitro ADCC assay can be performed, for example, the in vitro ADCC assay described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta).
  • Useful effector cells for such assays include PBMC and NK cells.
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement.
  • the activation of the typical complement pathway is initiated by combining the first component of the complement system (C1q) with an antibody (of the appropriate subclass) that binds to its corresponding antigen.
  • a CDC assay can be performed, such as the CDC assay described in Gazzano-Santoro et al., J. Immunol Methods 202:163 (1996).
  • polypeptide variants with an altered Fc region amino acid sequence polypeptides with a variant Fc region
  • polypeptide variants with enhanced or reduced C1q binding are described.
  • the present invention firstly uses recombinant human VEGFR2 protein to immunize mice, and then obtains the high-binding scFv antibody clone VEGFR2-MK19 which specifically binds to recombinant human VEGFR2 protein through phage display library screening. Then, the nucleotide sequences encoding the heavy chain and light chain variable regions of the VEGFR2-MK19 scFv antibody were inserted into the pSTEP2 vector with the human IgG1 constant region or the human kappa constant region nucleotide sequence, respectively, for culture expression. Purification by protein A purification column to obtain high-purity human-mouse chimeric antibody. The ELISA test shows that the anti-VEGFR2 chimeric antibody can block the binding of VEGFR2 to its ligand and the proliferation of HUVEC cells.
  • the light chain or heavy chain variable region of the human antibody that is closest to the mouse light chain or heavy chain variable region is selected as the template, and each of the mouse antibody light chain/heavy chain
  • the three CDRs were inserted into the corresponding positions of the above-mentioned human template to obtain humanized light chain variable region (VL) and heavy chain variable region (VH) sequences. Since the key points of the mouse-derived framework region are essential to support the activity of the CDR, the key points were backmutated to the sequence of the mouse antibody.
  • the light chain/heavy chain signal peptide sequence, the back-mutated humanized antibody light chain/heavy chain variable region sequence, and the human IgG1 heavy chain constant region/human kappa light chain constant region sequence were spliced in sequence to obtain the humanization
  • the invention also relates to nucleic acid molecules encoding the antibodies of the invention or parts thereof.
  • the sequences of these nucleic acid molecules include but are not limited to SEQ ID NO: 3-7, 26-33, and 36-39.
  • nucleic acid molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof.
  • the variants of the present invention can be described with reference to their physical characteristics in hybridization. Those skilled in the art will recognize that using nucleic acid hybridization techniques, nucleic acids can be used to identify their complements and their equivalents or homologs. It will also be recognized that hybridization can occur with less than 100% complementarity. However, considering the proper selection of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural correlation with specific probes.
  • the invention also provides a recombinant construct comprising one or more nucleotide sequences of the invention.
  • the recombinant construct of the present invention is constructed by inserting a nucleic acid molecule encoding the antibody of the present invention into a vector, such as a plasmid, phagemid, phage, or viral vector.
  • the antibody of the present invention can be prepared by recombinantly expressing nucleotide sequences encoding the light chain and the heavy chain or parts thereof in a host cell.
  • one or more recombinant expression vectors carrying the nucleotide sequence encoding the light chain and/or heavy chain or part thereof can be used to transfect the host cell so that the light chain and the heavy chain are in the Expressed in host cells.
  • Standard recombinant DNA methodology is used to prepare and/or obtain nucleic acids encoding heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as Sambrook, Fritsch and Maniatis (eds.
  • Suitable host cells are prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells are bacteria
  • examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of the expression vector including the selection regulatory sequence is affected by many factors, such as the choice of host cell, the desired expression level of the protein, and whether the expression is constitutive or inducible.
  • a usable expression vector for bacteria By inserting the structural DNA sequence encoding the desired antibody together with suitable translation initiation and termination signals and a functional promoter into an operable reading frame, a usable expression vector for bacteria can be constructed.
  • the vector will contain one or more phenotypic selectable markers and an origin of replication to ensure the maintenance of the vector and provide amplification in the host as needed.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas, Streptomyces, and grapes. Multiple species in the genus Staphylococcus.
  • Bacterial vectors can be, for example, phage, plasmid or phagemid based. These vectors may contain a selection marker and a bacterial origin of replication, which are derived from commercially available plasmids that usually contain elements of the well-known cloning vector pBR322 (ATCC 37017). After transforming an appropriate host strain and growing the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by an appropriate method (for example, temperature change or chemical induction), and the cells are cultured for an additional time. The cells are usually harvested by centrifugation, broken down by physical or chemical methods, and the resulting crude extract is retained for further purification.
  • an appropriate method for example, temperature change or chemical induction
  • a variety of expression vectors can be advantageously selected according to the intended use of the expressed protein. For example, when a large number of such proteins are to be produced for antibody production or for screening peptide libraries, for example, a vector that directs high-level expression of a fusion protein product that is easy to purify may be required.
  • Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (e.g. CMV promoter/enhancer) Promoter), simian virus 40 (SV40) promoter and/or enhancer (e.g. SV40 promoter/enhancer), adenovirus promoter and/or enhancer (e.g. adenovirus major late promoter (AdMLP)) and Polyoma virus promoter and/or enhancer.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • AdMLP adenovirus major late promoter
  • Polyoma virus promoter and/or enhancer e.g. adenovirus major late promoter (AdMLP)
  • the recombinant expression vector may also include an origin of replication and a selection marker (see, for example, U.S. 4,399,216, U.S. 4,634,665 and U.S. 5,179,017 of Axel et al.).
  • Suitable selection markers include genes that confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced.
  • drugs such as G418, hygromycin
  • methotrexate to host cells into which the vector has been introduced.
  • the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate
  • the neo gene confers resistance to G418.
  • Transfection of the expression vector into host cells can be performed using standard techniques such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection.
  • Suitable mammalian host cells for expressing the antibodies provided herein include Chinese Hamster Ovary (CHO cells) [including dhfr-CHO cells, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216 In -4220, DHFR selection markers are used, such as those described in RJ Kaufman and PASharp (1982) Mol. Biol. 159:601-621], NSO myeloma cells, COS cells, and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • DHFR selection markers are used, such as those described in RJ Kaufman and PASharp (1982) Mol. Biol. 159:601-621]
  • NSO myeloma cells such as those described in RJ Kaufman and PASharp (1982) Mol. Biol. 159:601-621
  • NSO myeloma cells such as those described in RJ Kaufman and PASharp (1982) Mol. Biol. 159
  • the antibody of the present invention can be recovered and purified from recombinant cell culture by known methods, including but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion Or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.
  • High performance liquid chromatography (“HPLC”) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley&Sons, NY, NY, (1997-2001), such as Chapters 1, 4, 6, 8, 9, and 10, each of which is fully cited Include this article.
  • VEGFR2-HK19 Characterization and function analysis of the humanized VEGFR2-HK19 antibody of the present invention are performed.
  • the analysis results show that the antibody of the present invention has the following advantages: (1) It can specifically bind to VEGFR2 antigen with high affinity and has a low dissociation rate, thereby providing good antitumor efficacy; (2) Comparing with ramucirumab It has a higher binding affinity for VEGFR2; (Example 4) (3) VEGFR2-HK19 has a similar ability to block VEGF165 protein from binding to VEGFR2; (Example 4.2.1) (4) VEGFR2- HK19 has a stronger ability to block the binding of VEGFR2-Fc recombinant protein to VEGF-C or VEGF-D than ramucirumab; (Example 4.2.3) (5) VEGFR2-HK19 blocks VEGF165, VEGF-C, and VEGF- The EC50 of C+VEGF-D or VEGF-A+VEGF-C
  • the antibodies of the present invention can be used to treat melanoma.
  • the antibodies of the present invention can also be used to prepare drugs for treating the diseases.
  • the antibody of the present invention and at least one other agent can be prepared into a pharmaceutical composition, which includes the antibody of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition may include additional therapeutic agents.
  • the invention also relates to a pharmaceutical package and a kit comprising one or more containers containing the aforementioned pharmaceutical composition of the invention. It is accompanied by a reminder of the form prescribed by the government agency that regulates the production, use or sale of drugs or biological products, which reflects that the drug has been approved by the above-mentioned agencies for human administration.
  • the pharmaceutical composition of the present invention can be prepared in a manner known in the art, for example, by conventional mixing, dissolving, granulating, tablet preparation, grinding, emulsifying, coating, embedding or freeze-drying methods.
  • compositions containing the compound of the present invention formulated in an acceptable carrier After the pharmaceutical composition containing the compound of the present invention formulated in an acceptable carrier has been prepared, they can be placed in an appropriate container and labeled for the treatment of the indicated condition.
  • labels would include the amount, frequency, and method of administration.
  • composition containing the antibody of the present invention is also combined with one or more other therapeutic agents, such as anti-tumor agents, wherein the resulting combination does not cause unacceptable adverse effects.
  • Example 1 Screening of murine antibodies that specifically block the binding of VEGFR2 to VEGF
  • Recombinant human VEGFR2 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., UniProtKB-P35968/NP_002244.1) was used to immunize Balb/c mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.).
  • the amino acid sequence of the extracellular region Met1-Glu764 of the recombinant human VEGFR2 protein is SEQ ID NO:1.
  • Mouse spleen tissue was extracted with TriPureHisolationReagent reagent (Roche, Cat.No.11667165001), and RNA was reverse-transcribed with reverse transcription reagent to obtain cDNA library, using overlap extension splicing PCR method [14-15], using reference 2
  • TriPureHisolationReagent reagent Gibcos, Cat.No.11667165001
  • RNA was reverse-transcribed with reverse transcription reagent to obtain cDNA library, using overlap extension splicing PCR method [14-15], using reference 2
  • the nucleotide sequences encoding the light chain and heavy chain variable regions of the mouse antibody are spliced into scFv encoding the antibody
  • the light and heavy chain variable regions are connected by a linker (SEQ ID NO: 2), and the resulting DNA fragment is digested with the restriction enzyme Sfi I and connected to the phage vector pComb3x (Beijing Yiqiao Shenzhou Technology Co., Ltd. Company), electrotransformed X-Blue competent to obtain mouse phage display scFv antibody library.
  • the second to third domain protein VEGFR2-domain 2&3 of recombinant human VEGFR2 (Beijing Yiqiao Shenzhou Technology Co., Ltd., 10012-H08H3, NP_002244.1, Asp120-Lys327 )) Coating on an ELISA plate, adding antibody phage to incubate, washing out unbound phage, recovering bound phage, and repeating this procedure. After multiple rounds of screening and enrichment, an anti-VEGFR2 positive antibody phage library is obtained. Monoclonal phages were selected from the enriched library for expression, and the binding to recombinant human VEGFR2 protein was detected by ELISA method.
  • ScFv antibodies that specifically bind to recombinant human VEGFR2 were screened from multiple monoclonals, and the monoclonals were obtained by sequencing The nucleotide sequence of the antibody, wherein the nucleotide sequence encoding the monoclonal scFv antibody VEGFR2-MK19 is SEQ ID NO: 3).
  • VEGFR2 monoclonal chimeric antibody was produced.
  • VEGFR2-MK19scFv The heavy chain nucleotide sequence of the VEGFR2-MK19 scFv antibody was amplified by PCR and inserted into the heavy chain signal peptide (SEQ ID NO: 28) and human IgG1 constant region (SEQ ID NO: 6) by in-fusion method
  • SEQ ID NO: 28 The heavy chain signal peptide
  • SEQ ID NO: 6 human IgG1 constant region
  • the light chain nucleotide sequence of the VEGFR2-MK19 scFv antibody was amplified by PCR and inserted into the light chain signal peptide (SEQ ID NO: 29) and human kappa constant region (SEQ ID NO: 7) by in-fusion method
  • the human-mouse chimeric light chain (SEQ ID NO: 37) expression vector was obtained from the pSTEP2 vector digested with ScaI/BsiWI (Fermentas).
  • 293E cells were passaged with SCD4-4-TC2 medium (Beijing Yiqiao Shenzhou Technology Co., Ltd.) to 200mL/bottle, the initial seeding density was 0.3 ⁇ 0.4*10 6 cells/mL, and the temperature was 37°C and the rotation speed was 175rpm CO 2 after culture shaker, until the cell density reached 1.5 to 3 * 10 6 cells / mL, 1: 1 mixture of plasmid DNA after the light and heavy chain plasmid DNA, 100 ug were mixed and added to 800 ⁇ L of culture flasks TF2 The transfection reagent was placed in a shaker with a temperature of 37°C and a rotating speed of 175 rpm and continued to be cultured until the 7th day of material collection.
  • SCD4-4-TC2 medium Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • the culture solution was centrifuged at 4000 rpm for 25 minutes, the supernatant was collected, and 1/5 of the volume of the supernatant was added to stock buffer. Equilibrate the protein A chromatography column with PBS for 5-10 times the column volume, add the filtered culture supernatant to the chromatography column, equilibrate again with 5-10 times the column volume, and elute the sample with sodium acetate buffer. After the sample is eluted, it is neutralized to neutrality with Tris to obtain a high-purity chimeric antibody for later use.
  • Example 2 Function test of VEGFR2 chimeric antibody
  • the recombinant human VEGFR2-His protein at a concentration of 0.1 ⁇ g/mL was coated on a 96-well plate with 100 ⁇ L per well and coated overnight at 4°C. Wash the plate the next day and block at room temperature for 1 hour, add 100 ⁇ L 2 ⁇ g/mL anti-VEGFR2 chimeric antibody and incubate for 1 hour, wash the plate to remove unbound antibody, add the detection secondary antibody goat anti-human IgG Fc/HRP, incubate and repeat the plate washing, add The substrate color developing solution is used for color development. After the display is terminated, the microplate reader reads OD450.
  • 293FT-VEGFR2 logarithmic growth phase VEGFR2 stably expressing cell line 293FT-VEGFR2-13-13 (hereinafter referred to as 293FT-VEGFR2) as the experimental material.
  • 293FT-VEGFR2 logarithmic growth phase VEGFR2 stably expressing cell line 293FT-VEGFR2-13-13
  • 293FT-VEGFR2 cells and 293FT cells were divided into 5 ⁇ 10 5 cells/tube with a volume of 50 ⁇ L, 10 ⁇ L of anti-VEGFR2 chimeric antibody diluted to a concentration of 0.1 ⁇ g/ ⁇ L was added, mixed and incubated at 4°C, washed with PBS, centrifuged to remove To bind the antibody, add goat anti-human IgG Fc-FITC secondary antibody and incubate at 4°C, repeat washing and centrifugation to remove the supernatant, remove the unbound secondary antibody, and finally add 200 ⁇ L PBS to resuspend the cells, and filter into the flow tube with a 400 mesh filter , And then use flow cytometry to detect.
  • the results are shown in Figure 2.
  • the chimeric antibody mhK19, as well as the chimeric antibodies mhT06, mhW03, mhX09 and mhX12 have good specific binding on 293FT-VEGFR2 cells.
  • VEGF165 is the most classic splicing body of VEGF-A
  • concentration 0.5 ⁇ g/mL on a 96-well plate, 100 ⁇ L per well, 4°C Coated overnight.
  • the plate was washed the next day and blocked at room temperature for 1 hour, then 100 ⁇ L of 1 ⁇ g/mL recombinant VEGFR2-His protein was added, and then the VEGFR2 chimeric antibody at the concentration of 2 ⁇ g/mL and 0.4 ⁇ g/mL was added to incubate together for 1 hour, where only VEGFR2 was set -The wells without antibody added to His protein are positive wells, wash the plate to remove unbound antibody, add C-His-R023/HRP detection antibody and incubate repeatedly, wash the plate again, add substrate color development solution for color development, use enzyme label after termination
  • the instrument reads OD450, and calculates the competitive inhibition rate% of different concentrations of antibody blocking the binding of recombinant human VEGFR2-His protein and human VEGF165 protein according to the OD450 reading.
  • the antibody competition inhibition rate % (positive well OD450-add the corresponding concentration of VEGFR2 antibody OD450) /Positive wells OD450 ⁇ 100%.
  • the results are shown in Figure 3.
  • Chimeric antibodies such as mhK19, mhT06, mhU11, mhW03, mhX09 and mhX12 can all block the binding of recombinant human VEGFR2-His protein to human VEGF165 protein, and the inhibition rate of mhK19 can reach at a concentration of 2 ⁇ g/mL. 96%.
  • Human umbilical vein endothelial cells HUVEC were seeded into 96-well cell culture plates at 4 ⁇ 10 3 cells/well, cultured in M199 medium (Gibco, 11043023) containing 10% FBS and 5% L-Gln for 4 h, and then 50 ⁇ L Add corresponding concentration of antibody to each well, then add VEGF165 at a final concentration of 10ng/mL at 10 ⁇ L/well, set up detection blank well B (no cells), negative control group M (seeding cells, no sample, add VEGF165) and M '(Inoculated cells, no sample and VEGF165).
  • the mouse antibody VEGFR2-MK19 was selected for humanized transformation and production.
  • VEGFR2-MK19scFv antibody nucleotide sequence determined in Example 1.3 was deduced to obtain the VEGFR2-MK19scFv antibody heavy chain and light chain variable region amino acid sequences, see SEQ ID NO: 8/9.
  • Mouse antibody humanization adopts the classic CDR transplantation method [18-19], and selects those that are more than 50% similar to the mouse light chain and heavy chain variable regions, and the heavy chain variable region and light chain variable region Antibodies whose framework regions are more than 50% similar in amino acid sequence to the framework regions of the heavy chain variable region and light chain variable region of the antibody to be modified are used as pre-humanized templates.
  • the human antibody with the highest spatial structural similarity in the variable region of the modified antibody is used as the humanized template.
  • the three CDR sequences of the light chain or heavy chain of the mouse antibody are replaced with the corresponding CDR amino acid sequences in the humanized template, and the HCDR2 P at position 60 is changed to A. .
  • the human template for the light chain variable region transplantation of VEGFR2-MK19 is IGKV1-NL1*01, which has 70.5% homology with the light chain of VEGFR2-MK19, and the human template for the heavy chain variable region is IGHV3 -21*01, the template has 81.6% homology with the heavy chain of VEGFR2-MK19.
  • the key points of the mouse-derived framework region play a crucial role in maintaining the stability of the CDR spatial structure
  • the key points were backmutated to the corresponding amino acids of the mouse antibody until the antibody with stable spatial structure was obtained and the following mutations were carried out: Kabat numbering, back mutated position 43 of the light chain to S, position 45 back mutated to Q, and position 70 back mutated to H; back mutated position 42 to D and back mutated position 44 to R in the heavy chain.
  • the 49th position is back-mutated to A.
  • Humanized antibody VEGFR2-HK19 was obtained by CDR humanization transplantation and framework region back mutation.
  • the amino acid sequences are shown in SEQ ID NO: 16/17, respectively; its heavy chain and light chain containing signal peptide
  • the amino acid sequences are shown in SEQ ID NO: 18/19, respectively, which respectively include the heavy chain/light chain signal peptide amino acid sequence (SEQ ID NO: 20/21) connected in sequence;
  • the humanized antibody heavy chain/light chain can be Amino acid sequence of the variable region (SEQ ID NO: 22/23);
  • the constant region of the humanized antibody is the human IgG1 heavy chain constant region/human kappa light chain constant region sequence (SEQ ID NO: 24/25).
  • the modified antibody has similar affinities and achieves complete humanization.
  • the CDR sequences after humanization are shown in Table 2.
  • PCR amplifies the nucleotide sequence (SEQ ID NO: 27) encoding the light chain of the VEGFR2-HK19 antibody containing the signal peptide, which contains the nucleotide sequence of the light chain signal peptide (SEQ ID NO: 29), human source
  • the nucleotide sequence of the variable region of the light chain of the antibody (SEQ ID NO: 31) and the nucleotide sequence of the constant region of the human kappa light chain (SEQ ID NO: 33) were inserted into the self-developed KpnI through the in-fusion method.
  • the pGS vector after double digestion with XbaI was verified by sequencing to obtain the correct plasmid.
  • PCR amplifies the nucleotide sequence (SEQ ID NO: 26) encoding the heavy chain of the VEGFR2-HK19 antibody, which contains the nucleotide sequence of the heavy chain signal peptide (SEQ ID NO: 28), humanized
  • the nucleotide sequence of the variable region of the antibody heavy chain (SEQ ID NO: 30) and the nucleotide sequence of the constant region of the human IgG1 heavy chain (SEQ ID NO: 32) are inserted into the correct containing light In the pGS vector (digested by NheI and NotI), the correct VEGFR2-HK19 light and heavy chain expression vector was verified by sequencing.
  • the expression vector is a eukaryotic cell expression vector containing GS screening genes and expression elements of antibody light and heavy chains.
  • the expression vector was transfected into CHO-K1-GS-deficient cells, and VEGFR2-HK19 high-expressing cell lines were obtained by MSX screening. High-expressing clones are selected by ELISA detection, and stable high-expressing cell strains are selected by combining the cell growth status and the key quality attribute analysis results of antibody drugs.
  • the CHO cell line producing VEGFR2-HK19 was cultured in a serum-free feeding suspension culture method, and high-quality VEGFR2-HK19 antibody was purified and obtained.
  • the recombinant human VEGFR2-His protein was coated on a 96-well plate with 100 ⁇ L per well and coated overnight at 4°C.
  • FACS was used to detect the binding of humanized antibodies VEGFR2-HK19 and ramucirumumab (Eli Lilly, C839381C) on VEGFR2-expressing cells.
  • H7N9-R1 was used as a negative control antibody.
  • 293FT-VEGFR2 cells were divided into 3 ⁇ 10 5 cells/tube with a volume of 50 ⁇ L, and different concentrations (2nM, 6nM, 17nM, 51nM, 154nM, 463nM and 1389nM) of VEGFR2-HK19, ramucirumab and 10 ⁇ L each of H7N9-R1 antibody, mixed and incubated at 4°C, washed with PBS washing solution, centrifuged to remove unbound antibody, added goat anti-human IgG Fc-FITC secondary antibody and incubated at 4°C, washed repeatedly and centrifuged to remove the supernatant to remove unbound Anti-, finally add 200 ⁇ L PBS to resuspend the cells, filter with 400 mesh
  • the Octet biomolecule interaction analysis system (model: Octet RED, manufacturer: Fortebio) was used to determine multiple concentration gradients (0.42nM, 0.90nM, 1.74nM, 3.47nM, 6.94nM) of VEGFR2-HK19, and ramucirumab (Eli Lilly, C839381C) Affinity with biotinylated VEGFR2-biotin.
  • the KD value of VEGFR2-HK19 binding to recombinant human VEGFR2-Biotin protein is 1.06*10 -11 M
  • the binding rate constant kon value is 8.26E+05M -1 s -1
  • the dissociation rate constant kdis value is 8.75E-06s -1
  • the binding affinity KD value of ramucirumumab and VEGFR2 protein is 4.58*10 -11 M
  • the binding rate constant kon value is 3.86E+05M -1 s -1
  • the dissociation rate constant kdis value is 1.77E-05s -1 .
  • VEGFR2-HK19 The affinity of VEGFR2-HK19 is about 4.32 times that of ramucirumab, and VEGFR2-HK19 has a slower dissociation rate, so VEGFR2-HK19 has a stronger ability to bind to VEGFR2-biotin protein than ramucirumab .
  • OCTET detects the binding of VEGFR2-HK19 and VEGFR2-biotin
  • Recombinant human VEGFR2-His protein, recombinant mouse mKDR-His protein and recombinant rat ratVEGFR2-His protein were coated on a 96-well plate at a concentration of 0.04 ⁇ g/mL, 10 ⁇ g/mL, and 10 ⁇ g/mL respectively, with 100 ⁇ L per well , Coated overnight at 4°C.
  • VEGFR2-HK19 and ramucirumab molecules specifically bind to recombinant human VEGFR2-His protein, but do not have cross-binding with recombinant mouse mKDR-His protein and recombinant rat ratVEGFR2-His protein.
  • VEGFR2-HK19 blocks the binding of VEGFR2-His recombinant protein to VEGF165
  • the recombinant VEGF165 protein at a concentration of 0.5 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 4°C. The plate was washed the next day and blocked at room temperature for 1 hour, then 100 ⁇ L of recombinant VEGFR2-His protein at a concentration of 1 ⁇ g/mL was added, and 100 ⁇ L of different concentrations (1.37ng/mL, 4.12ng/mL, 12.35ng/mL, 37.04ng /mL, 111.11ng/mL, 333.33ng/mL, 1000ng/mL, 3000ng/mL and 9000ng/mL) VEGFR2-HK19, ramucirumab ((Eli Lilly, C839381C)) and negative control antibody H7N9-R1 And set only the wells with VEGFR2-His protein and no antibody added as positive wells.
  • VEGFR2-HK19 blocks the binding of 293FT-VEGFR2 cell line to VEGF165
  • FACS was used to detect the effects of humanized antibodies VEGFR2-HK19 and ramucirumab (Eli Lilly, C839381C) on the binding of VEGFR2 overexpressing cells to recombinant protein VEGF165.
  • H7N9-R1 was used as a negative control antibody.
  • 293FT-VEGFR2 cells were divided into 3 ⁇ 10 5 cells/tube with a volume of 50 ⁇ L, and different concentrations (2777.8nM, 925.9nM, 308.6nM, 102.9nM, 34.3nM, 11.4nM and 3.8nM) of VEGFR2-HK19 were added 10 ⁇ L each of ramucirumab and H7N9-R1 antibody, incubate at 4°C for 20min, add 2.5 ⁇ g of in vivo biotin-labeled VEGF165 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11066-H27H-B), mix and incubate, set Cell wells with no antibody added and only VEGF165 protein are positive control wells.
  • Inhibition rate (%) (MFI positive- MFI sample )/MFI positive * 100%, MFI positive means that there is no antibody and only the MFI value of VEGF165 protein binding to VEGFR2 cells.
  • the MFI sample is the MFI value of VEGF165 protein binding to VEGFR2 cells after adding antibody.
  • the results are shown in Figure 9, the VEGFR2-HK19 antibody can block the binding of VEGF165 protein to VEGFR2 overexpressing cells.
  • VEGFR2-HK19 blocks the binding of VEGFR2-Fc recombinant protein to VEGF-C or VEGF-D
  • VEGFR2-Fc (Beijing Yiqiao Shenzhou Technology Co., Ltd., 10012-H02H) protein at a concentration of 5 ⁇ g/mL was coated on a 96-well plate with 100 ⁇ L per well and coated overnight at 4°C. The plate was washed the next day and sealed at room temperature for 1 hour, and then 100 ⁇ L of recombinant VEGF-C (Beijing Yiqiao Shenzhou Technology Co., Ltd.) protein at a concentration of 0.5 ⁇ g/mL or 2 ⁇ g/mL recombinant VEGF-D (Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added.
  • VEGF-C Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • GraphPad Prism 6.0 software was used to respectively fit the S-shaped curve and analyze the EC 50 of the antibody blocking the binding of VEGFR2-Fc protein to VEGF-C or VEGF-D.
  • VEGFR2-HK19 is more capable of blocking the binding of VEGFR2-Fc recombinant protein to VEGF-C or VEGF-D. Strong monoclonal antibody.
  • VEGFR2-HK19 blocks the proliferation of VEGF165 or VEGF-C on HUVEC cells
  • HUVEC cells were seeded into 96-well cell culture plates at 4 ⁇ 10 3 cells/well, cultured in M199 medium (Gibco, 11043023) containing 10% FBS and 5% L-Gln for 4 hours, and then 50 ⁇ L/well was added to the corresponding Then add VEGF165 with a final concentration of 10ng/mL or VEGF-C with a final concentration of 1000ng/mL at 10 ⁇ L/well, set up detection blank well B (without cells) and negative control group M (seeding cells without adding For samples, add VEGF165 or VEGF-C) and M'(seeding cells, do not add samples and VEGF165 or VEGF-C).
  • the neutralization rate% (negative control M group OD value-sample OD value)/(negative control) M group OD value-M'group OD value) ⁇ 100%, using the automatic analysis function of the statistical software GraphPad Prism to calculate the standard curve, the abscissa is the sample concentration, the ordinate is the neutralization rate, and the four-parameter regression equation is used to fit the standard curve. S" curve, calculate the half effective concentration (EC50) of the sample. The results are shown in Figures 12, 13 and Table 4.
  • VEGFR2-HK19 and ramucirumab (Eli Lilly, C839381C) block the maximum neutralization rate of VEGF165 or VEGF-C on HUVEC cell proliferation, the maximum neutralization rate of VEGFR2-HK19 The EC50 concentration is less than ramucirumab, suggesting that VEGFR2-HK19 has better growth inhibitory activity.
  • VEGFR2-HK19 blocks the proliferation effect of different subtypes of VEGF composition on HUVEC cells
  • HUVEC cells were seeded into 96-well cell culture plates at 4 ⁇ 10 3 cells/well, cultured in M199 medium (Gibco, 11043023) containing 10% FBS and 5% L-Gln for 4 hours, and then 50 ⁇ L/well was added to the corresponding concentration VEGF-C (1000ng/mL) and VEGF-D (8181ng/mL) mixture, or VEGF165 (25ng/mL) and VEGF-C (1000ng/mL) and VEGF-D ( 5455ng/mL).
  • M199 medium Gibco, 11043023
  • VEGF165 25ng/mL
  • VEGF-C 1000ng/mL
  • VEGF-D 5455ng/mL
  • Neutralization rate% (negative control M group OD value-sample OD value) / (negative control M Group OD value-M' group OD value) ⁇ 100%, using the automatic analysis function of the statistical software GraphPad Prism to calculate the standard curve, the abscissa is the sample concentration, the ordinate is the neutralization rate, and the four-parameter regression equation is used to fit the standard curve. ”Type curve, calculate the half effective concentration (EC50) of the sample. The results are shown in Figures 14, 15 and Table 5.
  • VEGFR2-HK19 and ramucirumab (Eli Lilly, C839381C) blocked the maximum neutralization rate of VEGF-C+VEGF-D on HUVEC cell proliferation, and VEGFR2-
  • the maximum neutralization rate of HK19 blocking VEGF-A+VEGF-C+VEGF-D on HUVEC cell proliferation is slightly less than ramucirumab, but the EC50 concentration of VEGFR2-HK19 is less than ramucirumab, suggesting that VEGFR2- HK19 has better growth inhibitory activity.
  • the recombinant CD16A reporter gene system method was used to determine the ADCC effect mediated by VEGFR2-HK19.
  • the effector cell is Jurkat-NFAT-Luc2p-CD16A
  • the target cell is 293FT-VEGFR2.
  • VEGFR2 is added.
  • the Fab segment of VEGFR2-HK19 binds to VEGFR2 overexpressed on target cells, and its Fc segment can bind to effector cells overexpressing Fc ⁇ III receptor (CD16A), thereby activating effector cells Jurkat-NFAT-Luc2p-CD16A And promote NFAT-RE-mediated bioluminescence.
  • the target cell 293FT-VEGFR2 was seeded on a 96-well plate at 2 ⁇ 10 4 cells/well, cultured in DMEM medium containing 10% FBS overnight, the supernatant was removed, and 0.5g/L PF68( Wash twice with the phenol red-free RPMI 1640 medium of Beijing Yiqiao Shenzhou Technology Co., Ltd., and then add the corresponding concentration of antibody at 40 ⁇ L/well, and then add 1 ⁇ 10 5 effector cells Jurkat-NFAT-Luc2p- at 40 ⁇ L/well.
  • CD16A each group has 3 multiple wells, and the target cells, effector cells and negative control group (with target cells and effector cells, no sample).
  • ramucirumab (Eli Lilly, C839381C) has a weaker ability to mediate ADCC effects, while VEGFR2-HK19 has strong ADCC effects.
  • Example 4.2 it was confirmed that VEGFR2-HK19 can block the binding of ligand VEGF165 and VEGFR2, which proved that the epitope of VEGFR2-HK19 is similar to the ligand, and the literature reported that VEGF165 binds to the second and third domains of the extracellular domain of VEGFR2 [ 2].
  • VEGFR2 domain protein VEGFR2-domain 2&3
  • extracellular region recombinant protein VEGFR2-His to VEGFR2-HK19 was detected by ELISA in this example. The results are shown in the table. 6.
  • VEGFR2-HK19 binds to the second to third domains of VEGFR2, and there may be epitope overlap with the ligand VEGF165 in the binding of VEGFR2 .
  • VEGFR2-HK19 and VEGFR2 protein interface In order to understand the interaction between VEGFR2-HK19 and VEGFR2 protein interface, molecular simulation and docking method were used in this example, and the Antidody model program in DS 4.0 (Accelrys Software Inc.) was used to model the homology of VEGFR2-HK19 from PDB.
  • the three-dimensional structure of VEGFR2 protein (PDB ID: 3V2A) was extracted from the database and initialized through the Protein Preparation program.
  • the VEGFR2-HK19 model and the VEGFR2 structure are docked through the ZDOCK program. According to the docking results, the top ten docking functions of the scoring function are optimized by RDOCK, and the optimal model ( Figure 17) is further analyzed by the Protein Interface Analysis program.
  • the docking model interface shows that the key binding peptide sequences of VEGFR2-HK19 and VEGFR2 proteins are 137 YITENK 142 in domain 2 and 255 GIDFNWEY 262 and 310 SSGLMTKK 317 in domain 3, respectively.
  • VEGFR2 mutant protein verifies that the humanized antibody VEGFR2-HK19 binds to key sites
  • VEGFR2-HK19 binding In order to further confirm the key sites of VEGFR2-HK19 binding, according to the predicted key binding peptide sequence of VEGFR2-HK19 and VEGFR2 protein in the above 4.5.2 example, a series of VEGFR2 protein mutants were designed and produced. The mutants and VEGFR2 were determined by ELISA. -HK19, the binding ability of the listed antibody ramucirumab (Eli Lilly, C839381C) and the ligand VEGF-A. The results showed that the binding of VEGFR2-HK19 to VEGFR2 protein mutants was reduced to varying degrees (Table 7).
  • VEGFR2-HK19 mainly binds to key sites such as Y137A/K142A, D257A, S311A/G312A, L313A, and T315A. This result is consistent with 4.5. 2
  • the analysis results of the docking model in the embodiment are basically consistent, verifying the accuracy of the model.
  • the key sites of VEGF and ramucirumab antibody are basically consistent with the crystal structures reported in the literature [20-21]. Y137A/K142A, R164S/Y165A, D257A, S311A/G312A, etc.
  • Example 5 Anti-tumor effect of humanized antibody VEGFR2-HK19 on B16-F1 melanoma subcutaneously transplanted tumor model in KDR humanized mice
  • mice (China Institute for Food and Drug Control) were subcutaneously inoculated with B16-F1 tumor masses of about 2 ⁇ 2 ⁇ 2 (mm 3 ) on the right flank, and 39 mice (20 females, males) were inoculated 19).
  • the tumor volume is about 100mm 3 or so, the mice will be administered in groups, and mice will be selected according to the individual tumor volume.
  • the animals will be randomly divided into 4 groups according to the tumor volume using excel software, each with 6 (3 male and female), the first group It is a blank control group, the second group is the experimental group (VEGFR2-HK19), and the third and fourth groups are other anti-VEGFR2 antibodies that are not related to the application, so relevant data are not presented.
  • Intraperitoneal injection (IP) administration was started on the day of grouping.
  • the dose of the experimental group was 15 mg/kg, and the blank control group (Vehicle) was injected with an equal volume of solvent, administered twice a week for 5 consecutive times.
  • the specific dosing schedule is shown in the following table 8.
  • the anti-tumor effect of the drug is evaluated by calculating the tumor growth inhibition rate TGI (%): TGI (%) ⁇ 60% is invalid; TGI (%) ⁇ 60%, and the tumor volume of the experimental group is significantly lower than the solvent control group after statistical processing (P ⁇ 0.05) is effective, that is, it has a significant inhibitory effect on tumor growth.
  • TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100, where:
  • T0 The mean tumor volume of the experimental group on day 0 of administration
  • V0 The mean tumor volume of the solvent control group on day 0 of administration.
  • the dosage volume is calculated based on the weight of the experimental animal as 1 mL/kg.
  • test antibody VEGFR2-HK19 binding to KDR epitope is effective in the B16-F1 melanoma subcutaneous transplantation tumor model, and it has a significant inhibitory effect on the B16-F1 melanoma subcutaneous transplantation tumor at the dose level of 15mg/kg.
  • Example 6 Study on the pharmacokinetics and immunogenicity of humanized antibody VEGFR2-HK19 in mice
  • the mouse pharmacokinetics of the antibody VEGFR2-HK19 was investigated in CD1 mice: CD-1 mice were administered at a dose of 5 mg/kg via tail vein and subcutaneous injection, 6 mice in each group, half male and half, before the drug, Blood was collected 0.5h (IV group), 2h, 4h, 6h, 24h, 48h, 72h, 96h, 168h, 336h, 504h, 672h after the drug.
  • the indirect ELISA method was used to detect the concentration of VEGFR2-HK19 antibody in the serum, and the pharmacokinetic parameters were calculated by the non-compartmental model (NCA) in the Phoenix-WinNonlin6.4 software.
  • NCA non-compartmental model
  • the bridged ELISA method was used for all animals after injection and administration. Blood samples at 168, 336, 504, and 672 hours were tested for anti-drug antibodies.
  • the concentration of VEGFR2-HK19 antibody in the serum of CD-1 mice was detected by ELISA.
  • the plasma concentration-time curve is shown in Figure 21, and the pharmacokinetic parameters are shown in Table 11.
  • the 5mg/kg dose of VEGFR2-HK19 antibody was administered intravenously and subcutaneously to CD-1 mice. There was no significant difference between male and female animals.
  • the C max of intravenous and subcutaneous administration were 77.77 and 32.00 ⁇ g/mL, respectively, and the AUC last were 11702.39 and 11702.39 and respectively. 11033.42h* ⁇ g/mL, the absolute bioavailability of subcutaneous administration is 94.28%.
  • the parameters such as t 1/2 , Vz and Cl are basically the same under the two administration methods.
  • Ferrara N The role of vascular endothelial growth factor in pathological angiogenesis[J]. Breast Cancer Res Treat, 1995, 36(2): 127-137.
  • VEGF Vascular endothelial growth factor

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Abstract

The present invention pertains to the field of tumor immunotherapy, and provides a recombinant humanized anti-VEGFR2 antibody drug. The present invention further provides a nucleic acid sequence encoding the antibody, a vector containing the nucleic acid sequence, a pharmaceutical composition, and a kit. The antibody provided by the present invention can specifically bind to tumor cells and block the effects of VEGF-A, VEGF-C, and VEGF-D at the same time, and thus has a better inhibitory effect on angiogenesis. The antibody can also activate NK cells to produce antibody-dependent cytotoxicity reactions and inhibit the growth and metastasis of tumors, and can be used for the clinical treatment of melanoma.

Description

一种人源化VEGFR2抗体及其应用A humanized VEGFR2 antibody and its application 技术领域Technical field
本发明属于肿瘤免疫治疗领域，涉及一种重组人源化的抗VEGFR2抗体及其应用。The invention belongs to the field of tumor immunotherapy, and relates to a recombinant humanized anti-VEGFR2 antibody and its application.
背景技术Background technique
肿瘤血管为肿瘤的发生发展输送充足的营养物质，并提供肿瘤细胞的逃逸通道。靶向血管新生的药物可阻断肿瘤的营养供给，从而达到“饿杀”肿瘤的效果。然而，肿瘤血管的新生受肿瘤微环境中多种生长因子、受体及下游信号通路的综合调控。其中，血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)通过与其受体(vascular endothelial growth factor receptor,VEGFR)结合，在生理性和病理性血管形成过程中发挥重要作用[1]。Tumor blood vessels transport sufficient nutrients for the occurrence and development of tumors and provide escape channels for tumor cells. Drugs that target angiogenesis can block the nutrient supply of tumors, thereby achieving the effect of "starving" the tumor. However, tumor angiogenesis is comprehensively regulated by a variety of growth factors, receptors and downstream signaling pathways in the tumor microenvironment. Among them, vascular endothelial growth factor (vascular endothelial growth factor, VEGF) binds to its receptor (vascular endothelial growth factor receptor, VEGFR), and plays an important role in the process of physiological and pathological blood vessel formation [1].
VEGFR2(也称为FLK-1，或KDR)，是VEGFR家族的主要成员，属于III型受体酪氨酸激酶，主要分布在血管及***内皮细胞膜表面。VEGFR2的胞外区含7个免疫球蛋白样结构域(即D1-D7)，包括配体VEGF结合结构域(D2-D3)和二聚化结构域(D4-D7)，其胞内区含酪氨酸激酶结构域[2-4]。VEGF(包括VEGF-A、C、D、E等)在正常人血浆中的浓度约为137±7.7pg/mL[5]，胰腺癌、卵巢癌等患者血清中VEGF浓度显著上调，为200-400pg/mL，而肿瘤组织或者肿瘤囊肿积液中，VEGF的浓度则高达0.5-20ng/mL[5-7]。当肿瘤微环境中高浓度的VEGF二聚体结合VEGFR2后，诱导VEGFR2受体形成同源二聚体(主要)，亦可与VEGFR1或者VEGFR3形成异源二聚体(次要)，引起VEGFR2胞内区酪氨酸残基自磷酸化，活化下游磷脂酶C(PLC)等信号通路，促进肿瘤血管内皮细胞增殖和存活、介导肿瘤细胞迁移、改变血管通透性，最终引起肿瘤血管增生[2]。VEGFR2 (also known as FLK-1, or KDR), is the main member of the VEGFR family, is a type III receptor tyrosine kinase, and is mainly distributed on the surface of the endothelial cell membrane of blood vessels and lymphatic vessels. The extracellular domain of VEGFR2 contains 7 immunoglobulin-like domains (ie D1-D7), including the ligand VEGF binding domain (D2-D3) and dimerization domain (D4-D7), and its intracellular domain contains Tyrosine kinase domain [2-4]. The concentration of VEGF (including VEGF-A, C, D, E, etc.) in normal human plasma is about 137±7.7 pg/mL [5], and the concentration of VEGF in serum of patients with pancreatic cancer and ovarian cancer is significantly increased to 200- 400pg/mL, while the concentration of VEGF in tumor tissue or tumor cyst effusion is as high as 0.5-20ng/mL[5-7]. When the high concentration of VEGF dimer in the tumor microenvironment binds to VEGFR2, it induces VEGFR2 receptors to form homodimers (primary), and can also form heterodimers with VEGFR1 or VEGFR3 (secondary), causing VEGFR2 intracellular Autophosphorylation of tyrosine residues in the region, activate downstream phospholipase C (PLC) and other signaling pathways, promote tumor vascular endothelial cell proliferation and survival, mediate tumor cell migration, change vascular permeability, and ultimately cause tumor angiogenesis[2 ].
VEGFR2抗体类药物可阻断VEGFR2与VEGF结合，抑制血管 生成，降低血管通透性，在短时间内使肿瘤微环境重新获得平衡，从而获得良好的抑癌效果。礼来(Eli Lilly)公司研发的第一个抗VEGFR2肿瘤治疗药物雷莫芦单抗(CYRAMZA)[8]，已在化疗/放疗失败后的胃癌/胃食管连接处癌[9]、化疗失败后的非小细胞肺癌[10]、化疗/放疗联合Avastin(VEGF抗体)失败后的结直肠癌[11]和转移性乳腺癌[12]等多种肿瘤中显示出良好的治疗效果，特别是在胃癌的二线治疗中显示出比VEGF单抗药物Avastin更佳的疗效。此外，由于VEGF在正常生理代谢中的重要作用，尽管Avastin在多种疾病中耐受良好，临床研究中发现Avastin具有胃穿孔等严重副作用[13]，而靶向VEGFR-2可保留VEGF结合VEGFR1同源二聚体的功能，保留了人正常组织新生血管的部分功能，因此，预期靶向VEGFR-2导致的胃肠道穿孔等***性毒副作用更低，具有更好的安全性优势，而临床更需要兼顾安全和疗效的药物。VEGFR2 antibody drugs can block the binding of VEGFR2 and VEGF, inhibit angiogenesis, reduce vascular permeability, and restore the balance of the tumor microenvironment in a short period of time, thereby obtaining a good tumor suppressor effect. Eli Lilly, the first anti-VEGFR2 tumor treatment drug ramucirumab (CYRAMZA) [8], has been used for gastric cancer/gastroesophageal junction cancer after chemotherapy/radiotherapy failure [9], chemotherapy failure After non-small cell lung cancer [10], colorectal cancer [11] and metastatic breast cancer [12] after the failure of chemotherapy/radiotherapy combined with Avastin (VEGF antibody), it has shown good therapeutic effects, especially In the second-line treatment of gastric cancer, it shows better efficacy than the VEGF monoclonal antibody drug Avastin. In addition, due to the important role of VEGF in normal physiological metabolism, although Avastin is well tolerated in many diseases, clinical studies have found that Avastin has serious side effects such as gastric perforation [13], and targeting VEGFR-2 can retain VEGF binding to VEGFR1 The function of homodimer retains part of the function of human normal tissue neovascularization. Therefore, it is expected that the systemic toxic side effects such as gastrointestinal perforation caused by targeting VEGFR-2 are lower and have better safety advantages. There is a need for drugs with both safety and efficacy in clinical practice.
目前为止，雷莫芦单抗是唯一一款获FDA批准上市用于晚期胃癌、结肠癌及非小细胞肺癌的二线治疗的VEGFR2单克隆抗体药物，而且其尚未在国内批准上市，因此，国内急需研发更安全有效的针对VEGFR2靶点的创新型单克隆抗体药物以填补这一空白。本发明公开的抗体能与肿瘤细胞特异性结合，并同时阻断VEGF-A，VEGF-C和VEGF-D的作用，因而具有更佳的抑制血管生成作用。所述抗体还能激活NK细胞发生抗体依赖性细胞毒性(ADCC)反应，抑制肿瘤的生长和转移，可用于临床治疗黑色素瘤。So far, ramucirumab is the only VEGFR2 monoclonal antibody drug approved by the FDA for the second-line treatment of advanced gastric cancer, colon cancer and non-small cell lung cancer, and it has not yet been approved for marketing in China. There is an urgent need to develop safer and more effective innovative monoclonal antibody drugs against the VEGFR2 target to fill this gap. The antibody disclosed in the present invention can specifically bind to tumor cells and block the effects of VEGF-A, VEGF-C and VEGF-D at the same time, thus having a better effect of inhibiting angiogenesis. The antibody can also activate the antibody-dependent cytotoxicity (ADCC) reaction of NK cells, inhibit tumor growth and metastasis, and can be used for clinical treatment of melanoma.
发明内容Summary of the invention
在一个方面，本发明提供了一种分离的抗VEGFR2抗体或其抗原结合片段，其包含具有SEQ ID NO：13所示的氨基酸序列的重链CDR1域、具有SEQ ID NO：14/50所示的氨基酸序列的重链CDR2域和具有SEQ ID NO：15所示的氨基酸序列的重链CDR3域的重链可变区，和具有SEQ ID NO：10所示的氨基酸序列的轻链CDR1域、具有SEQ ID NO：11所示的氨基酸序列的轻链CDR2域和具有SEQ  ID NO：12所示的氨基酸序列的轻链CDR3域的轻链可变区。In one aspect, the present invention provides an isolated anti-VEGFR2 antibody or antigen-binding fragment thereof, which comprises a heavy chain CDR1 domain having the amino acid sequence shown in SEQ ID NO: 13, and having a heavy chain CDR1 domain as shown in SEQ ID NO: 14/50. The heavy chain CDR2 domain of the amino acid sequence and the heavy chain CDR3 domain of the heavy chain CDR3 domain with the amino acid sequence shown in SEQ ID NO: 15, and the light chain CDR1 domain with the amino acid sequence of SEQ ID NO: 10, The light chain CDR2 domain having the amino acid sequence shown in SEQ ID NO: 11 and the light chain CDR3 domain having the amino acid sequence shown in SEQ ID NO: 12 are light chain variable regions.
在一个实施方式中，所述抗VEGFR2抗体或其抗原结合片段包含具有如SEQ ID NO：8所示的氨基酸序列或与SEQ ID NO：8具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列的重链可变区和具有如SEQ ID NO：9所示的氨基酸序列或与SEQ ID NO：9具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列的轻链可变区。In one embodiment, the anti-VEGFR2 antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 8 or at least 90%, 92%, 95%, 98%, or at least 90% to SEQ ID NO: 8 The heavy chain variable region of the amino acid sequence with 99% sequence identity and the amino acid sequence shown in SEQ ID NO: 9 or at least 90%, 92%, 95%, 98% or 99% with SEQ ID NO: 9 The light chain variable region of the amino acid sequence of sequence identity.
在一个实施方式中，所述抗VEGFR2抗体或其抗原结合片段是人源化抗体或嵌合抗体。In one embodiment, the anti-VEGFR2 antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody.
在一个实施方式中，所述抗VEGFR2抗体或其抗原结合片段包含具有如SEQ ID NO：22所示的氨基酸序列或与SEQ ID NO：22具有至少85％、90％、95％或99％序列同一性的氨基酸序列的重链可变区和具有如SEQ ID NO：23所示的氨基酸序列或与SEQ ID NO：23具有至少85％、90％、95％或99％序列同一性的轻链可变区。In one embodiment, the anti-VEGFR2 antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 22 or at least 85%, 90%, 95% or 99% sequence with SEQ ID NO: 22 The heavy chain variable region of the identical amino acid sequence and the light chain having the amino acid sequence shown in SEQ ID NO: 23 or having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 23 Variable region.
在一个实施方式中，所述抗体进一步包含轻链恒定区和重链恒定区,优选地所述轻链恒定区为氨基酸序列为SEQ ID NO:25的kappa轻链恒定区的氨基酸序列或与SEQ ID NO:25具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述重链恒定区为与氨基酸序列为SEQ ID NO:24的IgG1重链恒定区的氨基酸序列或与SEQ ID NO:24具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。In one embodiment, the antibody further comprises a light chain constant region and a heavy chain constant region. Preferably, the light chain constant region is the amino acid sequence of the kappa light chain constant region of SEQ ID NO: 25 or the amino acid sequence of the kappa light chain constant region. ID NO: 25 has an amino acid sequence with at least 90%, 92%, 95%, 98% or 99% sequence identity, and/or the heavy chain constant region is an IgG1 heavy chain with the amino acid sequence of SEQ ID NO: 24 The amino acid sequence of the constant region or the amino acid sequence having at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 24.
在一个实施方式中，所述抗VEGFR2抗体或其抗原结合片段为IgG抗体，优选为IgG1抗体。In one embodiment, the anti-VEGFR2 antibody or antigen-binding fragment thereof is an IgG antibody, preferably an IgG1 antibody.
在一个实施方式中，所述抗VEGFR2抗体或其抗原结合片段为单克隆抗体。In one embodiment, the anti-VEGFR2 antibody or antigen-binding fragment thereof is a monoclonal antibody.
在一个实施方式中，所述抗VEGFR2抗体或其抗原结合片段与重组VEGFR2的结合亲和力K D为1-100pM，优选5-50pM,更优选10.6pM。 In one embodiment, the binding affinity K D of the anti-VEGFR2 antibody or antigen-binding fragment thereof to recombinant VEGFR2 is 1-100 pM, preferably 5-50 pM, more preferably 10.6 pM.
在一个实施方式中，所述抗原结合片段为Fv、Fab、Fab′、Fab′-SH、 F(ab′)2、Fd片段、Fd'片段、单链抗体分子或单域抗体；其中单链抗体分子优选为scFv、di-scFv、tri-scFv、双体抗体或scFab。In one embodiment, the antigen binding fragment is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecule or single-domain antibody; The antibody molecule is preferably scFv, di-scFv, tri-scFv, diabody or scFab.
在一个实施方式中，所述抗VEGFR2抗体或其抗原结合片段，其结合表位是VEGFR2的Y137/K142、R164/Y165、D257、S311/G312。In one embodiment, the binding epitope of the anti-VEGFR2 antibody or antigen-binding fragment thereof is Y137/K142, R164/Y165, D257, S311/G312 of VEGFR2.
在另一方面，本发明提供一种抗VEGFR2抗体或其抗原结合片段，其与本发明的抗VEGFR2抗体或其抗原结合片段结合抗原上的相同的表位。In another aspect, the present invention provides an anti-VEGFR2 antibody or antigen-binding fragment thereof, which binds to the same epitope on the antigen as the anti-VEGFR2 antibody or antigen-binding fragment thereof of the present invention.
在另一方面，本发明提供一种VEGFR2分子表位，其是VEGFR2的Y137/K142、R164/Y165、D257、S311/G312。In another aspect, the present invention provides a molecular epitope of VEGFR2, which is Y137/K142, R164/Y165, D257, S311/G312 of VEGFR2.
在另一方面，本发明提供一种抗体-药物缀合物，其包含如权利要求1-9任一项所述的抗VEGFR2抗体或其抗原结合片段和另外的治疗剂，优选地所述抗VEGFR2抗体或其抗原结合片段和另外的治疗剂通过接头连接。In another aspect, the present invention provides an antibody-drug conjugate comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-9 and another therapeutic agent, preferably the anti-VEGFR2 antibody The VEGFR2 antibody or antigen-binding fragment thereof and the other therapeutic agent are connected by a linker.
在另一方面，本发明提供一种核酸，其编码根据本发明的抗VEGFR2抗体或其抗原结合片段。In another aspect, the present invention provides a nucleic acid encoding the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention.
在一个实施方式中，所述核酸包含如SEQ ID NO：4所示的核苷酸序列和/或如SEQ ID NO：5所示的核苷酸序列，或者包含如SEQ ID NO：30所示的核苷酸序列和/或如SEQ ID NO：31所示的核苷酸序列。In one embodiment, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO: 4 and/or a nucleotide sequence as shown in SEQ ID NO: 5, or comprises a nucleotide sequence as shown in SEQ ID NO: 30 The nucleotide sequence of and/or the nucleotide sequence shown in SEQ ID NO: 31.
在另一方面，本发明提供一种表达载体，其包含根据本发明的核酸。In another aspect, the invention provides an expression vector comprising the nucleic acid according to the invention.
在另一方面，本发明提供一种宿主细胞，其包含根据本发明的核酸或表达载体。In another aspect, the present invention provides a host cell comprising the nucleic acid or expression vector according to the present invention.
在另一方面，本发明提供一种用于产生本发明的抗VEGFR2抗体或其抗原结合片段的方法，包括在适合于抗体表达的条件下培养所述的宿主细胞，和从培养基中回收表达的抗体。In another aspect, the present invention provides a method for producing the anti-VEGFR2 antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell under conditions suitable for antibody expression, and recovering the expression from the culture medium. Of antibodies.
在另一方面，本发明提供一种药物组合物，包含根据本发明的抗VEGFR2抗体或其抗原结合片段或根据本发明的抗体-药物缀合物或根据本发明的核酸或表达载体，及药学上可接受的载体。In another aspect, the present invention provides a pharmaceutical composition comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the nucleic acid or expression vector according to the present invention, and a pharmaceutical composition Acceptable carrier.
在另一方面，本发明涉及抗VEGFR2抗体或其抗原结合片段或抗体-药物缀合物或药物组合物，其用于治疗黑色素瘤。In another aspect, the present invention relates to an anti-VEGFR2 antibody or antigen-binding fragment thereof or antibody-drug conjugate or pharmaceutical composition for the treatment of melanoma.
在另一方面，本发明提供一种用于治疗黑色素瘤的方法，包括向需要的受试者施用治疗有效量的根据本发明的抗VEGFR2抗体或其抗原结合片段或根据本发明的抗体-药物缀合物或根据本发明的药物组合物，从而治疗所述黑色素瘤。In another aspect, the present invention provides a method for treating melanoma, comprising administering to a subject in need a therapeutically effective amount of the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug according to the present invention A conjugate or a pharmaceutical composition according to the present invention, thereby treating the melanoma.
在另一方面，本发明涉及根据本发明的抗VEGFR2抗体或其抗原结合片段或根据本发明的抗体-药物缀合物或根据本发明的药物组合物在制备用于治疗黑色素瘤的药物中的用途。In another aspect, the present invention relates to an anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or an antibody-drug conjugate according to the present invention or a pharmaceutical composition according to the present invention in the preparation of a medicament for the treatment of melanoma use.
在另一方面，本发明提供一种药物组合，其包含根据本发明的抗VEGFR2抗体或其抗原结合片段或根据本发明的抗体-药物缀合物或根据本发明的药物组合物及一种或多种另外的治疗剂。In another aspect, the present invention provides a pharmaceutical combination comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the pharmaceutical composition according to the present invention and one or A variety of additional therapeutic agents.
在另一方面，本发明提供一种试剂盒，其包含根据本发明的抗VEGFR2抗体或其抗原结合片段或根据本发明的抗体-药物缀合物或根据本发明的药物组合物,优选地,所述试剂盒进一步包含给药的装置。In another aspect, the present invention provides a kit comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the pharmaceutical composition according to the present invention, preferably, The kit further includes a device for administration.
附图说明Description of the drawings
本发明结合附图进行说明，附图中：The present invention is described in conjunction with the accompanying drawings, in which:
图1显示了通过ELISA检测的抗VEGFR2嵌合抗体与重组蛋白VEGFR2-His的结合。Figure 1 shows the binding of the anti-VEGFR2 chimeric antibody to the recombinant protein VEGFR2-His detected by ELISA.
图2显示了通过FACS检测的抗VEGFR2嵌合抗体在293FT-VEGFR2细胞株上的结合。Figure 2 shows the binding of anti-VEGFR2 chimeric antibody on 293FT-VEGFR2 cell line detected by FACS.
图3显示了通过ELISA检测的抗VEGFR2嵌合抗体阻断重组人VEGFR2-His与人VEGF165重组蛋白的结合。Figure 3 shows that the anti-VEGFR2 chimeric antibody detected by ELISA blocks the binding of recombinant human VEGFR2-His to human VEGF165 recombinant protein.
图4显示了抗VEGFR2嵌合抗体阻断VEGF165对HUVEC细胞的增殖作用。Figure 4 shows that the anti-VEGFR2 chimeric antibody blocks the proliferation effect of VEGF165 on HUVEC cells.
图5显示了通过ELSIA检测VEGFR2-HK19与重组人VEGFR2-His的结合。Figure 5 shows the detection of the binding of VEGFR2-HK19 to recombinant human VEGFR2-His by ELSIA.
图6显示了通过FACS检测的VEGFR2-HK19在293FT-VEGFR2细胞株上的结合。Figure 6 shows the binding of VEGFR2-HK19 on the 293FT-VEGFR2 cell line detected by FACS.
图7显示了通过ELISA检测的VEGFR2-HK19的种属交叉结合。Figure 7 shows the species cross-binding of VEGFR2-HK19 detected by ELISA.
图8显示了通过ELISA检测的VEGFR2-HK19阻断VEGF165与VEGFR2的结合。Figure 8 shows that VEGFR2-HK19 blocked the binding of VEGF165 to VEGFR2 detected by ELISA.
图9显示了通过FACS检测的VEGFR2-HK19阻断VEGF165与293FT-VEGFR2的结合。Figure 9 shows that VEGFR2-HK19 blocked the binding of VEGF165 to 293FT-VEGFR2 detected by FACS.
图10显示了通过ELISA检测的VEGFR2-HK19阻断VEGFR2-Fc重组蛋白与VEGF-C的结合。Figure 10 shows that VEGFR2-HK19 blocked the binding of VEGFR2-Fc recombinant protein to VEGF-C detected by ELISA.
图11显示了通过ELISA检测的VEGFR2-HK19阻断VEGFR2-Fc重组蛋白与VEGF-D的结合。Figure 11 shows that VEGFR2-HK19 blocked the binding of VEGFR2-Fc recombinant protein to VEGF-D detected by ELISA.
图12显示了VEGFR2-HK19阻断VEGF165对HUVEC细胞的增殖作用。Figure 12 shows that VEGFR2-HK19 blocks the proliferation effect of VEGF165 on HUVEC cells.
图13显示了VEGFR2-HK19阻断VEGF-C对HUVEC细胞的增殖作用。Figure 13 shows that VEGFR2-HK19 blocks the proliferation effect of VEGF-C on HUVEC cells.
图14显示了VEGFR2-HK19阻断VEGF-C+VEGF-D对HUVEC细胞的增殖作用。Figure 14 shows that VEGFR2-HK19 blocks the proliferation effect of VEGF-C+VEGF-D on HUVEC cells.
图15显示了VEGFR2-HK19阻断VEGF-A+VEGF-C+VEGF-D对HUVEC细胞的增殖作用。Figure 15 shows that VEGFR2-HK19 blocks the proliferation effect of VEGF-A+VEGF-C+VEGF-D on HUVEC cells.
图16显示了通过CD16A重组报告基因检测的VEGFR2-HK19的ADCC作用。Figure 16 shows the ADCC effect of VEGFR2-HK19 detected by the CD16A recombinant reporter gene.
图17显示了VEGFR2-HK19同源建模与VEGFR2的晶体结构对接。Figure 17 shows the docking of VEGFR2-HK19 homology modeling with the crystal structure of VEGFR2.
图18显示了VEGFR2-HK19和VEGF-A与VEGFR2结合的表位。Figure 18 shows the epitopes that VEGFR2-HK19 and VEGF-A bind to VEGFR2.
图19显示了VEGFR2-HK19对B16-F1黑色素瘤移植KDR人源化小鼠体重的影响。Figure 19 shows the effect of VEGFR2-HK19 on the body weight of B16-F1 melanoma transplanted KDR humanized mice.
图20显示了VEGFR2-HK19对B16-F1黑色素瘤移植KDR人源化小鼠肿瘤体积的影响。Figure 20 shows the effect of VEGFR2-HK19 on the tumor volume of B16-F1 melanoma transplanted KDR humanized mice.
图21显示了CD-1小鼠单次给予VEGFR2-HK19抗体的药物浓 度-时间曲线。Figure 21 shows the drug concentration-time curve of a single administration of VEGFR2-HK19 antibody to CD-1 mice.
具体实施方式Detailed ways
本发明的各个方面涉及分离的抗VEGFR2抗体或其抗原结合片段、包含该抗体或其抗原结合片段的抗体-药物缀合物、编码该抗体或其抗原结合片段的核酸和表达载体、包含该核酸或表达载体的宿主细胞、产生该抗VEGFR2抗体或其抗原结合片段的方法、包含该抗VEGFR2抗体或其抗原结合片段的药物组合物以及使用该抗VEGFR2抗体或其抗原结合片段治疗黑色素瘤的方法。Various aspects of the present invention relate to isolated anti-VEGFR2 antibodies or antigen-binding fragments thereof, antibody-drug conjugates comprising the antibodies or antigen-binding fragments thereof, nucleic acids and expression vectors encoding the antibodies or antigen-binding fragments thereof, and containing the nucleic acids Or a host cell expressing a vector, a method for producing the anti-VEGFR2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising the anti-VEGFR2 antibody or an antigen-binding fragment thereof, and a method for treating melanoma using the anti-VEGFR2 antibody or the antigen-binding fragment thereof .
定义definition
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为了本发明的目的，定义以下术语，以同本技术领域通常理解的含义保持一致。Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the technical field to which the present invention belongs. For the purpose of the present invention, the following terms are defined to be consistent with the meanings commonly understood in the technical field.
当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。When used herein and in the appended claims, the singular forms "a", "an", "another" and "the" include plural referents unless the context clearly dictates otherwise.
术语“抗体”意指免疫球蛋白分子，是指表现所需生物学活性的抗体的任何形式。包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体),甚至包括抗体片段。典型地，全长抗体结构优选包含4条多肽链，通常通过二硫键相互连接的2条重(H)链和2条轻(L)链。每条重链包含重链可变区和重链恒定区。每条轻链包含轻链可变区和轻链恒定区。在此典型全长抗体结构外,其结构还包括其他衍生形式。The term "antibody" means an immunoglobulin molecule, and refers to any form of antibody that exhibits the desired biological activity. Including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (such as bispecific antibodies), and even antibody fragments. Typically, the full-length antibody structure preferably contains 4 polypeptide chains, usually 2 heavy (H) chains and 2 light (L) chains connected to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, its structure also includes other derivative forms.
所述重链可变区和轻链可变区可进一步细分为更保守的区域(称为框架区(FR))和穿插其中的高变区(称为互补决定区(CDR))。The heavy chain variable region and light chain variable region can be further subdivided into more conservative regions (called framework regions (FR)) and hypervariable regions interspersed (called complementarity determining regions (CDR)).
术语“互补决定区”(CDR，例如CDR1、CDR2和CDR3)是指抗体可变区的这样一些氨基酸残基，其存在对于抗原结合来说是必需的。每个可变区通常具有3个被鉴别为CDR1、CDR2和CDR3的CDR区域。每个互补决定区可包含来自如Kabat所定义的“互补决定区”的氨基酸残基(Kabat等,Sequences of Proteins of Immulological  Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD.1991))和/或来自“高变环”的那些残基(Chothia和Lesk；J Mol Biol 196:901-917(1987))。The term "complementarity determining region" (CDR, such as CDR1, CDR2, and CDR3) refers to the amino acid residues of the variable region of an antibody, the presence of which is necessary for antigen binding. Each variable region usually has 3 CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity determining region can contain amino acid residues from the "complementarity determining region" defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. 1991 )) and/or those residues from the "hypervariable loop" (Chothia and Lesk; J Mol Biol 196:901-917 (1987)).
术语“构架”或“FR”残基是如本文中所定义的CDR残基之外的那些可变区残基。The term "framework" or "FR" residues are those variable region residues other than the CDR residues as defined herein.
每个重链可变区和轻链可变区通常包含3个CDR和最多达4个FR，所述CDR和FR从氨基末端至羧基末端以例如以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。Each heavy chain variable region and light chain variable region usually contains 3 CDRs and up to 4 FRs. The CDRs and FRs are arranged in the following order from the amino terminal to the carboxy terminal, for example: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
给定抗体的互补性决定区(CDR)和框架区(FR)可以使用Kabat体系标识(Kabat等:Sequences of Proteins of Immunological Interest，第5版，美国卫生和公众服务部，PHS，NIH，NIH出版编号91-3242，1991)。The complementarity determining region (CDR) and framework region (FR) of a given antibody can be identified using the Kabat system (Kabat et al.: Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, PHS, NIH, NIH publication No. 91-3242, 1991).
术语“恒定区”是指抗体的轻链和重链上的这样一些氨基酸序列，不直接参与抗体与抗原的结合，但展现出多种效应子功能，例如抗体依赖性细胞毒性。The term "constant region" refers to such amino acid sequences on the light chain and heavy chain of an antibody that do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as antibody-dependent cytotoxicity.
根据其恒定区的氨基酸序列的抗原性差异，抗体的重链可以被分为α、δ、ε、γ和μ五类，当其与轻链组成完整的抗体，可被分为五类：IgA、IgD、IgE、IgG和IgM，这些类中的若干还可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。基于其恒定结构域的氨基酸序列，抗体的轻链可归入κ和λ。According to the antigenic difference of the amino acid sequence of its constant region, the heavy chain of an antibody can be divided into five categories: α, δ, ε, γ, and μ. When it forms a complete antibody with the light chain, it can be divided into five categories: IgA , IgD, IgE, IgG and IgM, several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be classified into κ and λ.
“抗体的抗原结合片段”包含完整抗体分子的一部分，其保留母体抗体的至少某些结合特异性，通常包括至少部分母体抗体的抗原结合区或可变区(例如一个或多个CDR)。抗原结合片段的实例包括但不限于Fv、Fab、Fab′、Fab′-SH、F(ab′)2、Fd片段、Fd'片段、单链抗体分子(例如scFv，di-scFv或tri-scFv、双体抗体或scFab)、单域抗体。An "antigen-binding fragment of an antibody" includes a portion of a complete antibody molecule that retains at least some of the binding specificity of the parent antibody, and usually includes at least a portion of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecules (e.g., scFv, di-scFv or tri-scFv , Diabody or scFab), single domain antibody.
术语“抗体片段”是指保留母体抗体的至少某些生物学特性的非完整抗体分子，其实例除上述“抗原结合片段”所述及的那些之外，还包括但不限于Fc片段。The term "antibody fragment" refers to an incomplete antibody molecule that retains at least some biological properties of the parent antibody, and examples thereof include, but are not limited to, Fc fragments in addition to those mentioned in the above-mentioned "antigen-binding fragments".
术语“抗体-药物缀合物”或“ADC”是指与一种或多种化学药物化学连接的结合蛋白如抗体或其抗原结合片段，其可以任选地是治疗剂或细胞毒性剂(如一种或多种细胞因子或化疗药物)。在优选的实施方案中，ADC包括抗体、细胞毒性或治疗药物，以及能够使药物与抗体连接或缀合的接头。ADC通常具有与抗体缀合的1至8个中任一值的药物，包括2、4、6或8的载药物质。可以包含在ADC中的药物的非限制性实例是有丝***抑制剂、抗肿瘤抗生素、免疫调节剂、用于基因治疗载体、烷化剂、抗血管生成剂、抗代谢药、含硼药剂、化疗保护剂、激素、抗激素剂、皮质类固醇、光活性治疗剂、寡核苷酸、放射性核素剂、拓扑异构酶抑制剂、酪氨酸激酶抑制剂和放射致敏剂。The term "antibody-drug conjugate" or "ADC" refers to a binding protein chemically linked to one or more chemical drugs, such as an antibody or antigen-binding fragment thereof, which may optionally be a therapeutic agent or a cytotoxic agent (such as a One or more cytokines or chemotherapy drugs). In a preferred embodiment, the ADC includes an antibody, cytotoxic or therapeutic drug, and a linker that enables the drug to be linked or conjugated to the antibody. ADCs usually have any value of 1 to 8 drugs conjugated to antibodies, including 2, 4, 6, or 8 drug-loading substances. Non-limiting examples of drugs that can be included in the ADC are mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemotherapy protection Agents, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors and radiosensitizers.
术语“嵌合抗体”是指重链和/或轻链的一部分来源于特定来源或物种，而其余部分来源于不同来源或物种的抗体。“嵌合抗体”亦可以为如上定义的功能性的片段。“人源化抗体”是“嵌合抗体”的子集。The term "chimeric antibody" refers to an antibody in which a part of the heavy chain and/or light chain is derived from a specific source or species, and the remaining part is derived from a different source or species. The "chimeric antibody" may also be a functional fragment as defined above. "Humanized antibodies" are a subset of "chimeric antibodies."
术语“人源化抗体”或“人源化抗原结合片段”在本文中被定义为这样的抗体或抗体片段：(i)来源于非人来源(例如，携带异源免疫***的转基因小鼠)且基于人种系序列；或(ii)可变区是非人来源而恒定区是人来源的嵌合抗体；或者(iii)CDR移植的，其中可变区的CDR来自非人来源，而可变区的一个或多个构架区为人来源的，并且恒定区(如果有的话)是人来源的。“人源化”的目的是消除非人来源抗体在人体内的免疫原性,而同时最大可能地保留亲和力。选择与非人来源抗体构架序列最相似的人构架序列为模板进行人源化改造是有利的。在某些情况下，可能需要用非人构架中相应的残基替换人类构架序列中的一个或多个氨基酸，以避免亲和性的丧失。The term "humanized antibody" or "humanized antigen-binding fragment" is defined herein as an antibody or antibody fragment: (i) derived from a non-human source (for example, a transgenic mouse carrying a heterologous immune system) And based on human germline sequence; or (ii) the variable region is of non-human origin and the constant region is a chimeric antibody of human origin; or (iii) CDR grafted, wherein the CDR of the variable region is derived from a non-human source, and the variable One or more framework regions of the region are of human origin, and the constant region (if any) is of human origin. The purpose of "humanization" is to eliminate the immunogenicity of non-human source antibodies in the human body, while retaining the greatest possible affinity. It is advantageous to select a human framework sequence that is most similar to the framework sequence of a non-human source antibody as a template for humanization. In some cases, it may be necessary to replace one or more amino acids in the human framework sequence with corresponding residues in the non-human framework to avoid loss of affinity.
“单克隆抗体”是指获自基本上同质的抗体群体的抗体，即，所述包含单一抗体的群体除了可能以极少量存在的可能突变(例如天然突变)之外是相同的。因此，所述术语“单克隆”表明所述抗体的性质，即不是不相关抗体的混合物。与通常包括针对不同决定簇(表 位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂的每个单克隆抗体均针对抗原上的单独一个决定簇。除了其特异性之外，单克隆抗体制剂的优点在于它们通常不会被其他抗体污染。所述术语“单克隆”不应被理解为需要通过任何特定的方法产生所述抗体。A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the population comprising a single antibody is identical except for possible mutations (such as natural mutations) that may be present in very small amounts. Therefore, the term "monoclonal" indicates the nature of the antibody, that is, it is not a mixture of unrelated antibodies. In contrast to polyclonal antibody preparations which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibody preparations is that they are generally not contaminated by other antibodies. The term "monoclonal" should not be understood as requiring the production of the antibody by any specific method.
抗体“特异性结合”目的抗原例如肿瘤相关的多肽抗原靶(本文中，VEGFR2)，即以足够的亲和力结合所述抗原以使得所述抗体可用作治疗性试剂，靶向表达所述抗原的细胞或组织，并且与其他蛋白质无显著交叉反应或者与除了上文提到的抗原靶的同源体和变体(例如突变形式、剪接变体，或蛋白水解作用截短的形式)以外的蛋白质无显著交叉反应。The antibody "specifically binds" to an antigen of interest, such as a tumor-associated polypeptide antigen target (herein, VEGFR2), that is, binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent to target a target expressing the antigen Cells or tissues, and have no significant cross-reactivity with other proteins or with proteins other than homologs and variants (such as mutant forms, splice variants, or truncated forms of proteolysis) of the antigen target mentioned above No significant cross reaction.
术语“结合亲和力”是指分子的单个结合位点与其结合伴侣之间非共价相互作用总和的强度。除非另有说明，用于本文时“结合亲和力”是指固有的结合亲和力，其反映结合对(例如抗体和抗原)的成员之间1:1的相互作用。如本文所用，术语“KD”是指抗体-抗原相互作用的平衡解离常数。如本文所用，术语“kon”是指抗体与抗原结合的速率常数。如本文所用，术语“koff”是指抗体与抗体/抗原复合物解离的速率常数。“KD”、“结合速率常数k on”和“解离速率常数k off”通常用于描述分子(例如抗体)与其结合伴侣(例如抗原)之间的亲和力，即，配体结合特定蛋白的紧密程度。结合亲和力受非共价分子间相互作用的影响，例如氢键，静电相互作用，两个分子之间的疏水和范德华力。另外，配体与其靶分子之间的结合亲和力可能受到其他分子的存在的影响。亲和力可通过本领域中已知的常规方法来分析，包括本文描述的ELISA。 The term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise stated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). As used herein, the term "KD" refers to the equilibrium dissociation constant of the antibody-antigen interaction. As used herein, the term "kon" refers to the rate constant at which an antibody binds to an antigen. As used herein, the term "koff" refers to the rate constant at which the antibody dissociates from the antibody/antigen complex. "KD", "association rate constant k on "and "dissociation rate constant k off " are usually used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen), that is, how tightly a ligand binds to a specific protein. degree. Binding affinity is affected by interactions between non-covalent molecules, such as hydrogen bonds, electrostatic interactions, hydrophobicity and van der Waals forces between two molecules. In addition, the binding affinity between the ligand and its target molecule may be affected by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including the ELISA described herein.
术语“表位”包括能够特异性结合至抗体或T细胞受体的任何蛋白质决定簇。表位决定簇通常由分子的化学活性表面基团(例如氨基酸或糖侧链，或其组合)组成，并且通常具有特定三维结构特征以及特定的电荷特征。The term "epitope" includes any protein determinant capable of specifically binding to an antibody or T cell receptor. Epitope determinants usually consist of chemically active surface groups of molecules (for example, amino acids or sugar side chains, or combinations thereof), and usually have specific three-dimensional structural characteristics and specific charge characteristics.
术语“分离的”抗体是已经被鉴别并且从表达它的细胞的组分中分离的抗体。分离的抗体包括重组细胞内的原位抗体，所述抗体的 天然环境中的至少一种组分是不存在的。然而，通常情况下，分离的抗体是通过至少一个纯化步骤进行制备。The term "isolated" antibody is an antibody that has been identified and isolated from a component of the cell that expresses it. Isolated antibodies include antibodies in situ in recombinant cells where at least one component of the antibody's natural environment is absent. However, usually, the isolated antibody is prepared through at least one purification step.
两条多肽或核酸序列之间的“序列同一性”表示所述序列之间相同的残基的数目占残基总数的百分比，且基于比较的分子中较小者的大小来计算。在计算同一性百分数时，将正在比较的序列以产生序列之间最大匹配的方式比对，通过特定算法解决比对中的空位(如果存在的话)。确定两个序列之间同一性的优选计算机程序方法包括，但不限于，GCG程序包，包括GAP、BLASTP、BLASTN和FASTA(Altschul等人，1990，J.Mol.Biol.215：403-410)。上述程序可以公开地从国际生物技术信息中心(NCBI)和其他来源得到。熟知的Smith Waterman算法也可用于确定同一性。The "sequence identity" between two polypeptide or nucleic acid sequences means the number of identical residues between the sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller of the compared molecules. When calculating the percent identity, the sequences being compared are aligned in a way that produces the largest match between the sequences, and the gaps in the alignment (if any) are resolved by a specific algorithm. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410) . The above program can be publicly obtained from the International Center for Biotechnology Information (NCBI) and other sources. The well-known Smith Waterman algorithm can also be used to determine identity.
术语“Fc受体”或“FcR”指与抗体Fc区结合的受体。优选天然序列的人FcR，且优选与IgG抗体结合的受体(γ受体)，其包括FcγRI，FcγRII和FcγRIII亚型，以及这些受体的变体。其它FcR均被包含在术语“FcR”中。该术语也包括新生儿受体(FcRn)其负责将母体的IgG转运至胎儿(Guyer等，免疫学杂志117：587(1976)和Kim等，免疫学杂志24：249(1994))。The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Human FcR of natural sequence is preferred, and receptors (γ receptors) that bind to IgG antibodies are preferred, which include FcγRI, FcγRII and FcγRIII subtypes, and variants of these receptors. Other FcRs are included in the term "FcR". The term also includes the neonatal receptor (FcRn) which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunology 117:587 (1976) and Kim et al., J. Immunology 24:249 (1994)).
术语“新生儿Fc受体”、简称“FcRn”，其结合IgG抗体Fc区。新生儿Fc受体(FcRn)在体内IgG类抗体的代谢命运中起重要作用。FcRn行使功能以从溶酶体降解途径营救IgG，从而降低其在血清中的清除率并加长半衰期。因此，IgG体外FcRn结合性质/特征指示它在血液循环中的体内药代动力学性质。The term "neonatal Fc receptor", or "FcRn" for short, binds to the Fc region of an IgG antibody. The neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG antibodies in the body. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its clearance in serum and increasing its half-life. Therefore, the in vitro FcRn binding properties/characteristics of IgG indicate its in vivo pharmacokinetic properties in the blood circulation.
术语“效应子功能”指可归因于抗体的Fc区的那些生物学活性，其随抗体同种型而不同。抗体效应子功能的实例包括：C1q结合和依赖补体的细胞毒性(CDC)、Fc受体结合、依赖抗体的细胞毒性(ADCC)、依赖抗体的吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞对抗原的摄取、细胞表面受体(例如B细胞受体)的下调和B细胞激活。The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody, which differ by antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complexes Mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors), and B cell activation.
术语“效应细胞”指表达一种或多种FcR并行使效应子功能的白 细胞。在一个方面，所述效应细胞至少表达FcγRIII并执行ADCC效应子功能。介导ADCC的人白细胞的实例包括外周血单核细胞(PBMC)、自然杀伤(NK)细胞、单核细胞、细胞毒性T细胞和嗜中性粒细胞。效应细胞可以从天然来源，例如，血液中分离。效应细胞通常是与效应子阶段相关联的淋巴细胞，并发挥作用，以产生细胞因子(辅助T细胞)、杀死被病原体感染的细胞(细胞毒性T细胞)或分泌抗体(分化的B细胞)。The term "effector cells" refers to leukocytes that express one or more FcRs and perform effector functions. In one aspect, the effector cell at least expresses FcyRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, for example, blood. Effector cells are usually lymphocytes associated with the effector stage and function to produce cytokines (helper T cells), kill cells infected by pathogens (cytotoxic T cells) or secrete antibodies (differentiated B cells) .
"免疫细胞"包括具有造血的起源并在免疫应答中起作用的细胞。免疫细胞包括：淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。"Immune cells" include cells that have hematopoietic origin and play a role in immune responses. Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
“抗体依赖性细胞介导的细胞毒性”或“ADCC”是指一种细胞毒性形式，其中结合到在某些细胞毒性细胞(例如NK细胞、嗜中性粒细胞和巨噬细胞)上存在的Fcγ受体上的分泌Ig使得这些细胞毒性效应细胞能够特异性结合至承载抗原的靶细胞，随后使用例如细胞毒素杀死所述靶细胞。为了评估目的抗体的ADCC活性，可进行体外ADCC测定法，例如记载于美国专利No.5,500,362或5,821,337或美国专利No.6,737,056(Presta)中的体外ADCC测定法。用于这类测定法的有用效应细胞包括PBMC和NK细胞。"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity that binds to certain cytotoxic cells (such as NK cells, neutrophils, and macrophages) The secreted Ig on the Fcγ receptor enables these cytotoxic effector cells to specifically bind to the target cell carrying the antigen, and then kill the target cell using, for example, a cytotoxin. In order to evaluate the ADCC activity of the antibody of interest, an in vitro ADCC assay can be performed, for example, the in vitro ADCC assay described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Useful effector cells for such assays include PBMC and NK cells.
“补体依赖性细胞毒性”或“CDC”是指在补体的存在下靶细胞的裂解。典型的补体途径的活化是通过将补体***的第一组分(C1q)与结合至其相应抗原的(适当亚类的)抗体结合来起始。为了评估补体活化，可进行CDC测定法，例如记载于Gazzano-Santoro等,J.Immunol Methods 202:163(1996)中的CDC测定法。例如在美国专利No.6,194,551B1和WO1999/51642中描述了具有改变的Fc区氨基酸序列的多肽变体(具有变体Fc区的多肽)和具有增强或降低的C1q结合的多肽变体。"Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. The activation of the typical complement pathway is initiated by combining the first component of the complement system (C1q) with an antibody (of the appropriate subclass) that binds to its corresponding antigen. In order to evaluate complement activation, a CDC assay can be performed, such as the CDC assay described in Gazzano-Santoro et al., J. Immunol Methods 202:163 (1996). For example, in U.S. Patent No. 6,194,551B1 and WO1999/51642, polypeptide variants with an altered Fc region amino acid sequence (polypeptides with a variant Fc region) and polypeptide variants with enhanced or reduced C1q binding are described.
本发明的抗体的氨基酸序列和核苷酸序列Amino acid sequence and nucleotide sequence of the antibody of the present invention
本发明首先采用重组人VEGFR2蛋白来免疫小鼠，然后通过噬 菌体展示文库筛选获得与重组人VEGFR2蛋白特异性结合的高结合力scFv抗体克隆VEGFR2-MK19。之后采用PCR方法将编码VEGFR2-MK19 scFv抗体的重链和轻链可变区的核苷酸序列分别***带人IgG1恒定区或人kappa恒定区核苷酸序列的pSTEP2载体中，进行培养表达。采用蛋白A纯化柱进行纯化获得高纯度人鼠嵌合抗体。ELISA测试表明，该抗VEGFR2嵌合抗体能够很好地阻断VEGFR2与其配体的结合及对HUVEC细胞的增殖作用。The present invention firstly uses recombinant human VEGFR2 protein to immunize mice, and then obtains the high-binding scFv antibody clone VEGFR2-MK19 which specifically binds to recombinant human VEGFR2 protein through phage display library screening. Then, the nucleotide sequences encoding the heavy chain and light chain variable regions of the VEGFR2-MK19 scFv antibody were inserted into the pSTEP2 vector with the human IgG1 constant region or the human kappa constant region nucleotide sequence, respectively, for culture expression. Purification by protein A purification column to obtain high-purity human-mouse chimeric antibody. The ELISA test shows that the anti-VEGFR2 chimeric antibody can block the binding of VEGFR2 to its ligand and the proliferation of HUVEC cells.
然后，采用经典的人源化方式CDR移植方法，选择与鼠轻链或重链可变区最接近的人抗体轻链或重链可变区为模板，将鼠抗体轻链/重链的各3个CDR分别***到上述人源模板的相应位置，获得人源化的轻链可变区(VL)和重链可变区(VH)序列。由于鼠源框架区的关键位点对于支撑CDR的活性至关重要，因此将关键点回复突变为鼠抗体的序列。分别将轻链/重链信号肽序列、回复突变的人源化抗体轻链/重链的可变区序列、人IgG1重链恒定区/人kappa轻链恒定区序列依次拼接，获得人源化抗体PD1-H944的氨基酸序列和核苷酸序列。Then, using the classical humanization method of CDR transplantation, the light chain or heavy chain variable region of the human antibody that is closest to the mouse light chain or heavy chain variable region is selected as the template, and each of the mouse antibody light chain/heavy chain The three CDRs were inserted into the corresponding positions of the above-mentioned human template to obtain humanized light chain variable region (VL) and heavy chain variable region (VH) sequences. Since the key points of the mouse-derived framework region are essential to support the activity of the CDR, the key points were backmutated to the sequence of the mouse antibody. The light chain/heavy chain signal peptide sequence, the back-mutated humanized antibody light chain/heavy chain variable region sequence, and the human IgG1 heavy chain constant region/human kappa light chain constant region sequence were spliced in sequence to obtain the humanization The amino acid sequence and nucleotide sequence of antibody PD1-H944.
本发明的核酸Nucleic acid of the invention
本发明还涉及编码本发明的抗体或其部分的核酸分子。这些核酸分子的序列包括但不限于SEQ ID NO:3-7、26-33和36-39。The invention also relates to nucleic acid molecules encoding the antibodies of the invention or parts thereof. The sequences of these nucleic acid molecules include but are not limited to SEQ ID NO: 3-7, 26-33, and 36-39.
本发明的核酸分子不限于本文公开的序列，还包括其变体。本发明中变体可以参照它们在杂交中的物理特性来描述。本领域技术人员会认识到利用核酸杂交技术，核酸可用于鉴别其互补物以及其等同物或同系物。还会认识到杂交可以以低于100％互补性发生。然而，考虑到条件的适当选择，杂交技术可用于基于DNA序列与特定探针的结构相关性来区分所述DNA序列。对于这类条件的指导参见Sambrook等,Molecular Cloning:A Laboratory Manual,2nd Ed.；Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989和Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Sedman,J.G.,Smith,J.A.,&Struhl,K.eds.(1995).Current Protocols in Molecular  Biology.New York:John Wiley and Sons。The nucleic acid molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof. The variants of the present invention can be described with reference to their physical characteristics in hybridization. Those skilled in the art will recognize that using nucleic acid hybridization techniques, nucleic acids can be used to identify their complements and their equivalents or homologs. It will also be recognized that hybridization can occur with less than 100% complementarity. However, considering the proper selection of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural correlation with specific probes. For guidance on such conditions, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons.
重组载体和表达Recombinant vector and expression
本发明还提供了包含本发明的一个或多个核苷酸序列的重组构建体。通过将编码本发明的抗体的核酸分子***载体例如质粒、噬粒、噬菌体或病毒载体中构建本发明的重组构建体。The invention also provides a recombinant construct comprising one or more nucleotide sequences of the invention. The recombinant construct of the present invention is constructed by inserting a nucleic acid molecule encoding the antibody of the present invention into a vector, such as a plasmid, phagemid, phage, or viral vector.
本发明的抗体可通过在宿主细胞中重组表达编码轻链和重链或其部分的核苷酸序列来制备。为了以重组方法表达抗体，可用携带编码轻链和/或重链或其部分的核苷酸序列的一个或多个重组表达载体转染宿主细胞，以使得所述轻链和重链在所述宿主细胞中表达。标准重组DNA方法学被用于制备和/或获得编码重链和轻链的核酸、将这些核酸纳入重组表达载体中并且将所述载体引入至宿主细胞中，例如Sambrook,Fritsch and Maniatis(eds.),Molecular Cloning；A Laboratory Manual,Second Edition,Cold Spring Harbor,N.Y.,(1989)、Ausubel,F.M.等(eds.)Current Protocols in Molecular Biology,Greene Publishing Associates,(1989)和Boss等的美国专利No.4,816,397中记载的那些。The antibody of the present invention can be prepared by recombinantly expressing nucleotide sequences encoding the light chain and the heavy chain or parts thereof in a host cell. In order to express the antibody in a recombinant method, one or more recombinant expression vectors carrying the nucleotide sequence encoding the light chain and/or heavy chain or part thereof can be used to transfect the host cell so that the light chain and the heavy chain are in the Expressed in host cells. Standard recombinant DNA methodology is used to prepare and/or obtain nucleic acids encoding heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as Sambrook, Fritsch and Maniatis (eds. ), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel, FM, etc. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and Boss, etc. U.S. Patent Nos . Those recorded in 4,816,397.
合适的宿主细胞为原核细胞和真核细胞。原核宿主细胞的实例为细菌，真核宿主细胞的实例为酵母、昆虫或哺乳动物细胞。应理解，包括选择调节序列的表达载体的设计受到多种因素的影响，例如宿主细胞的选择、所需的蛋白质的表达水平以及表达是组成型的还是可诱导型的。Suitable host cells are prokaryotic cells and eukaryotic cells. Examples of prokaryotic host cells are bacteria, and examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of the expression vector including the selection regulatory sequence is affected by many factors, such as the choice of host cell, the desired expression level of the protein, and whether the expression is constitutive or inducible.
细菌表达Bacterial expression
通过将编码所需抗体的结构DNA序列连同合适的翻译起始和终止信号以及有功能的启动子***可操作阅读框中，来构建可用于细菌的可用表达载体。所述载体会包含一个或多个表型选择标记以及复制起点以确保维持所述载体，以及根据需要在宿主内提供扩增。用于转化的合适的原核宿主包括大肠杆菌(E.coli)、枯草芽孢杆菌(Bacillus subtilis)、鼠伤寒沙门氏菌(Salmonella typhimurium)以及假单胞菌属(Pseudomonas)、链霉菌属(Streptomyces)和葡萄 球菌属(Staphylococcus)中的多个物种。By inserting the structural DNA sequence encoding the desired antibody together with suitable translation initiation and termination signals and a functional promoter into an operable reading frame, a usable expression vector for bacteria can be constructed. The vector will contain one or more phenotypic selectable markers and an origin of replication to ensure the maintenance of the vector and provide amplification in the host as needed. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas, Streptomyces, and grapes. Multiple species in the genus Staphylococcus.
细菌载体可以是例如基于噬菌体、质粒或噬粒的。这些载体可含有选择标记和细菌复制起点，其来源于通常含有公知的克隆载体pBR322(ATCC 37017)的元件的可商购的质粒。转化合适的宿主菌株并使所述宿主菌株生长至适当细胞密度之后，通过适当的方法(例如，温度变化或化学诱导)将所选择的启动子去阻遏/诱导，并且将细胞培养额外的时间。通常通过离心收获细胞，通过物理或化学方法使细胞破裂，并且保留所得的粗提取物用于进一步纯化。Bacterial vectors can be, for example, phage, plasmid or phagemid based. These vectors may contain a selection marker and a bacterial origin of replication, which are derived from commercially available plasmids that usually contain elements of the well-known cloning vector pBR322 (ATCC 37017). After transforming an appropriate host strain and growing the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by an appropriate method (for example, temperature change or chemical induction), and the cells are cultured for an additional time. The cells are usually harvested by centrifugation, broken down by physical or chemical methods, and the resulting crude extract is retained for further purification.
在细菌***中，根据所表达的蛋白的目的用途，可有利地选择多种表达载体。例如，当要生产大量这样的蛋白用于生产抗体或用于筛选肽文库时，例如，可能需要指导易于纯化的融合蛋白产物的高水平表达的载体。In a bacterial system, a variety of expression vectors can be advantageously selected according to the intended use of the expressed protein. For example, when a large number of such proteins are to be produced for antibody production or for screening peptide libraries, for example, a vector that directs high-level expression of a fusion protein product that is easy to purify may be required.
哺乳动物表达和纯化Mammalian expression and purification
用于哺乳动物宿主细胞表达的优选调节序列包括在哺乳动物细胞中指导高水平蛋白表达的病毒元件，例如源于巨细胞病毒(CMV)的启动子和/或增强子(例如CMV启动子/增强子)、猿猴病毒40(SV40)的启动子和/或增强子(例如SV40启动子/增强子)、腺病毒的启动子和/或增强子(例如腺病毒主要晚期启动子(AdMLP))和多瘤病毒的启动子和/或增强子。对病毒调节元件及其序列的进一步描述参见例如，Stinski的U.S.5,168,062、Bell等的U.S.4,510,245和Schaffner等的U.S.4,968,615。重组表达载体还可以包括复制起点和选择标记(参见例如，Axel等的U.S.4,399,216、U.S.4,634,665和U.S.5,179,017)。合适的选择标记包括赋予已经引入所述载体的宿主细胞对药物例如G418、潮霉素或甲氨蝶呤的抗性的基因。例如，二氢叶酸还原酶(DHFR)基因赋予对甲氨蝶呤的抗性，而neo基因赋予对G418的抗性。Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (e.g. CMV promoter/enhancer) Promoter), simian virus 40 (SV40) promoter and/or enhancer (e.g. SV40 promoter/enhancer), adenovirus promoter and/or enhancer (e.g. adenovirus major late promoter (AdMLP)) and Polyoma virus promoter and/or enhancer. For a further description of viral regulatory elements and their sequences, see, for example, U.S. 5,168,062 to Stinski, U.S. 4,510,245 to Bell et al., and U.S. 4,968,615 to Schaffner et al. The recombinant expression vector may also include an origin of replication and a selection marker (see, for example, U.S. 4,399,216, U.S. 4,634,665 and U.S. 5,179,017 of Axel et al.). Suitable selection markers include genes that confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, while the neo gene confers resistance to G418.
将所述表达载体至宿主细胞中的转染可以利用标准技术例如电穿孔、磷酸钙沉淀和DEAE-葡聚糖转染来进行。Transfection of the expression vector into host cells can be performed using standard techniques such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection.
用于表达本文提供的抗体的合适的哺乳动物宿主细胞包括中国 仓鼠卵巢(CHO细胞)[包括dhfr-CHO细胞，记载于Urlaub和Chasin,(1980)Proc.Natl.Acad.Sci.USA 77:4216-4220中，使用DHFR选择标记，例如记载于R.J.Kaufman和P.A.Sharp(1982)Mol.Biol.159:601-621中]、NSO骨髓瘤细胞、COS细胞和SP2细胞。Suitable mammalian host cells for expressing the antibodies provided herein include Chinese Hamster Ovary (CHO cells) [including dhfr-CHO cells, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216 In -4220, DHFR selection markers are used, such as those described in RJ Kaufman and PASharp (1982) Mol. Biol. 159:601-621], NSO myeloma cells, COS cells, and SP2 cells.
本发明的抗体可通过公知方法从重组细胞培养物回收和纯化，所述公知方法包括但不限于，硫酸铵或乙醇沉淀、酸提取、蛋白A亲和层析、蛋白G亲和层析、阴离子或阳离子交换色谱法、磷酸纤维素色谱法、疏水相互作用色谱法、亲和色谱法、羟磷灰石色谱法以及凝集素色谱法。高效液相色谱法(“HPLC”)也可用于纯化。参见例如，Colligan,Current Protocols in Immunology或Current Protocols in Protein Science,John Wiley&Sons,NY,N.Y.,(1997-2001)，例如第1、4、6、8、9、10章，各自以引用的方式全文纳入本文。The antibody of the present invention can be recovered and purified from recombinant cell culture by known methods, including but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion Or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley&Sons, NY, NY, (1997-2001), such as Chapters 1, 4, 6, 8, 9, and 10, each of which is fully cited Include this article.
本发明的抗体的特性和功能Characteristics and functions of the antibody of the present invention
对本发明的人源化VEGFR2-HK19抗体进行特性分析和功能分析。分析结果表明，本发明的抗体具备以下优势：(1)能够与VEGFR2抗原高亲和力特异性结合，具有低解离速率，从而提供良好的抗肿瘤药效；(2)与雷莫芦单抗相比，对于VEGFR2具有更高的结合亲和力；(实施例4)(3)VEGFR2-HK19阻断VEGF165蛋白结合VEGFR2的能力与雷莫芦单抗相近；(实施例4.2.1)(4)VEGFR2-HK19阻断VEGFR2-Fc重组蛋白与VEGF-C或VEGF-D结合的能力比雷莫芦单抗强；(实施例4.2.3)(5)VEGFR2-HK19阻断VEGF165、VEGF-C、VEGF-C+VEGF-D或VEGF-A+VEGF-C+VEGF-D对HUVEC细胞增殖作用的EC50均小于雷莫芦单抗；(实施例4.3.1和4.3.2)(6)VEGFR2-HK19具有比雷莫芦单抗更强的ADCC作用；(实施例4.4)和(7)在人源化KDR小鼠B16-F1黑色素癌皮下移植瘤模型中对B16-F1黑色素癌皮下移植瘤有明显的抑制作用。(实施例5)Characterization and function analysis of the humanized VEGFR2-HK19 antibody of the present invention are performed. The analysis results show that the antibody of the present invention has the following advantages: (1) It can specifically bind to VEGFR2 antigen with high affinity and has a low dissociation rate, thereby providing good antitumor efficacy; (2) Comparing with ramucirumab It has a higher binding affinity for VEGFR2; (Example 4) (3) VEGFR2-HK19 has a similar ability to block VEGF165 protein from binding to VEGFR2; (Example 4.2.1) (4) VEGFR2- HK19 has a stronger ability to block the binding of VEGFR2-Fc recombinant protein to VEGF-C or VEGF-D than ramucirumab; (Example 4.2.3) (5) VEGFR2-HK19 blocks VEGF165, VEGF-C, and VEGF- The EC50 of C+VEGF-D or VEGF-A+VEGF-C+VEGF-D on HUVEC cell proliferation is less than that of ramucirumab; (Examples 4.3.1 and 4.3.2) (6) VEGFR2-HK19 has A stronger ADCC effect than ramucirumab; (Example 4.4) and (7) in the humanized KDR mouse B16-F1 melanoma subcutaneous xenograft model, there are obvious effects on the B16-F1 melanoma subcutaneous xenograft tumor Inhibition. (Example 5)
用途use
本发明的抗体可用于治疗黑色素瘤。本发明的抗体还可用于制备治疗所述病症的药物。The antibodies of the present invention can be used to treat melanoma. The antibodies of the present invention can also be used to prepare drugs for treating the diseases.
药物组合物Pharmaceutical composition
可将本发明的抗体与至少一种其他试剂(例如稳定化合物)制备成药物组合物，其包括本发明的抗体和一种或多种药学上可接受的载体、稀释剂或赋形剂。任选地，所述药物组合物可包含另外的治疗剂。The antibody of the present invention and at least one other agent (such as a stabilizing compound) can be prepared into a pharmaceutical composition, which includes the antibody of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients. Optionally, the pharmaceutical composition may include additional therapeutic agents.
试剂盒Reagent test kit
本发明还涉及药物包装和包含一个或多个容器的试剂盒，所述容器含有上文提到的本发明的药物组合物。其上附有管理药物或生物制品的生产、使用或销售的政府机构规定形式提示，其反映该药物被上述机构批准用于人类给药。The invention also relates to a pharmaceutical package and a kit comprising one or more containers containing the aforementioned pharmaceutical composition of the invention. It is accompanied by a reminder of the form prescribed by the government agency that regulates the production, use or sale of drugs or biological products, which reflects that the drug has been approved by the above-mentioned agencies for human administration.
制备和储存Preparation and storage
本发明的药物组合物可以以本领域中已知的方式制备，例如通过常规的混合、溶解、造粒、锭剂制备、研磨、乳化、包裹、包埋或冻干方法。The pharmaceutical composition of the present invention can be prepared in a manner known in the art, for example, by conventional mixing, dissolving, granulating, tablet preparation, grinding, emulsifying, coating, embedding or freeze-drying methods.
在已经制备包含配制于可接受的载体中的本发明化合物的药物组合物之后，可以将它们放置在适当的容器中并贴上标签用于治疗所标明的病症。这类标签会包括给药的量、频率和方法。After the pharmaceutical composition containing the compound of the present invention formulated in an acceptable carrier has been prepared, they can be placed in an appropriate container and labeled for the treatment of the indicated condition. Such labels would include the amount, frequency, and method of administration.
药物组合Drug combination
上述包含本发明的抗体的药物组合物还与一种或多种其他治疗剂，例如抗肿瘤剂组合，其中所得组合不会引起不可接受的不利影响。The above-mentioned pharmaceutical composition containing the antibody of the present invention is also combined with one or more other therapeutic agents, such as anti-tumor agents, wherein the resulting combination does not cause unacceptable adverse effects.
以下的实施例便于更好地理解本发明，但并不限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂公司购买。The following examples facilitate a better understanding of the present invention, but do not limit the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies.
实施例Example
实施例1：筛选特异阻断VEGFR2与VEGF结合的鼠源抗体Example 1: Screening of murine antibodies that specifically block the binding of VEGFR2 to VEGF
1.1 小鼠免疫1.1 Mouse immunity
采用重组人VEGFR2蛋白(北京义翘神州科技有限公司，UniProtKB-P35968/NP_002244.1)免疫Balb/c小鼠(北京维通利华实验动物技术有限公司)。该重组人VEGFR2蛋白的胞外区Met1-Glu764氨基酸序列为SEQ ID NO:1。Recombinant human VEGFR2 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., UniProtKB-P35968/NP_002244.1) was used to immunize Balb/c mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). The amino acid sequence of the extracellular region Met1-Glu764 of the recombinant human VEGFR2 protein is SEQ ID NO:1.
具体方法：将重组人VEGFR2蛋白与弗氏完全佐剂混合，使用混合物进行三次皮下注射免疫，每次免疫的蛋白剂量为50μg，免疫间隔依次为2周，3周。从第三次免疫起，免疫后七天经眼眶内眦静脉丛采血。采用ELISA方法，包被重组人VEGFR2蛋白以检测小鼠抗VEGFR2的血清效价。第三次免疫血清8000倍稀释后滴度达标(ELISA OD>1.0)后，间隔84天，使用25μg重组人VEGFR2蛋白进行静脉注射加强，4天之后处死小鼠，取小鼠的脾脏组织冻存于液氮中。Specific method: Mix the recombinant human VEGFR2 protein with Freund's complete adjuvant, and use the mixture for three subcutaneous injections of immunization. The protein dose for each immunization is 50 μg, and the immunization interval is 2 weeks and 3 weeks. From the third immunization, blood was collected from the orbital intracanthal venous plexus seven days after immunization. The ELISA method was used to coat recombinant human VEGFR2 protein to detect the serum titer of mouse anti-VEGFR2. After the third immunized serum was diluted 8000 times and the titer reached the target (ELISA OD>1.0), 25μg of recombinant human VEGFR2 protein was used for intravenous injection at an interval of 84 days. After 4 days, the mice were sacrificed and the spleen tissues of the mice were frozen and stored In liquid nitrogen.
1.2 噬菌体抗体展示文库构建1.2 Construction of phage antibody display library
小鼠脾组织用TriPureHisolationReagent试剂(Roche，Cat.No.11667165001)提取RNA，利用反转录试剂将RNA反转录获得cDNA文库，利用重叠延伸拼接PCR法[14-15]，采用参考文献中2对引物扩增鼠抗体的轻链可变区，用另1对引物扩增重链可变区序列，将编码鼠抗体轻链和重链可变区序列的核苷酸序列拼接成编码抗体scFv的核苷酸序列，轻重链可变区通过接头接头(SEQ ID NO:2)连接，产生的DNA片段经限制性内切酶Sfi I酶切后连接到噬菌体载体pComb3x(北京义翘神州科技有限公司)中，电转X-Blue感受态获得小鼠的噬菌体展示scFv抗体库。Mouse spleen tissue was extracted with TriPureHisolationReagent reagent (Roche, Cat.No.11667165001), and RNA was reverse-transcribed with reverse transcription reagent to obtain cDNA library, using overlap extension splicing PCR method [14-15], using reference 2 Use the primers to amplify the variable region of the mouse antibody light chain, and use another pair of primers to amplify the sequence of the variable region of the heavy chain. The nucleotide sequences encoding the light chain and heavy chain variable regions of the mouse antibody are spliced into scFv encoding the antibody The light and heavy chain variable regions are connected by a linker (SEQ ID NO: 2), and the resulting DNA fragment is digested with the restriction enzyme Sfi I and connected to the phage vector pComb3x (Beijing Yiqiao Shenzhou Technology Co., Ltd. Company), electrotransformed X-Blue competent to obtain mouse phage display scFv antibody library.
1.3 VEGFR2单克隆抗体筛选1.3 Screening of VEGFR2 monoclonal antibodies
参考噬菌体抗体淘选方法及流程[16]，将重组人VEGFR2的第2到第3结构域蛋白VEGFR2-结构域2&3(北京义翘神州科技有限公司，10012-H08H3，NP_002244.1，Asp120-Lys327))包被在ELISA板上，加入抗体噬菌体孵育，洗去未结合的噬菌体，回收结合的噬菌体，如此重复，经多轮筛选富集获得抗VEGFR2阳性抗体噬菌体 文库。从富集的文库中挑取单克隆噬菌体进行表达，用ELISA方法检测与重组人VEGFR2蛋白的结合，从多株单克隆中筛选获得与重组人VEGFR2特异性结合的scFv抗体，单克隆通过测序获得抗体的核苷酸序列，其中编码单克隆scFv抗体VEGFR2-MK19的核苷酸序列为SEQ ID NO:3)。Refer to the phage antibody panning method and process [16], the second to third domain protein VEGFR2-domain 2&3 of recombinant human VEGFR2 (Beijing Yiqiao Shenzhou Technology Co., Ltd., 10012-H08H3, NP_002244.1, Asp120-Lys327 )) Coating on an ELISA plate, adding antibody phage to incubate, washing out unbound phage, recovering bound phage, and repeating this procedure. After multiple rounds of screening and enrichment, an anti-VEGFR2 positive antibody phage library is obtained. Monoclonal phages were selected from the enriched library for expression, and the binding to recombinant human VEGFR2 protein was detected by ELISA method. ScFv antibodies that specifically bind to recombinant human VEGFR2 were screened from multiple monoclonals, and the monoclonals were obtained by sequencing The nucleotide sequence of the antibody, wherein the nucleotide sequence encoding the monoclonal scFv antibody VEGFR2-MK19 is SEQ ID NO: 3).
1.4 VEGFR2单克隆嵌合抗体的生产1.4 Production of VEGFR2 monoclonal chimeric antibody
基于1.3中筛选得到的与重组人VEGFR2特异性结合的scFv抗体生产VEGFR2单克隆嵌合抗体。以VEGFR2-MK19scFv为例，详述生产过程。通过PCR扩增VEGFR2-MK19 scFv抗体的重链核苷酸序列，通过in-fusion方法***到带重链信号肽(SEQ ID NO:28)和人IgG1恒定区(SEQ ID NO:6)的经过ScaI/NheI(Fermentas)双酶切的pSTEP2载体中，获得人鼠嵌合重链(SEQ ID NO:36)表达载体。同理，PCR扩增VEGFR2-MK19 scFv抗体的轻链核苷酸序列，通过in-fusion方法***到带轻链信号肽(SEQ ID NO:29)和人kappa恒定区(SEQ ID NO:7)的经过ScaI/BsiWI(Fermentas)酶切的pSTEP2载体中获得人鼠嵌合轻链(SEQ ID NO:37)表达载体。Based on the scFv antibody specifically binding to recombinant human VEGFR2 screened in 1.3, a VEGFR2 monoclonal chimeric antibody was produced. Take VEGFR2-MK19scFv as an example to describe the production process. The heavy chain nucleotide sequence of the VEGFR2-MK19 scFv antibody was amplified by PCR and inserted into the heavy chain signal peptide (SEQ ID NO: 28) and human IgG1 constant region (SEQ ID NO: 6) by in-fusion method In the pSTEP2 vector digested with ScaI/NheI (Fermentas), a human-mouse chimeric heavy chain (SEQ ID NO: 36) expression vector was obtained. Similarly, the light chain nucleotide sequence of the VEGFR2-MK19 scFv antibody was amplified by PCR and inserted into the light chain signal peptide (SEQ ID NO: 29) and human kappa constant region (SEQ ID NO: 7) by in-fusion method The human-mouse chimeric light chain (SEQ ID NO: 37) expression vector was obtained from the pSTEP2 vector digested with ScaI/BsiWI (Fermentas).
扩增重链可变区引物：Amplification of heavy chain variable region primers:
F1F1 GCTACCAGGGTGCTGAGTGAAGTGAAGCTGGTGGAGGCTACCAGGGTGCTGAGTGAAGTGAAGCTGGTGGAG
R1R1 TGGGCCCTTGGTGCTAGCTGCAGAGACAGTGACCAGTGGGCCCTTGGTGCTAGCTGCAGAGACAGTGACCAG
扩增轻链可变区引物：Amplification of the light chain variable region primers:
F2F2 GCCACAGGAGTGCATAGTGACATCAAAATGACTCAGGCCACAGGAGTGCATAGTGACATCAAAATGACTCAG
R2R2 TGGTGCAGCCACCGTACGTTTGATTTCCAGCTTGGTTGGTGCAGCCACCGTACGTTTGATTTCCAGCTTGGT
293E细胞用SCD4-4-TC2培养基(北京义翘神州科技有限公司)传代至200mL/瓶，起始接种密度在0.3～0.4*10 6个细胞/mL，放入温度为37℃转速为175rpm的CO 2摇床中培养，待细胞密度达到1.5～3*10 6个细胞/mL后，按1:1混合轻重链质粒DNA，分别向培养 瓶中加入100μg混合后的质粒DNA和800μL的TF2转染试剂，放入温度为37℃转速为175rpm的摇床中继续培养至第7天收料。培养液4000rpm离心25min，收集上清，加入1/5上清液体积的stock缓冲液。用PBS将蛋白A层析柱平衡5-10倍柱体积，将过滤后的培养上清加入层析柱，再次平衡5-10倍柱体积后，用醋酸钠缓冲液洗脱样品。样品洗脱后用Tris中和至中性，获得高纯度嵌合抗体备用。 293E cells were passaged with SCD4-4-TC2 medium (Beijing Yiqiao Shenzhou Technology Co., Ltd.) to 200mL/bottle, the initial seeding density was 0.3～0.4*10 6 cells/mL, and the temperature was 37℃ and the rotation speed was 175rpm CO 2 after culture shaker, until the cell density reached 1.5 to 3 * 10 6 cells / mL, 1: 1 mixture of plasmid DNA after the light and heavy chain plasmid DNA, 100 ug were mixed and added to 800μL of culture flasks TF2 The transfection reagent was placed in a shaker with a temperature of 37°C and a rotating speed of 175 rpm and continued to be cultured until the 7th day of material collection. The culture solution was centrifuged at 4000 rpm for 25 minutes, the supernatant was collected, and 1/5 of the volume of the supernatant was added to stock buffer. Equilibrate the protein A chromatography column with PBS for 5-10 times the column volume, add the filtered culture supernatant to the chromatography column, equilibrate again with 5-10 times the column volume, and elute the sample with sodium acetate buffer. After the sample is eluted, it is neutralized to neutrality with Tris to obtain a high-purity chimeric antibody for later use.
实施例2：VEGFR2嵌合抗体功能检测Example 2: Function test of VEGFR2 chimeric antibody
2.1 ELISA方法检测嵌合抗体与重组蛋白VEGFR2-His结合2.1 ELISA method to detect the binding of chimeric antibody and recombinant protein VEGFR2-His
将浓度为0.1μg/mL的重组人VEGFR2-His蛋白包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h后，分别加入100μL 2μg/mL的抗VEGFR2嵌合抗体孵育1h，洗板去除未结合抗体，加入检测二抗山羊抗人IgG Fc/HRP孵育后重复洗板，加入底物显色液进行显色，终止显示后，酶标仪读取OD450。以重组人VEGFR2抗体为横坐标，OD450读数为纵坐标，利用GraphPad Prism 6.0软件分析并绘制柱形图。结果见图1，其前述的衍生于VEGFR2-MK19 scFv的嵌合抗体mhK19和另一嵌合抗体mhX09与重组蛋白VEGFR2-His有较好的结合。The recombinant human VEGFR2-His protein at a concentration of 0.1 μg/mL was coated on a 96-well plate with 100 μL per well and coated overnight at 4°C. Wash the plate the next day and block at room temperature for 1 hour, add 100 μL 2 μg/mL anti-VEGFR2 chimeric antibody and incubate for 1 hour, wash the plate to remove unbound antibody, add the detection secondary antibody goat anti-human IgG Fc/HRP, incubate and repeat the plate washing, add The substrate color developing solution is used for color development. After the display is terminated, the microplate reader reads OD450. Using the recombinant human VEGFR2 antibody as the abscissa and the OD450 reading as the ordinate, use GraphPad Prism 6.0 software to analyze and draw a bar graph. The results are shown in Figure 1. The aforementioned chimeric antibody mhK19 derived from VEGFR2-MK19 scFv and another chimeric antibody mhX09 have good binding to the recombinant protein VEGFR2-His.
2.2 FACS检测嵌合抗体在VEGFR2过表达细胞系上的结合2.2 FACS detection of the binding of chimeric antibodies on VEGFR2 overexpressing cell lines
以对数生长期的VEGFR2稳定表达细胞株293FT-VEGFR2-13-13(以下简称为293FT-VEGFR2)为实验材料，通过流式细胞仪FACS检测嵌合抗体在293FT-VEGFR2上的结合，同时设定293FT细胞株为阴性对照细胞进行检测。293FT-VEGFR2细胞和293FT细胞分别分装为5×10 5个细胞/管，体积50μL，加入浓度稀释为0.1μg/μL的抗VEGFR2嵌合抗体10μL，4℃混合孵育之后PBS清洗，离心去除未结合抗体，加入山羊抗人IgG Fc-FITC二抗4℃孵育，重复清洗并离心去上清，除去未结合的二抗，最后加入200μL PBS重悬细胞，400目滤网过滤至流式管中，随后用流式细胞仪检测。结果见图2，嵌合抗体mhK19、以及嵌合抗体mhT06、mhW03、mhX09和mhX12在293FT-VEGFR2细胞上有较好的特异性结合。 Using the logarithmic growth phase VEGFR2 stably expressing cell line 293FT-VEGFR2-13-13 (hereinafter referred to as 293FT-VEGFR2) as the experimental material, the binding of the chimeric antibody to 293FT-VEGFR2 was detected by flow cytometry FACS. Determine the 293FT cell line as a negative control cell for detection. 293FT-VEGFR2 cells and 293FT cells were divided into 5×10 5 cells/tube with a volume of 50μL, 10μL of anti-VEGFR2 chimeric antibody diluted to a concentration of 0.1μg/μL was added, mixed and incubated at 4℃, washed with PBS, centrifuged to remove To bind the antibody, add goat anti-human IgG Fc-FITC secondary antibody and incubate at 4°C, repeat washing and centrifugation to remove the supernatant, remove the unbound secondary antibody, and finally add 200μL PBS to resuspend the cells, and filter into the flow tube with a 400 mesh filter , And then use flow cytometry to detect. The results are shown in Figure 2. The chimeric antibody mhK19, as well as the chimeric antibodies mhT06, mhW03, mhX09 and mhX12 have good specific binding on 293FT-VEGFR2 cells.
2.3 嵌合抗体阻断VEGF165结合VEGFR2-His2.3 Chimeric antibody blocks VEGF165 from binding to VEGFR2-His
将浓度为0.5μg/mL的重组VEGF165蛋白(北京义翘神州科技有限公司，11066-HNAB，VEGF165是VEGF-A最经典的剪切体)包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h后，加入100μL 1μg/mL的重组VEGFR2-His蛋白，再加入浓度分别为2μg/mL和0.4μg/mL的VEGFR2嵌合抗体共同孵育1h，其中设定仅加入VEGFR2-His蛋白未加入抗体的孔为阳性孔，洗板去除未结合抗体，加入C-His-R023/HRP检测抗体孵育后重复洗板，加入底物显色液进行显色，终止后用酶标仪读取OD450，根据OD450读值计算不同浓度抗体阻断重组人VEGFR2-His蛋白与人VEGF165蛋白结合的竞争抑制率％，抗体竞争抑制率％＝(阳性孔OD450-加入相应浓度VEGFR2抗体OD450)/阳性孔OD450×100％。结果见图3，mhK19、mhT06、mhU11、mhW03、mhX09和mhX12等嵌合抗体均能阻断重组人VEGFR2-His蛋白与人VEGF165蛋白的结合，mhK19在浓度为2μg/mL条件下抑制率能达到96％。Coat the recombinant VEGF165 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11066-HNAB, VEGF165 is the most classic splicing body of VEGF-A) with a concentration of 0.5μg/mL on a 96-well plate, 100μL per well, 4℃ Coated overnight. The plate was washed the next day and blocked at room temperature for 1 hour, then 100 μL of 1 μg/mL recombinant VEGFR2-His protein was added, and then the VEGFR2 chimeric antibody at the concentration of 2 μg/mL and 0.4 μg/mL was added to incubate together for 1 hour, where only VEGFR2 was set -The wells without antibody added to His protein are positive wells, wash the plate to remove unbound antibody, add C-His-R023/HRP detection antibody and incubate repeatedly, wash the plate again, add substrate color development solution for color development, use enzyme label after termination The instrument reads OD450, and calculates the competitive inhibition rate% of different concentrations of antibody blocking the binding of recombinant human VEGFR2-His protein and human VEGF165 protein according to the OD450 reading. The antibody competition inhibition rate %=(positive well OD450-add the corresponding concentration of VEGFR2 antibody OD450) /Positive wells OD450×100%. The results are shown in Figure 3. Chimeric antibodies such as mhK19, mhT06, mhU11, mhW03, mhX09 and mhX12 can all block the binding of recombinant human VEGFR2-His protein to human VEGF165 protein, and the inhibition rate of mhK19 can reach at a concentration of 2 μg/mL. 96%.
2.4 嵌合抗体阻断VEGF165对HUVEC细胞的增殖作用2.4 Chimeric antibodies block the proliferation of VEGF165 on HUVEC cells
人脐静脉内皮细胞HUVEC按4×10 3个细胞/孔接种到96孔细胞培养板，在含有10％FBS及5％L-Gln的M199培养基(Gibco，11043023)中培养4h，然后按50μL/孔加入相应浓度的抗体，随后以10μL/孔加入终浓度为10ng/mL的VEGF165，设置检测空白孔B(无细胞)、阴性对照组M(接种细胞，不加样品，加VEGF165)和M’(接种细胞，不加样品及VEGF165)。将96孔板置于37℃、5％CO 2细胞培养箱内培养3天后，按10μL/孔加入WST-8显色液(南京旋光科技有限公司，GLT008)，将96孔板置CO2培养箱，显色稳定后用酶标仪测定450nm、630nm的吸光度。结果以OD450减去OD630得到样品检测值，各孔检测值减去空白孔B扣除背景后计算中和率，中和率％＝(阴性对照M组OD值-样品OD值)/(阴性对照M组OD值-M’组OD值)×100％。结果见图4，嵌合抗体mhK19、mhW03和mhX09均具有较强的阻断VEGF165对HUVEC细胞增殖 的作用。 Human umbilical vein endothelial cells HUVEC were seeded into 96-well cell culture plates at 4×10 3 cells/well, cultured in M199 medium (Gibco, 11043023) containing 10% FBS and 5% L-Gln for 4 h, and then 50 μL Add corresponding concentration of antibody to each well, then add VEGF165 at a final concentration of 10ng/mL at 10μL/well, set up detection blank well B (no cells), negative control group M (seeding cells, no sample, add VEGF165) and M '(Inoculated cells, no sample and VEGF165). Place the 96-well plate in a 37°C, 5% CO 2 cell incubator for 3 days, then add 10 μL/well of WST-8 chromogenic solution (Nanjing Rotation Technology Co., Ltd., GLT008), and place the 96-well plate in a CO2 incubator After the color is stable, the absorbance at 450nm and 630nm is measured with a microplate reader. Results OD450 was subtracted from OD630 to obtain the sample detection value. The detection value of each well was subtracted from the blank well B and the background was subtracted to calculate the neutralization rate. Neutralization rate% = (negative control M group OD value-sample OD value) / (negative control M Group OD value-M'group OD value)×100%. The results are shown in Figure 4. The chimeric antibodies mhK19, mhW03 and mhX09 all have a strong blocking effect of VEGF165 on the proliferation of HUVEC cells.
实施例3：鼠抗体人源化改造及生产Example 3: Humanized modification and production of mouse antibodies
根据实施例2中嵌合抗体功能检测结果，选取鼠抗体VEGFR2-MK19进行人源化改造及生产。According to the results of the chimeric antibody function test in Example 2, the mouse antibody VEGFR2-MK19 was selected for humanized transformation and production.
3.1 鼠抗体轻链及重链CDR的确定3.1 Determination of the CDR of the mouse antibody light chain and heavy chain
将实施例1.3中测定的VEGFR2-MK19scFv抗体核苷酸序列，推导得到VEGFR2-MK19scFv抗体的重链和轻链可变区氨基酸序列，见SEQ ID NO:8/9。参考Kabat[17]以及IMGT编号方式，确定抗体VEGFR2-MK19scFv轻链及重链各3个CDR的氨基酸序列，见表1中SEQ ID NO:10-15，除HCDR2中第60位P突变为A外，上述的轻链及重链各3个CDR在后续人源化步骤中被移植且保留在最终获得的人源化抗体VEGFR2-HK19scFv中，见实施例3.2和3.3。The VEGFR2-MK19scFv antibody nucleotide sequence determined in Example 1.3 was deduced to obtain the VEGFR2-MK19scFv antibody heavy chain and light chain variable region amino acid sequences, see SEQ ID NO: 8/9. Refer to Kabat[17] and IMGT numbering method to determine the amino acid sequence of each of the 3 CDRs of the light chain and heavy chain of the antibody VEGFR2-MK19scFv, see SEQ ID NO: 10-15 in Table 1, except that the 60th P mutation in HCDR2 is A In addition, the above three CDRs of the light chain and the heavy chain were transplanted in the subsequent humanization step and retained in the finally obtained humanized antibody VEGFR2-HK19scFv, see Examples 3.2 and 3.3.
表1：VEGFR2-MK19轻链及重链CDR序列Table 1: VEGFR2-MK19 light chain and heavy chain CDR sequences
名称name 序列sequence
LCDR1LCDR1 RASENIYSNLA(SEQIDNO:10)RASENIYSNLA (SEQIDNO: 10)
LCDR2LCDR2 SATDLAD(SEQ ID NO:11)SATDLAD(SEQ ID NO:11)
LCDR3LCDR3 QQYWSIPT(SEQ ID NO:12)QQYWSIPT(SEQ ID NO:12)
HCDR1HCDR1 GFTFSSYSMS(SEQ ID NO:13)GFTFSSYSMS(SEQ ID NO: 13)
HCDR2HCDR2 SISSGGSYIYYPDSVKG(SEQ ID NO:14)SISSGGSYIYYPDSVKG (SEQ ID NO: 14)
HCDR3HCDR3 SRVDEGFAY(SEQ ID NO:15)SRVDEGFAY (SEQ ID NO: 15)
3.2 鼠抗体CDR移植3.2 Mouse antibody CDR transplantation
鼠抗体人源化采用经典的CDR移植方法[18-19]，选择分别与鼠轻链和重链可变区相似性均在50％以上，且重链可变区和轻链可变区的框架区分别与待改造抗体重链可变区和轻链可变区的框架区的氨基酸序列相似性在50％以上的抗体做为预人源化模板，从预人源化模板中选择与待改造抗体的可变区空间结构相似性最高的人源抗体做为人源化模板，将鼠抗体轻链或重链的3个CDR序列置换人源 化模板中相应的CDR氨基酸序列，将HCDR2中第60位P突变为A。。用于VEGFR2-MK19的轻链可变区移植的人源模板为IGKV1-NL1*01，该模板与VEGFR2-MK19轻链的同源性为70.5％，重链可变区的人源模板为IGHV3-21*01，该模板与VEGFR2-MK19重链的同源性为81.6％。Mouse antibody humanization adopts the classic CDR transplantation method [18-19], and selects those that are more than 50% similar to the mouse light chain and heavy chain variable regions, and the heavy chain variable region and light chain variable region Antibodies whose framework regions are more than 50% similar in amino acid sequence to the framework regions of the heavy chain variable region and light chain variable region of the antibody to be modified are used as pre-humanized templates. The human antibody with the highest spatial structural similarity in the variable region of the modified antibody is used as the humanized template. The three CDR sequences of the light chain or heavy chain of the mouse antibody are replaced with the corresponding CDR amino acid sequences in the humanized template, and the HCDR2 P at position 60 is changed to A. . The human template for the light chain variable region transplantation of VEGFR2-MK19 is IGKV1-NL1*01, which has 70.5% homology with the light chain of VEGFR2-MK19, and the human template for the heavy chain variable region is IGHV3 -21*01, the template has 81.6% homology with the heavy chain of VEGFR2-MK19.
3.3 回复突变人源化可变区序列的框架区3.3 Back-mutating the framework region of the humanized variable region sequence
由于鼠源框架区的关键点对于维持CDR空间结构的稳定性具有至关重要的作用，因此将关键点回复突变为鼠抗体的相应氨基酸，直至获得空间结构稳定的抗体共进行了以下突变：按照Kabat编号，将轻链的第43位回复突变为S，第45位回复突变为Q，第70位回复突变为H；重链的第42位回复突变为D，第44位回复突变为R，第49位回复突变为A。经CDR人源化移植和框架区回复突变获得人源化抗体VEGFR2-HK19，其重链和轻链氨基酸序列分别如SEQ ID NO:16/17所示；其含有信号肽的重链和轻链氨基酸序列分别如SEQ ID NO:18/19所示，分别包含依次连接的重链/轻链信号肽氨基酸序列(SEQ ID NO:20/21)；其人源化抗体重链/轻链的可变区氨基酸序列(SEQ ID NO:22/23)；其人源化抗体的恒定区为人IgG1重链恒定区/人kappa轻链恒定区序列(SEQ ID NO:24/25)。改造后的抗体与嵌合抗体相比，其亲和力相近，并达到完全的人源化，人源化后的CDR序列，详见表2。Since the key points of the mouse-derived framework region play a crucial role in maintaining the stability of the CDR spatial structure, the key points were backmutated to the corresponding amino acids of the mouse antibody until the antibody with stable spatial structure was obtained and the following mutations were carried out: Kabat numbering, back mutated position 43 of the light chain to S, position 45 back mutated to Q, and position 70 back mutated to H; back mutated position 42 to D and back mutated position 44 to R in the heavy chain. The 49th position is back-mutated to A. Humanized antibody VEGFR2-HK19 was obtained by CDR humanization transplantation and framework region back mutation. Its heavy chain and light chain amino acid sequences are shown in SEQ ID NO: 16/17, respectively; its heavy chain and light chain containing signal peptide The amino acid sequences are shown in SEQ ID NO: 18/19, respectively, which respectively include the heavy chain/light chain signal peptide amino acid sequence (SEQ ID NO: 20/21) connected in sequence; the humanized antibody heavy chain/light chain can be Amino acid sequence of the variable region (SEQ ID NO: 22/23); the constant region of the humanized antibody is the human IgG1 heavy chain constant region/human kappa light chain constant region sequence (SEQ ID NO: 24/25). Compared with the chimeric antibody, the modified antibody has similar affinities and achieves complete humanization. The CDR sequences after humanization are shown in Table 2.
表2：VEGFR2-HK19轻链及重链CDR序列Table 2: VEGFR2-HK19 light chain and heavy chain CDR sequences
名称name 序列sequence
LCDR1LCDR1 RASENIYSNLA(SEQIDNO:10)RASENIYSNLA (SEQIDNO: 10)
LCDR2LCDR2 SATDLAD(SEQ ID NO:11)SATDLAD(SEQ ID NO:11)
LCDR3LCDR3 QQYWSIPT(SEQ ID NO:12)QQYWSIPT(SEQ ID NO:12)
HCDR1HCDR1 GFTFSSYSMS(SEQ ID NO:13)GFTFSSYSMS(SEQ ID NO: 13)
HCDR2HCDR2 SISSGGSYIYYADSVKG(SEQ ID NO:50)SISSGGSYIYYADSVKG (SEQ ID NO: 50)
HCDR3HCDR3 SRVDEGFAY(SEQ ID NO:15)SRVDEGFAY (SEQ ID NO: 15)
3.4 人源化抗体的生产3.4 Production of humanized antibodies
PCR扩增编码含有信号肽的VEGFR2-HK19抗体轻链核苷酸序列(SEQ ID NO:27)，其中包含依次连接的轻链信号肽的核苷酸序列(SEQ ID NO:29)、人源化抗体轻链可变区的核苷酸序列(SEQ ID NO:31)和人kappa轻链恒定区的核苷酸序列(SEQ ID NO:33)，通过in-fusion方法***自主研发的经KpnI和XbaI双酶切后的pGS载体，通过测序验证获得正确的质粒。同样的，PCR扩增编码VEGFR2-HK19抗体重链核苷酸序列(SEQ ID NO:26)，其中包含依次连接的重链信号肽的核苷酸序列(SEQ ID NO:28)、人源化抗体重链可变区的核苷酸序列(SEQ ID NO:30)和人IgG1重链恒定区的核苷酸序列(SEQ ID NO:32)，通过in-fusion方法***已经构建正确的包含轻链的pGS载体(经NheI和NotI双酶切)中，通过测序验证获得正确的VEGFR2-HK19轻重链表达载体。该表达载体是包含GS筛选基因和抗体轻、重链的表达元件的真核细胞表达载体。将该表达载体转染至CHO-K1-GS缺陷的细胞中，经MSX筛选获得VEGFR2-HK19高表达细胞株。采用ELISA检测选取高表达的克隆，并结合细胞生长的状态和抗体药物的关键质量属性分析结果筛选获得高表达细胞稳定株。采用无血清加料悬浮培养的方式培养生产VEGFR2-HK19的CHO细胞株，纯化获得高质量VEGFR2-HK19抗体。PCR amplifies the nucleotide sequence (SEQ ID NO: 27) encoding the light chain of the VEGFR2-HK19 antibody containing the signal peptide, which contains the nucleotide sequence of the light chain signal peptide (SEQ ID NO: 29), human source The nucleotide sequence of the variable region of the light chain of the antibody (SEQ ID NO: 31) and the nucleotide sequence of the constant region of the human kappa light chain (SEQ ID NO: 33) were inserted into the self-developed KpnI through the in-fusion method. The pGS vector after double digestion with XbaI was verified by sequencing to obtain the correct plasmid. Similarly, PCR amplifies the nucleotide sequence (SEQ ID NO: 26) encoding the heavy chain of the VEGFR2-HK19 antibody, which contains the nucleotide sequence of the heavy chain signal peptide (SEQ ID NO: 28), humanized The nucleotide sequence of the variable region of the antibody heavy chain (SEQ ID NO: 30) and the nucleotide sequence of the constant region of the human IgG1 heavy chain (SEQ ID NO: 32) are inserted into the correct containing light In the pGS vector (digested by NheI and NotI), the correct VEGFR2-HK19 light and heavy chain expression vector was verified by sequencing. The expression vector is a eukaryotic cell expression vector containing GS screening genes and expression elements of antibody light and heavy chains. The expression vector was transfected into CHO-K1-GS-deficient cells, and VEGFR2-HK19 high-expressing cell lines were obtained by MSX screening. High-expressing clones are selected by ELISA detection, and stable high-expressing cell strains are selected by combining the cell growth status and the key quality attribute analysis results of antibody drugs. The CHO cell line producing VEGFR2-HK19 was cultured in a serum-free feeding suspension culture method, and high-quality VEGFR2-HK19 antibody was purified and obtained.
实施例4：人源化抗体特性分析Example 4: Analysis of Humanized Antibody Characteristics
4.1 人源化抗体与VEGFR2抗原结合的亲和力分析4.1 The affinity analysis of the binding of humanized antibody to VEGFR2 antigen
4.1.1人源化抗体VEGFR2-HK19与重组人VEGFR2-His蛋白的结合4.1.1 The binding of humanized antibody VEGFR2-HK19 to recombinant human VEGFR2-His protein
将不同浓度(0.5ng/mL、1.4ng/mL、4.1ng/mL、12.3ng/mL、37ng/mL、111.1ng/mL、333.3ng/mL、1000ng/mL、3000ng/mL和9000ng/mL)的重组人VEGFR2-His蛋白包被于96孔板上，每孔100 μL，4℃包被过夜。次日洗板，室温封闭1h后，分别加入100μL 2μg/mL的VEGFR2-HK19、雷莫芦单抗(Eli Lilly,C839381C)和阴性对照抗体(H7N9-R1)孵育1h，之后洗板去除未结合抗体，加入二抗山羊抗人IgG Fc/HRP孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD450。以重组人VEGFR2-His蛋白浓度为横坐标，OD450读数为纵坐标，利用GraphPad Prism 6.0软件拟合S型曲线并分析抗体与重组人VEGFR2-His蛋白结合的EC 50。结果见图5，人源化分子VEGFR2-HK19与重组人VEGFR2-His的特异性结合EC 50为191.5ng/mL，R 2＝0.999；雷莫芦单抗结合的EC 50为162.1ng/mL，R 2＝0.997；VEGFR2-HK19结合重组人VEGFR2-His蛋白的能力与雷莫芦单抗相近。 Different concentrations (0.5ng/mL, 1.4ng/mL, 4.1ng/mL, 12.3ng/mL, 37ng/mL, 111.1ng/mL, 333.3ng/mL, 1000ng/mL, 3000ng/mL and 9000ng/mL) The recombinant human VEGFR2-His protein was coated on a 96-well plate with 100 μL per well and coated overnight at 4°C. Wash the plate the next day and block for 1 hour at room temperature, add 100μL 2μg/mL VEGFR2-HK19, ramucirumumab (Eli Lilly, C839381C) and negative control antibody (H7N9-R1) and incubate for 1h, then wash the plate to remove unbound For the antibody, add the secondary antibody goat anti-human IgG Fc/HRP and incubate the plate repeatedly, then add the substrate color developing solution for color development, and read the OD450 by the microplate reader after termination. Recombinant human VEGFR2-His protein concentration as the abscissa, the ordinate of OD450 reading, using GraphPad Prism 6.0 software fitting S-shaped curve and analyzed for antibody to recombinant human VEGFR2-His protein binding to EC 50. The results are shown in Figure 5. The EC 50 of the specific binding of humanized molecule VEGFR2-HK19 and recombinant human VEGFR2-His is 191.5ng/mL, R 2 =0.999; the EC 50 of ramucirumab binding is 162.1ng/mL, R 2 =0.997; The ability of VEGFR2-HK19 to bind recombinant human VEGFR2-His protein is similar to ramucirumumab.
4.1.2 人源化抗体VEGFR2-HK19与重组293FT-VEGFR2细胞的结合4.1.2 The binding of humanized antibody VEGFR2-HK19 to recombinant 293FT-VEGFR2 cells
用流式细胞仪FACS检测人源化抗体VEGFR2-HK19及雷莫芦单抗(Eli Lilly,C839381C)在VEGFR2表达细胞上的结合，H7N9-R1为阴性对照抗体。293FT-VEGFR2细胞分别分装为3×10 5个细胞/管，体积为50μL，加入不同浓度(2nM、6nM、17nM、51nM、154nM、463nM和1389nM)的VEGFR2-HK19、雷莫芦单抗和H7N9-R1抗体各10μL，4℃混合孵育之后PBS洗液清洗，离心去除未结合抗体，加入山羊抗人IgG Fc-FITC二抗4℃孵育，重复清洗并离心去上清，除去未结合的二抗，最后加入200μL PBS重悬细胞，400目滤网过滤至流式管中，在流式细胞仪上进行检测。图6显示，VEGFR2-HK19抗体在293FT-VEGFR2细胞上的结合与雷莫芦单抗相近。 FACS was used to detect the binding of humanized antibodies VEGFR2-HK19 and ramucirumumab (Eli Lilly, C839381C) on VEGFR2-expressing cells. H7N9-R1 was used as a negative control antibody. 293FT-VEGFR2 cells were divided into 3×10 5 cells/tube with a volume of 50μL, and different concentrations (2nM, 6nM, 17nM, 51nM, 154nM, 463nM and 1389nM) of VEGFR2-HK19, ramucirumab and 10μL each of H7N9-R1 antibody, mixed and incubated at 4°C, washed with PBS washing solution, centrifuged to remove unbound antibody, added goat anti-human IgG Fc-FITC secondary antibody and incubated at 4°C, washed repeatedly and centrifuged to remove the supernatant to remove unbound Anti-, finally add 200μL PBS to resuspend the cells, filter with 400 mesh filter into the flow tube, and test on the flow cytometer. Figure 6 shows that the binding of VEGFR2-HK19 antibody on 293FT-VEGFR2 cells is similar to that of ramucirumab.
4.1.3 人源化抗体VEGFR2-HK19与重组人VEGFR2-生物素蛋白的亲和力4.1.3 Affinity between humanized antibody VEGFR2-HK19 and recombinant human VEGFR2-biotin protein
利用Octet生物分子相互作用分析***(型号：Octet RED,厂家：Fortebio)测定多个浓度梯度(0.42nM、0.90nM、1.74nM、3.47nM、6.94nM)的VEGFR2-HK19，和雷莫芦单抗(Eli Lilly,C839381C)与生物素化VEGFR2-生物素的亲和力。如表3，VEGFR2-HK19与重 组人VEGFR2-生物素蛋白结合的KD值为1.06*10 -11M，结合速率常数kon值为8.26E+05M -1s -1，解离速率常数kdis值为8.75E-06s -1；雷莫芦单抗与VEGFR2蛋白结合亲和力KD值为4.58*10 -11M，结合速率常数kon值为3.86E+05M -1s -1，解离速率常数kdis值为1.77E-05s -1。VEGFR2-HK19亲和力约为雷莫芦单抗亲和力的4.32倍，并且VEGFR2-HK19具有更慢的解离速率，因此VEGFR2-HK19比雷莫芦单抗具有更强的结合VEGFR2-生物素蛋白的能力。 The Octet biomolecule interaction analysis system (model: Octet RED, manufacturer: Fortebio) was used to determine multiple concentration gradients (0.42nM, 0.90nM, 1.74nM, 3.47nM, 6.94nM) of VEGFR2-HK19, and ramucirumab (Eli Lilly, C839381C) Affinity with biotinylated VEGFR2-biotin. As shown in Table 3, the KD value of VEGFR2-HK19 binding to recombinant human VEGFR2-Biotin protein is 1.06*10 -11 M, the binding rate constant kon value is 8.26E+05M -1 s -1 , and the dissociation rate constant kdis value is 8.75E-06s -1 ; The binding affinity KD value of ramucirumumab and VEGFR2 protein is 4.58*10 -11 M, the binding rate constant kon value is 3.86E+05M -1 s -1 , the dissociation rate constant kdis value is 1.77E-05s -1 . The affinity of VEGFR2-HK19 is about 4.32 times that of ramucirumab, and VEGFR2-HK19 has a slower dissociation rate, so VEGFR2-HK19 has a stronger ability to bind to VEGFR2-biotin protein than ramucirumab .
表3.OCTET检测VEGFR2-HK19与VEGFR2-生物素结合Table 3. OCTET detects the binding of VEGFR2-HK19 and VEGFR2-biotin
Figure PCTCN2020102559-appb-000001
Figure PCTCN2020102559-appb-000001
4.1.4 人源化抗体VEGFR2-HK19种属交叉结合4.1.4 Cross-binding of 19 species of humanized antibody VEGFR2-HK
将重组人VEGFR2-His蛋白、重组小鼠mKDR-His蛋白和重组大鼠ratVEGFR2-His蛋白分别按0.04μg/mL、10μg/mL、10μg/mL的浓度包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h，分别加入100μL浓度为2μg/mL的VEGFR2-HK19、雷莫芦单抗分子(北京义翘神州科技有限公司)、阴性对照抗体(H7N9-R1)孵育1h，洗板去除未结合抗体，加入检测二抗山羊抗人IgG Fc/HRP孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD450。以蛋白浓度为横坐标，OD450读数为纵坐标，利用GraphPad Prism 6.0软件做柱状图。结果如图7显示，VEGFR2-HK19和雷莫芦单抗分子与重组人VEGFR2-His蛋白有特异性结合，与重组小鼠mKDR-His蛋白和重组大鼠ratVEGFR2-His蛋白无交叉结合。Recombinant human VEGFR2-His protein, recombinant mouse mKDR-His protein and recombinant rat ratVEGFR2-His protein were coated on a 96-well plate at a concentration of 0.04μg/mL, 10μg/mL, and 10μg/mL respectively, with 100μL per well , Coated overnight at 4°C. Wash the plate the next day, block at room temperature for 1 hour, add 100μL of 2μg/mL VEGFR2-HK19, ramucirumab molecule (Beijing Yiqiao Shenzhou Technology Co., Ltd.), and negative control antibody (H7N9-R1), incubate for 1h, wash Remove the unbound antibody from the plate, add the detection secondary antibody goat anti-human IgG Fc/HRP and incubate the plate repeatedly, add the substrate color developing solution for color development, and read the OD450 by the microplate reader after termination. Take the protein concentration as the abscissa and the OD450 reading as the ordinate, and use the GraphPad Prism 6.0 software to make a histogram. The results are shown in Figure 7. VEGFR2-HK19 and ramucirumab molecules specifically bind to recombinant human VEGFR2-His protein, but do not have cross-binding with recombinant mouse mKDR-His protein and recombinant rat ratVEGFR2-His protein.
4.2 配体封闭4.2 Ligand blocking
4.2.1 人源化抗体VEGFR2-HK19阻断VEGFR2-His重组蛋白与VEGF165结合4.2.1 Humanized antibody VEGFR2-HK19 blocks the binding of VEGFR2-His recombinant protein to VEGF165
将浓度为0.5μg/mL的重组VEGF165蛋白包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h后，加入100μL 浓度为1μg/mL的重组VEGFR2-His蛋白，蛋白孔中同时加入100μL不同浓度(1.37ng/mL、4.12ng/mL、12.35ng/mL、37.04ng/mL、111.11ng/mL、333.33ng/mL、1000ng/mL、3000ng/mL和9000ng/mL)的VEGFR2-HK19、雷莫芦单抗((Eli Lilly,C839381C))和阴性对照抗体H7N9-R1，并且定仅加入VEGFR2-His蛋白未加入抗体的孔为阳性孔，样品室温孵育1h，洗板去除未结合抗体，加入C-His-R023/HRP检测抗体孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD450，根据OD450读值计算不同浓度抗体阻断重组人VEGFR2蛋白与人VEGF165蛋白结合的竞争抑制率％，抗体竞争抑制率％＝(阳性孔OD450-加入相应浓度VEGFR2抗体OD450)/阳性孔OD450×100％。以抗体浓度为横坐标，抑制率为纵坐标，利用GraphPad Prism 6.0软件拟合S型曲线并分析抗体阻断VEGFR2-His蛋白与VEGF165结合的EC 50。结果如图8显示，VEGFR2-HK19阻断VEGFR2重组蛋白与VEGF165结合的EC 50为283.4ng/mL，R 2＝0.994，雷莫芦单抗阻断VEGFR2重组蛋白与VEGF165结合的EC 50为275.2ng/mL，R 2＝0.988，VEGFR2-HK19阻断VEGF165蛋白结合VEGFR2的能力与雷莫芦单抗相近。 The recombinant VEGF165 protein at a concentration of 0.5 μg/mL was coated on a 96-well plate, 100 μL per well, and coated overnight at 4°C. The plate was washed the next day and blocked at room temperature for 1 hour, then 100 μL of recombinant VEGFR2-His protein at a concentration of 1 μg/mL was added, and 100 μL of different concentrations (1.37ng/mL, 4.12ng/mL, 12.35ng/mL, 37.04ng /mL, 111.11ng/mL, 333.33ng/mL, 1000ng/mL, 3000ng/mL and 9000ng/mL) VEGFR2-HK19, ramucirumab ((Eli Lilly, C839381C)) and negative control antibody H7N9-R1 And set only the wells with VEGFR2-His protein and no antibody added as positive wells. Incubate the sample for 1h at room temperature, wash the plate to remove unbound antibody, add C-His-R023/HRP detection antibody and incubate the plate repeatedly, add the substrate to display The color solution is developed. After termination, the microplate reader reads the OD450. According to the OD450 reading, the competitive inhibition rate% of different concentrations of antibody blocking the binding of recombinant human VEGFR2 protein and human VEGF165 protein is calculated, and the competition inhibition rate of antibody% = (positive hole OD450 -Add the corresponding concentration of VEGFR2 antibody (OD450)/positive well OD450×100%. Antibody concentration as the abscissa, ordinate inhibition rate using GraphPad Prism 6.0 software fitting S-shaped curve and analyzed for antibody to block VEGFR2-His EC 50 VEGF165 binding protein. The results are shown in Figure 8. The EC 50 of VEGFR2-HK19 blocking the binding of VEGFR2 recombinant protein to VEGF165 was 283.4ng/mL, R 2 = 0.994, and the EC 50 of ramucirumab blocking the binding of VEGFR2 recombinant protein to VEGF165 was 275.2ng /mL, R 2 =0.988, the ability of VEGFR2-HK19 to block VEGF165 protein binding to VEGFR2 is similar to ramucirumumab.
4.2.2 人源化抗体VEGFR2-HK19阻断293FT-VEGFR2细胞系与VEGF165结合4.2.2 Humanized antibody VEGFR2-HK19 blocks the binding of 293FT-VEGFR2 cell line to VEGF165
用流式细胞仪FACS检测人源化抗体VEGFR2-HK19及雷莫芦单抗(Eli Lilly,C839381C)对VEGFR2过表达细胞与重组蛋白VEGF165结合的影响，H7N9-R1为阴性对照抗体。293FT-VEGFR2细胞分别分装为3×10 5个细胞/管，体积为50μL，加入不同浓度(2777.8nM、925.9nM、308.6nM、102.9nM、34.3nM、11.4nM和3.8nM)的VEGFR2-HK19、雷莫芦单抗和H7N9-R1抗体各10μL，于4℃孵育20min之后加入2.5μg体内生物素标记的VEGF165蛋白(北京义翘神州科技有限公司，11066-H27H-B)混合孵育，设定未加入抗体仅有VEGF165蛋白的细胞孔为阳性对照孔，PBS洗液清洗，离心去除未结合抗体，加入Strepavidin Alexa fluor488二抗4 ℃孵育，重复清洗并离心去上清，除去未结合的二抗，最后加入200μL PBS重悬细胞，400目滤网过滤至流式管中，在流式细胞仪上检测。根据VEGF165蛋白与VEGFR2过表达细胞结合的平均荧光强度(MFI)分析抗体阻断二者结合的抑制率(％)，抑制率(％)＝(MFI 阳性-MFI 样品)/MFI 阳性*100％，MFI阳性为无抗体仅加入VEGF165蛋白与VEGFR2细胞结合的MFI值，MFI样品为加入抗体后VEGF165蛋白与VEGFR2细胞结合的MFI值。结果如图9所示，VEGFR2-HK19抗体能够阻断VEGF165蛋白与VEGFR2过表达细胞的结合。 FACS was used to detect the effects of humanized antibodies VEGFR2-HK19 and ramucirumab (Eli Lilly, C839381C) on the binding of VEGFR2 overexpressing cells to recombinant protein VEGF165. H7N9-R1 was used as a negative control antibody. 293FT-VEGFR2 cells were divided into 3×10 5 cells/tube with a volume of 50μL, and different concentrations (2777.8nM, 925.9nM, 308.6nM, 102.9nM, 34.3nM, 11.4nM and 3.8nM) of VEGFR2-HK19 were added 10μL each of ramucirumab and H7N9-R1 antibody, incubate at 4°C for 20min, add 2.5μg of in vivo biotin-labeled VEGF165 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11066-H27H-B), mix and incubate, set Cell wells with no antibody added and only VEGF165 protein are positive control wells. Wash with PBS washing solution, centrifuge to remove unbound antibody, add Strepavidin Alexa fluor488 secondary antibody and incubate at 4 ℃, repeat washing and centrifuge to remove the supernatant to remove unbound secondary antibody Finally, add 200μL of PBS to resuspend the cells, filter with a 400 mesh filter into the flow tube, and test on the flow cytometer. According to the average fluorescence intensity (MFI) of the binding between VEGF165 protein and VEGFR2 overexpressing cells, the inhibition rate (%) of the antibody blocking the binding of the two was analyzed. Inhibition rate (%) = (MFI positive- MFI sample )/MFI positive * 100%, MFI positive means that there is no antibody and only the MFI value of VEGF165 protein binding to VEGFR2 cells. The MFI sample is the MFI value of VEGF165 protein binding to VEGFR2 cells after adding antibody. The results are shown in Figure 9, the VEGFR2-HK19 antibody can block the binding of VEGF165 protein to VEGFR2 overexpressing cells.
4.2.3 人源化抗体VEGFR2-HK19阻断VEGFR2-Fc重组蛋白与VEGF-C或VEGF-D结合4.2.3 Humanized antibody VEGFR2-HK19 blocks the binding of VEGFR2-Fc recombinant protein to VEGF-C or VEGF-D
将浓度为5μg/mL的重组VEGFR2-Fc(北京义翘神州科技有限公司，10012-H02H)蛋白包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h后，分别加入100μL浓度为0.5μg/mL的重组VEGF-C(北京义翘神州科技有限公司)蛋白，或2μg/mL的重组VEGF-D(北京义翘神州科技有限公司)蛋白，蛋白孔中同时加入100μL不同浓度(4.12ng/mL、12.35ng/mL、37.04ng/mL、111.11ng/mL、333.33ng/mL、1000ng/mL、3000ng/mL和9000ng/mL)的VEGFR2-HK19、雷莫芦单抗(Eli Lilly,C839381C)和阴性对照抗体H7N9-R1，其中设定仅加入VEGF-C-His蛋白或VEGF-D-His未加入抗体的孔为阳性孔，样品室温孵育1h，洗板去除未结合抗体，加入C-His-R023/HRP检测抗体孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD450，根据OD450读值计算不同浓度抗体阻断重组人VEGFR2蛋白与人VEGF-C或VEGF-D蛋白结合的竞争抑制率％，抗体竞争抑制率％＝(阳性孔OD450-加入相应浓度VEGFR2抗体OD450)/阳性孔OD450×100％。以抗体浓度为横坐标，抑制率为纵坐标，利用GraphPad Prism 6.0软件分别拟合S型曲线并分析抗体阻断VEGFR2-Fc蛋白与VEGF-C或VEGF-D结合的EC 50。 The recombinant VEGFR2-Fc (Beijing Yiqiao Shenzhou Technology Co., Ltd., 10012-H02H) protein at a concentration of 5 μg/mL was coated on a 96-well plate with 100 μL per well and coated overnight at 4°C. The plate was washed the next day and sealed at room temperature for 1 hour, and then 100 μL of recombinant VEGF-C (Beijing Yiqiao Shenzhou Technology Co., Ltd.) protein at a concentration of 0.5 μg/mL or 2 μg/mL recombinant VEGF-D (Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added. Co., Ltd.) protein, 100μL of different concentrations (4.12ng/mL, 12.35ng/mL, 37.04ng/mL, 111.11ng/mL, 333.33ng/mL, 1000ng/mL, 3000ng/mL and 9000ng/mL ) VEGFR2-HK19, ramucirumab (Eli Lilly, C839381C) and the negative control antibody H7N9-R1, where the positive hole is set to add only VEGF-C-His protein or VEGF-D-His without antibody Incubate the sample for 1 hour at room temperature, wash the plate to remove unbound antibodies, add C-His-R023/HRP detection antibody and incubate the plate repeatedly, add the substrate color solution for color development, after termination, the microplate reader reads OD450, according to OD450 Calculate the competitive inhibition rate of different concentrations of antibody blocking the binding of recombinant human VEGFR2 protein and human VEGF-C or VEGF-D protein, %, antibody competition inhibition rate% = (positive hole OD450-adding corresponding concentration of VEGFR2 antibody OD450)/positive hole OD450×100%. Using the antibody concentration as the abscissa and the inhibition rate as the ordinate, GraphPad Prism 6.0 software was used to respectively fit the S-shaped curve and analyze the EC 50 of the antibody blocking the binding of VEGFR2-Fc protein to VEGF-C or VEGF-D.
结果见图10和图11，VEGFR2-HK19阻断VEGFR2-Fc重组蛋 白与VEGF-C结合的EC 50为57.3ng/mL，R 2＝0.999，雷莫芦单抗阻断VEGFR2重组蛋白与VEGF-C结合的EC 50为70.9ng/mL，R 2＝0.999；VEGFR2-HK19阻断VEGFR2重组蛋白与VEGF-D结合的EC 50为76.7ng/mL，R 2＝0.998，雷莫芦单抗阻断VEGFR2重组蛋白与VEGF-D结合的EC 50为85.9ng/mL，R 2＝0.998；综上，VEGFR2-HK19阻断VEGFR2-Fc重组蛋白与VEGF-C或VEGF-D结合的能力比雷莫芦单抗强。 The results are shown in Figure 10 and Figure 11. The EC 50 of VEGFR2-HK19 blocking the binding of VEGFR2-Fc recombinant protein to VEGF-C is 57.3ng/mL, R 2 =0.999, ramucirumab blocks the combination of VEGFR2 recombinant protein and VEGF-C. EC 50 C bound to 70.9ng / mL, R 2 = 0.999 ; VEGFR2-HK19 VEGFR2 blockade of recombinant VEGF-D binding protein and the EC 50 of 76.7ng / mL, R 2 = 0.998 , Lu Remo blocking mAb The EC 50 of VEGFR2 recombinant protein binding to VEGF-D is 85.9ng/mL, R 2 =0.998; In summary, VEGFR2-HK19 is more capable of blocking the binding of VEGFR2-Fc recombinant protein to VEGF-C or VEGF-D. Strong monoclonal antibody.
4.3 人源化抗体VEGFR2-HK19的生长抑制作用4.3 The growth inhibitory effect of humanized antibody VEGFR2-HK19
4.3.1 人源化抗体VEGFR2-HK19阻断VEGF165或VEGF-C对HUVEC细胞的增殖作用4.3.1 Humanized antibody VEGFR2-HK19 blocks the proliferation of VEGF165 or VEGF-C on HUVEC cells
HUVEC细胞按4×10 3个细胞/孔接种到96孔细胞培养板，在含有10％FBS及5％L-Gln的M199培养基(Gibco，11043023)中培养4h，然后按50μL/孔加入相应浓度的抗体，随后以10μL/孔加入终浓度为10ng/mL的VEGF165或者终浓度为1000ng/mL的VEGF-C，设置检测空白孔B(无细胞)、阴性对照组M(接种细胞，不加样品，加VEGF165或VEGF-C)和M’(接种细胞，不加样品及VEGF165或VEGF-C)。将96孔板置于37℃、5％CO2细胞培养箱内培养3天后，按10μL/孔加入WST-8显色液(南京旋光科技有限公司，GLT008)，将96孔板置CO 2培养箱，显色稳定后用酶标仪测定450nm、630nm的吸光度。结果以OD450减去OD630得到样品检测值，各孔检测值减去空白孔B以扣除背景后计算中和率，中和率％＝(阴性对照M组OD值-样品OD值)/(阴性对照M组OD值-M’组OD值)×100％，采用统计软件GraphPad Prism的自动分析功能计算标准曲线，横坐标为样品浓度，纵坐标为中和率，用四参数回归方程拟合得到“S”型曲线，计算样品半数有效浓度(EC50)。结果如图12、13和表4，虽然VEGFR2-HK19与雷莫芦单抗(Eli Lilly,C839381C)阻断VEGF165或VEGF-C对HUVEC细胞增殖作用的最大中和率相当，但是VEGFR2-HK19的EC50浓度均小于雷莫芦单抗，提示VEGFR2-HK19的生长抑制活性更好。 HUVEC cells were seeded into 96-well cell culture plates at 4×10 3 cells/well, cultured in M199 medium (Gibco, 11043023) containing 10% FBS and 5% L-Gln for 4 hours, and then 50 μL/well was added to the corresponding Then add VEGF165 with a final concentration of 10ng/mL or VEGF-C with a final concentration of 1000ng/mL at 10μL/well, set up detection blank well B (without cells) and negative control group M (seeding cells without adding For samples, add VEGF165 or VEGF-C) and M'(seeding cells, do not add samples and VEGF165 or VEGF-C). Place the 96-well plate in a 37°C, 5% CO2 cell incubator for 3 days, add 10μL/well of WST-8 chromogenic solution (Nanjing Rotation Technology Co., Ltd., GLT008), and place the 96-well plate in a CO 2 incubator After the color is stable, the absorbance at 450nm and 630nm is measured with a microplate reader. Results OD450 was subtracted from OD630 to obtain the sample detection value. The detection value of each well was subtracted from the blank well B to calculate the neutralization rate after subtracting the background. The neutralization rate% = (negative control M group OD value-sample OD value)/(negative control) M group OD value-M'group OD value)×100%, using the automatic analysis function of the statistical software GraphPad Prism to calculate the standard curve, the abscissa is the sample concentration, the ordinate is the neutralization rate, and the four-parameter regression equation is used to fit the standard curve. S" curve, calculate the half effective concentration (EC50) of the sample. The results are shown in Figures 12, 13 and Table 4. Although VEGFR2-HK19 and ramucirumab (Eli Lilly, C839381C) block the maximum neutralization rate of VEGF165 or VEGF-C on HUVEC cell proliferation, the maximum neutralization rate of VEGFR2-HK19 The EC50 concentration is less than ramucirumab, suggesting that VEGFR2-HK19 has better growth inhibitory activity.
表4.VEGFR2-HK19中和VEGF165或VEGF-C对HUVEC细胞增殖作用的EC50及最大中和率Table 4. EC50 and maximum neutralization rate of VEGFR2-HK19 neutralizing VEGF165 or VEGF-C on HUVEC cell proliferation
Figure PCTCN2020102559-appb-000002
Figure PCTCN2020102559-appb-000002
4.3.2 人源化抗体VEGFR2-HK19阻断不同亚型VEGF组合物对HUVEC细胞的增殖作用4.3.2 Humanized antibody VEGFR2-HK19 blocks the proliferation effect of different subtypes of VEGF composition on HUVEC cells
HUVEC细胞按4×10 3个细胞/孔接种96孔细胞培养板，在含有10％FBS及5％L-Gln的M199培养基(Gibco，11043023)中培养4h，然后按50μL/孔加入相应浓度的抗体，随后以10μL/孔加入VEGF-C(1000ng/mL)与VEGF-D(8181ng/mL)的混合物，或者VEGF165(25ng/mL)与VEGF-C(1000ng/mL)及VEGF-D(5455ng/mL)的混合物。设置检测空白孔B(无细胞)、阴性对照组M(接种细胞，不加样品，加VEGF)和M’(接种细胞，不加样品及VEGF)。将96孔板置于37℃、5％CO 2细胞培养箱内培养3天后，按10μL/孔加入WST-8显色液(南京旋光科技有限公司，GLT008)，将96孔板置CO 2培养箱，显色稳定后用酶标仪测定450nm、630nm的吸光度。结果以OD450减去OD630得到样品检测值，各孔检测值减去空白孔B扣除背景后计算中和率，中和率％＝(阴性对照M组OD值-样品OD值)/(阴性对照M组OD值-M’组OD值)×100％，采用统计软件GraphPad Prism的自动分析功能计算标准曲线，横坐标为样品浓度，纵坐标为中和率，用四参数回归方程拟合得到“S”型曲线，计算样品半数有效浓度(EC50)。结果如图14、15和表5，尽管VEGFR2-HK19与雷莫芦单抗(Eli Lilly,C839381C)阻断 VEGF-C+VEGF-D对HUVEC细胞增殖作用的最大中和率相当，且VEGFR2-HK19阻断VEGF-A+VEGF-C+VEGF-D对HUVEC细胞增殖作用的最大中和率略小于雷莫芦单抗，但是VEGFR2-HK19的EC50浓度均小于雷莫芦单抗，提示VEGFR2-HK19的生长抑制活性更好。 HUVEC cells were seeded into 96-well cell culture plates at 4×10 3 cells/well, cultured in M199 medium (Gibco, 11043023) containing 10% FBS and 5% L-Gln for 4 hours, and then 50μL/well was added to the corresponding concentration VEGF-C (1000ng/mL) and VEGF-D (8181ng/mL) mixture, or VEGF165 (25ng/mL) and VEGF-C (1000ng/mL) and VEGF-D ( 5455ng/mL). Set up detection blank well B (no cells), negative control group M (seeding cells, no sample, plus VEGF) and M'(seeding cells, no sample and VEGF). Place the 96-well plate in a 37°C, 5% CO 2 cell incubator and culture for 3 days, add 10 μL/well of WST-8 color developing solution (Nanjing Rotation Technology Co., Ltd., GLT008), and place the 96-well plate in CO 2 for culture After the color is stable, the absorbance at 450nm and 630nm is measured with a microplate reader. Results OD450 was subtracted from OD630 to obtain the sample detection value. The detection value of each well was subtracted from the blank well B and the background was subtracted to calculate the neutralization rate. Neutralization rate% = (negative control M group OD value-sample OD value) / (negative control M Group OD value-M' group OD value)×100%, using the automatic analysis function of the statistical software GraphPad Prism to calculate the standard curve, the abscissa is the sample concentration, the ordinate is the neutralization rate, and the four-parameter regression equation is used to fit the standard curve. ”Type curve, calculate the half effective concentration (EC50) of the sample. The results are shown in Figures 14, 15 and Table 5. Although VEGFR2-HK19 and ramucirumab (Eli Lilly, C839381C) blocked the maximum neutralization rate of VEGF-C+VEGF-D on HUVEC cell proliferation, and VEGFR2- The maximum neutralization rate of HK19 blocking VEGF-A+VEGF-C+VEGF-D on HUVEC cell proliferation is slightly less than ramucirumab, but the EC50 concentration of VEGFR2-HK19 is less than ramucirumab, suggesting that VEGFR2- HK19 has better growth inhibitory activity.
表5.VEGFR2-HK19中和不同亚型的VEGF组合物对HUVEC细胞增殖作用的EC50及最大中和率Table 5. The EC50 and maximum neutralization rate of VEGFR2-HK19 neutralizing different subtypes of VEGF compositions on HUVEC cell proliferation
Figure PCTCN2020102559-appb-000003
Figure PCTCN2020102559-appb-000003
4.4 人源化抗体VEGFR2-HK19的ADCC作用4.4 ADCC effect of humanized antibody VEGFR2-HK19
本实施例中采用重组CD16A报告基因***方法测定VEGFR2-HK19介导的ADCC效应，效应细胞为Jurkat-NFAT-Luc2p-CD16A，而靶细胞为293FT-VEGFR2，当两种细胞共培养的同时加入VEGFR2-HK19后，VEGFR2-HK19的Fab段与靶细胞上过表达的VEGFR2结合，其Fc段可与过表达FcγIII型受体(CD16A)的效应细胞结合，从而激活效应细胞Jurkat-NFAT-Luc2p-CD16A并促进NFAT-RE介导的生物发光。具体而言，将靶细胞293FT-VEGFR2按2×10 4个细胞/孔接种于96孔板，在含有10％FBS的DMEM培养基中培养过夜，去掉上清，用含0.5g/L PF68(北京义翘神州科技有限公司)的无酚红RPMI 1640培养基清洗两遍，然后按40μL/孔加入相应浓度的抗体，随后以40μL/孔加入1×10 5个效应细胞Jurkat-NFAT-Luc2p-CD16A，每组设3个复孔，同 时设靶细胞、效应细胞和阴性对照组(加靶细胞及效应细胞，不加样品)。置于37℃、5％CO 2条件下培养4h后，按20μL/孔加入Passive Lysis 5X Buffer(Promega，E1941)，震荡混匀后每孔取20μL上清液转移至96孔白底板，通过LB960-微孔板式发光检测仪检测荧光信号。利用GraphPad Prism软件分析并绘制量效曲线，横坐标为样品的浓度，纵坐标为化学发光强度(RLU)，计算EC50及诱导倍数(样品RLU/阴性对照组RLU)。 In this example, the recombinant CD16A reporter gene system method was used to determine the ADCC effect mediated by VEGFR2-HK19. The effector cell is Jurkat-NFAT-Luc2p-CD16A, and the target cell is 293FT-VEGFR2. When the two cells are co-cultured, VEGFR2 is added. -After HK19, the Fab segment of VEGFR2-HK19 binds to VEGFR2 overexpressed on target cells, and its Fc segment can bind to effector cells overexpressing FcγIII receptor (CD16A), thereby activating effector cells Jurkat-NFAT-Luc2p-CD16A And promote NFAT-RE-mediated bioluminescence. Specifically, the target cell 293FT-VEGFR2 was seeded on a 96-well plate at 2×10 4 cells/well, cultured in DMEM medium containing 10% FBS overnight, the supernatant was removed, and 0.5g/L PF68( Wash twice with the phenol red-free RPMI 1640 medium of Beijing Yiqiao Shenzhou Technology Co., Ltd., and then add the corresponding concentration of antibody at 40μL/well, and then add 1×10 5 effector cells Jurkat-NFAT-Luc2p- at 40μL/well. CD16A, each group has 3 multiple wells, and the target cells, effector cells and negative control group (with target cells and effector cells, no sample). After incubating at 37℃ and 5% CO 2 for 4 hours, add Passive Lysis 5X Buffer (Promega, E1941) at a rate of 20μL/well, shake and mix well, transfer 20μL of supernatant from each well to a 96-well white bottom plate, and pass through LB960 -Microplate luminescence detector detects fluorescence signal. Use GraphPad Prism software to analyze and draw a dose-response curve. The abscissa is the concentration of the sample, and the ordinate is the chemiluminescence intensity (RLU). The EC50 and the induction factor (sample RLU/negative control group RLU) are calculated.
如图16和表6所示，雷莫芦单抗(Eli Lilly,C839381C)介导ADCC作用的能力较弱，而VEGFR2-HK19具有较强的ADCC作用。As shown in Figure 16 and Table 6, ramucirumab (Eli Lilly, C839381C) has a weaker ability to mediate ADCC effects, while VEGFR2-HK19 has strong ADCC effects.
表6.VEGFR2-HK19介导ADCC作用的EC50及最大诱导倍数Table 6. EC50 and maximum induction factor of ADCC mediated by VEGFR2-HK19
Figure PCTCN2020102559-appb-000004
Figure PCTCN2020102559-appb-000004
4.5 人源化抗体VEGFR2-HK19的表位4.5 Epitope of humanized antibody VEGFR2-HK19
4.5.1 人源化抗体VEGFR2-HK19与结构域蛋白结合4.5.1 Humanized antibody VEGFR2-HK19 binds to domain protein
实施例4.2中证实VEGFR2-HK19能够阻断配体VEGF165与VEGFR2的结合，证明VEGFR2-HK19的表位与配体相近，而文献报道VEGF165结合在VEGFR2胞外区的第2和第3结构域[2]。为了表征VEGFR2-HK19抗体的表位，本实施例中用ELISA法检测VEGFR2的结构域蛋白(VEGFR2-结构域2&3)和胞外区重组蛋白VEGFR2-His与VEGFR2-HK19的结合，结果详见表6，VEGFR2-HK19与VEGFR2-结构域2&3和VEGFR2-His无显著性差异，表明VEGFR2-HK19结合在VEGFR2的第2到第3结构域，且与配体VEGF165在VEGFR2的结合可能存在表位重叠。In Example 4.2, it was confirmed that VEGFR2-HK19 can block the binding of ligand VEGF165 and VEGFR2, which proved that the epitope of VEGFR2-HK19 is similar to the ligand, and the literature reported that VEGF165 binds to the second and third domains of the extracellular domain of VEGFR2 [ 2]. In order to characterize the epitope of the VEGFR2-HK19 antibody, the binding of VEGFR2 domain protein (VEGFR2-domain 2&3) and extracellular region recombinant protein VEGFR2-His to VEGFR2-HK19 was detected by ELISA in this example. The results are shown in the table. 6. There is no significant difference between VEGFR2-HK19 and VEGFR2-domain 2&3 and VEGFR2-His, indicating that VEGFR2-HK19 binds to the second to third domains of VEGFR2, and there may be epitope overlap with the ligand VEGF165 in the binding of VEGFR2 .
4.5.2 分子模拟预测人源化抗体VEGFR2-HK19表位4.5.2 Molecular simulation prediction of humanized antibody VEGFR2-HK19 epitope
为了深入了解VEGFR2-HK19与VEGFR2蛋白界面相互作用，本实施例中利用分子模拟及对接法，用DS 4.0(Accelrys Software Inc.)中的Antidody model程序对VEGFR2-HK19进行同源建模，从 PDB数据库中提取VEGFR2蛋白三维结构(PDB ID:3V2A)，经过Protein Preparation程序初始化。VEGFR2-HK19模型和VEGFR2结构通过ZDOCK程序进行对接，根据对接结果，对打分函数前十位的对接进行RDOCK优化，最优的模型(图17)经Protein Interface Analysis程序进一步分析。对接模型界面显示，VEGFR2-HK19与VEGFR2蛋白关键结合肽序列分别为结构域2的 137YITENK 142以及结构域3的 255GIDFNWEY 262、 310SSGLMTKK 317。 In order to understand the interaction between VEGFR2-HK19 and VEGFR2 protein interface, molecular simulation and docking method were used in this example, and the Antidody model program in DS 4.0 (Accelrys Software Inc.) was used to model the homology of VEGFR2-HK19 from PDB. The three-dimensional structure of VEGFR2 protein (PDB ID: 3V2A) was extracted from the database and initialized through the Protein Preparation program. The VEGFR2-HK19 model and the VEGFR2 structure are docked through the ZDOCK program. According to the docking results, the top ten docking functions of the scoring function are optimized by RDOCK, and the optimal model (Figure 17) is further analyzed by the Protein Interface Analysis program. The docking model interface shows that the key binding peptide sequences of VEGFR2-HK19 and VEGFR2 proteins are 137 YITENK 142 in domain 2 and 255 GIDFNWEY 262 and 310 SSGLMTKK 317 in domain 3, respectively.
4.5.3 VEGFR2突变蛋白验证人源化抗体VEGFR2-HK19结合关键位点4.5.3 VEGFR2 mutant protein verifies that the humanized antibody VEGFR2-HK19 binds to key sites
为了进一步确认VEGFR2-HK19结合的关键位点，根据上述4.5.2实施例中预测的VEGFR2-HK19与VEGFR2蛋白的关键结合肽序列，设计并生产一系列VEGFR2蛋白突变体，ELISA测定突变体与VEGFR2-HK19、上市抗体雷莫芦单抗(Eli Lilly,C839381C)和配体VEGF-A的结合能力。结果显示VEGFR2-HK19与VEGFR2蛋白突变体结合有不同程度的降低(表7)，VEGFR2-HK19主要结合在Y137A/K142A、D257A、S311A/G312A、L313A和T315A等关键位点，该结果与4.5.2实施例中对接模型分析结果基本吻合，验证了模型的准确性。VEGF与雷莫芦单抗抗体的关键位点文献报道的晶体结构基本吻合[20-21]。Y137A/K142A、R164S/Y165A、D257A、S311A/G312A等为同时影响VEGFR2-HK19和配体VEGF-A结合VEGFR2的关键点，证明VEGFR2-HK19抗体与VEGF-A存在表位交叠(图18)，由于抗体的亲和力大于配体，VEGFR2-HK19可通过直接阻断VEGF-A与VEGFR2的结合来发挥作用。此外，VEGFR2-HK19与上市药物雷莫芦单抗存在表位差异例如R222A为雷莫芦单抗的关键位点，但对VEGFR2-HK19结合并无影响，因此二者表位的不同可能对于解释ADCC功能的差异有重要的指导意义。In order to further confirm the key sites of VEGFR2-HK19 binding, according to the predicted key binding peptide sequence of VEGFR2-HK19 and VEGFR2 protein in the above 4.5.2 example, a series of VEGFR2 protein mutants were designed and produced. The mutants and VEGFR2 were determined by ELISA. -HK19, the binding ability of the listed antibody ramucirumab (Eli Lilly, C839381C) and the ligand VEGF-A. The results showed that the binding of VEGFR2-HK19 to VEGFR2 protein mutants was reduced to varying degrees (Table 7). VEGFR2-HK19 mainly binds to key sites such as Y137A/K142A, D257A, S311A/G312A, L313A, and T315A. This result is consistent with 4.5. 2 The analysis results of the docking model in the embodiment are basically consistent, verifying the accuracy of the model. The key sites of VEGF and ramucirumab antibody are basically consistent with the crystal structures reported in the literature [20-21]. Y137A/K142A, R164S/Y165A, D257A, S311A/G312A, etc. are the key points that simultaneously affect the binding of VEGFR2-HK19 and ligand VEGF-A to VEGFR2, proving that VEGFR2-HK19 antibody and VEGF-A have epitope overlap (Figure 18) Because the affinity of the antibody is greater than that of the ligand, VEGFR2-HK19 can directly block the binding of VEGF-A and VEGFR2. In addition, there are epitope differences between VEGFR2-HK19 and the marketed drug ramucirumab. For example, R222A is the key site of ramucirumab, but it has no effect on the binding of VEGFR2-HK19. Therefore, the difference between the epitopes of the two may explain The difference of ADCC function has important guiding significance.
Figure PCTCN2020102559-appb-000005
Figure PCTCN2020102559-appb-000005
实施例5：人源化抗体VEGFR2-HK19对KDR人源化小鼠B16-F1黑色素瘤皮下移植瘤模型的抗肿瘤作用研究Example 5: Anti-tumor effect of humanized antibody VEGFR2-HK19 on B16-F1 melanoma subcutaneously transplanted tumor model in KDR humanized mice
KDR人源化小鼠(中国食品药品检定研究院)右侧胁肋部皮下接种大小为约2×2×2(mm 3)的B16-F1瘤块，共接种39只(雌性20只，雄性19只)。当肿瘤体积约100mm 3左右时分组给药，根据个体肿瘤体积挑选小鼠入组，采用excel软件将动物按瘤体积随机分为4组，每组6只(雌雄各3只)，第1组为空白对照组，第2组为实验组(VEGFR2-HK19)，其中第3、4组为与本申请无关的其他抗VEGFR2抗体，因此不呈现相关数据。分组当天开始腹腔注射(I.P.)给药，实验组剂量为15mg/kg，空白对照组(Vehicle)注射等体积的溶剂，每周给药2次，连续给5次，具体给药方案见下表8。通过计算肿瘤生长抑制率TGI(％)评价药物的抗肿瘤作用：TGI(％)<60％为无效；TGI(％)≥60％，且经统计学处理实验组瘤体积显著低于溶剂对照组(P<0.05)为有效，即对肿瘤生长具有显著抑制作用。 KDR humanized mice (China Institute for Food and Drug Control) were subcutaneously inoculated with B16-F1 tumor masses of about 2×2×2 (mm 3 ) on the right flank, and 39 mice (20 females, males) were inoculated 19). When the tumor volume is about 100mm 3 or so, the mice will be administered in groups, and mice will be selected according to the individual tumor volume. The animals will be randomly divided into 4 groups according to the tumor volume using excel software, each with 6 (3 male and female), the first group It is a blank control group, the second group is the experimental group (VEGFR2-HK19), and the third and fourth groups are other anti-VEGFR2 antibodies that are not related to the application, so relevant data are not presented. Intraperitoneal injection (IP) administration was started on the day of grouping. The dose of the experimental group was 15 mg/kg, and the blank control group (Vehicle) was injected with an equal volume of solvent, administered twice a week for 5 consecutive times. The specific dosing schedule is shown in the following table 8. The anti-tumor effect of the drug is evaluated by calculating the tumor growth inhibition rate TGI (%): TGI (%) <60% is invalid; TGI (%) ≥ 60%, and the tumor volume of the experimental group is significantly lower than the solvent control group after statistical processing (P<0.05) is effective, that is, it has a significant inhibitory effect on tumor growth.
TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中：TGI(%)=[1-(Ti-T0)/(Vi-V0)]×100, where:
Ti：实验组在给药第i天的肿瘤体积均值，Ti: the mean tumor volume of the experimental group on the i day after administration,
T0：实验组在给药第0天的肿瘤体积均值，T0: The mean tumor volume of the experimental group on day 0 of administration,
Vi：溶剂对照组在给药第i天的肿瘤体积均值，Vi: the mean tumor volume of the solvent control group on the i day after administration,
V0：溶剂对照组在给药第0天的肿瘤体积均值。V0: The mean tumor volume of the solvent control group on day 0 of administration.
表8.试验分组及给药方案Table 8. Test groups and dosing schedule
Figure PCTCN2020102559-appb-000006
Figure PCTCN2020102559-appb-000006
a：给药容积依实验动物体重按1mL/kg计算。a: The dosage volume is calculated based on the weight of the experimental animal as 1 mL/kg.
除对照组因肿瘤体积过大出现部分动物死亡外，实验组动物在给药期间的活动和进食等状态良好，体重均有一定程度的上升，且实验组和对照组给药后动物体重无显著性差异(P>0.05)。所有动物体重变化情况见图19及表9。Except for the death of some animals in the control group due to the excessive tumor volume, the animals in the experimental group were in good activity and eating during the administration period, and their body weight increased to a certain extent, and the animal weight in the experimental group and the control group was not significant after administration Sexual difference (P>0.05). The body weight changes of all animals are shown in Figure 19 and Table 9.
表9.VEGFR2-HK19对B16-F1黑色素瘤皮下移植瘤模型中KDR人源化小鼠体重的影响Table 9. Effect of VEGFR2-HK19 on the body weight of KDR humanized mice in B16-F1 melanoma subcutaneous transplantation tumor model
Figure PCTCN2020102559-appb-000007
Figure PCTCN2020102559-appb-000007
a：均数±标准误；a: mean ± standard error;
b：实验组体重与溶剂对照组体重在给药17天后统计学比较，t检验；b: The weight of the experimental group and the weight of the solvent control group were statistically compared 17 days after administration, t test;
c：组1实验终点为2只小鼠。c: The end point of group 1 experiment is 2 mice.
末次给药4天后，受试动物均被安乐死，测量并计算肿瘤体积，结果见表10和图20。试验结束时，溶剂对照组平均肿瘤体积为6245±921mm 3，实验组(VEGFR2-HK19)平均肿瘤体积为1708±420mm 3，TGI为73.59％，与溶剂对照组的肿瘤体积相比有显著差异(P＝0.0021)。表明供试抗体VEGFR2-HK19结合KDR表位在B16-F1黑色素癌皮下移植瘤模型上是有效的，在15mg/kg剂量水平下对B16-F1黑色素癌皮下移植瘤有明显的抑制作用。 Four days after the last administration, all the test animals were euthanized, and the tumor volume was measured and calculated. The results are shown in Table 10 and Figure 20. At the end of the experiment, the average tumor volume of the solvent control group was 6245±921 mm 3 , the average tumor volume of the experimental group (VEGFR2-HK19) was 1708±420 mm 3 , and the TGI was 73.59%, which was significantly different from the tumor volume of the solvent control group ( P = 0.0021). It shows that the test antibody VEGFR2-HK19 binding to KDR epitope is effective in the B16-F1 melanoma subcutaneous transplantation tumor model, and it has a significant inhibitory effect on the B16-F1 melanoma subcutaneous transplantation tumor at the dose level of 15mg/kg.
表10.VEGFR2-HK19对KDR人源化小鼠B16-F1黑色素瘤皮下移植瘤模型中肿瘤体积的影响Table 10. The effect of VEGFR2-HK19 on tumor volume in B16-F1 melanoma subcutaneously transplanted tumor model in KDR humanized mice
Figure PCTCN2020102559-appb-000008
Figure PCTCN2020102559-appb-000008
Figure PCTCN2020102559-appb-000009
Figure PCTCN2020102559-appb-000009
a：实验组肿瘤体积与溶剂对照组肿瘤体积统计学比较，t检验。a: Statistical comparison of tumor volume between the experimental group and the solvent control group, t test.
实施例6：人源化抗体VEGFR2-HK19在小鼠体内的药代动力学和免疫原性研究Example 6: Study on the pharmacokinetics and immunogenicity of humanized antibody VEGFR2-HK19 in mice
抗体VEGFR2-HK19的小鼠药代动力学在CD1小鼠体内考察：以5mg/kg剂量分别经尾静脉和皮下注射给予CD-1小鼠，每组6只，雌雄各半，于药前、药后0.5h(i.v.组)、2h、4h、6h、24h、48h、72h、96h、168h、336h、504h、672h采血。采用间接ELISA法检测血清中VEGFR2-HK19抗体的浓度，并通过Phoenix-WinNonlin6.4软件中非房室模型(NCA)计算药代动力学参数，用桥联ELISA法对全部动物在注射给药后168、336、504、672h时的血样进行抗药抗体检测。The mouse pharmacokinetics of the antibody VEGFR2-HK19 was investigated in CD1 mice: CD-1 mice were administered at a dose of 5 mg/kg via tail vein and subcutaneous injection, 6 mice in each group, half male and half, before the drug, Blood was collected 0.5h (IV group), 2h, 4h, 6h, 24h, 48h, 72h, 96h, 168h, 336h, 504h, 672h after the drug. The indirect ELISA method was used to detect the concentration of VEGFR2-HK19 antibody in the serum, and the pharmacokinetic parameters were calculated by the non-compartmental model (NCA) in the Phoenix-WinNonlin6.4 software. The bridged ELISA method was used for all animals after injection and administration. Blood samples at 168, 336, 504, and 672 hours were tested for anti-drug antibodies.
(1)药代动力学结果(1) Pharmacokinetic results
通过ELISA法检测CD-1小鼠血清中VEGFR2-HK19抗体浓度，血药浓度时间曲线如图21所示，药代动力学参数如表11所示。5mg/kg剂量的VEGFR2-HK19抗体静脉及皮下给予CD-1小鼠，动物雌雄间未见明显差异，静脉和皮下给药的C max分别为77.77和32.00μg/mL，AUC last分别为11702.39和11033.42h*μg/mL，皮下给药的绝对生物利用度为94.28％。两种给药方式下t 1/2、Vz、Cl等参数基本一致。 The concentration of VEGFR2-HK19 antibody in the serum of CD-1 mice was detected by ELISA. The plasma concentration-time curve is shown in Figure 21, and the pharmacokinetic parameters are shown in Table 11. The 5mg/kg dose of VEGFR2-HK19 antibody was administered intravenously and subcutaneously to CD-1 mice. There was no significant difference between male and female animals. The C max of intravenous and subcutaneous administration were 77.77 and 32.00 μg/mL, respectively, and the AUC last were 11702.39 and 11702.39 and respectively. 11033.42h*μg/mL, the absolute bioavailability of subcutaneous administration is 94.28%. The parameters such as t 1/2 , Vz and Cl are basically the same under the two administration methods.
Figure PCTCN2020102559-appb-000010
Figure PCTCN2020102559-appb-000010
Figure PCTCN2020102559-appb-000011
Figure PCTCN2020102559-appb-000011
Figure PCTCN2020102559-appb-000012
Figure PCTCN2020102559-appb-000012
序列表Sequence Listing
Figure PCTCN2020102559-appb-000013
Figure PCTCN2020102559-appb-000013
Figure PCTCN2020102559-appb-000014
Figure PCTCN2020102559-appb-000014
Figure PCTCN2020102559-appb-000015
Figure PCTCN2020102559-appb-000015
Figure PCTCN2020102559-appb-000016
Figure PCTCN2020102559-appb-000016
Figure PCTCN2020102559-appb-000017
Figure PCTCN2020102559-appb-000017
Figure PCTCN2020102559-appb-000018
Figure PCTCN2020102559-appb-000018
Figure PCTCN2020102559-appb-000019
Figure PCTCN2020102559-appb-000019
Figure PCTCN2020102559-appb-000020
Figure PCTCN2020102559-appb-000020
Figure PCTCN2020102559-appb-000021
Figure PCTCN2020102559-appb-000021
Figure PCTCN2020102559-appb-000022
Figure PCTCN2020102559-appb-000022
Figure PCTCN2020102559-appb-000023
Figure PCTCN2020102559-appb-000023
Figure PCTCN2020102559-appb-000024
Figure PCTCN2020102559-appb-000024
Figure PCTCN2020102559-appb-000025
Figure PCTCN2020102559-appb-000025
Figure PCTCN2020102559-appb-000026
Figure PCTCN2020102559-appb-000026
Figure PCTCN2020102559-appb-000027
Figure PCTCN2020102559-appb-000027
Figure PCTCN2020102559-appb-000028
Figure PCTCN2020102559-appb-000028
Figure PCTCN2020102559-appb-000029
Figure PCTCN2020102559-appb-000029
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Claims (24)

  1. 一种分离的抗VEGFR2抗体或其抗原结合片段，包含具有SEQ ID NO：13所示的氨基酸序列的重链CDR1域、具有SEQ ID NO：14或SEQ ID NO：50所示的氨基酸序列的重链CDR2域和具有SEQ ID NO：15所示的氨基酸序列的重链CDR3域的重链可变区，和具有SEQ ID NO：10所示的氨基酸序列的轻链CDR1域、具有SEQ ID NO：11所示的氨基酸序列的轻链CDR2域和具有SEQ ID NO：12所示的氨基酸序列的轻链CDR3域的轻链可变区。An isolated anti-VEGFR2 antibody or antigen-binding fragment thereof, comprising a heavy chain CDR1 domain with the amino acid sequence shown in SEQ ID NO: 13, and a heavy chain with the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 50 The chain CDR2 domain and the heavy chain variable region of the heavy chain CDR3 domain having the amino acid sequence shown in SEQ ID NO: 15, and the light chain CDR1 domain having the amino acid sequence shown in SEQ ID NO: 10, having SEQ ID NO: The light chain CDR2 domain of the amino acid sequence shown in 11 and the light chain CDR3 domain of the light chain CDR3 domain having the amino acid sequence shown in SEQ ID NO: 12.
  2. 如权利要求1所述的抗VEGFR2抗体或其抗原结合片段，包含具有如SEQ ID NO：8所示的氨基酸序列或与SEQ ID NO：8具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列的重链可变区和具有如SEQ ID NO：9所示的氨基酸序列或与SEQ ID NO：9具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列的轻链可变区。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to claim 1, comprising an amino acid sequence as shown in SEQ ID NO: 8 or at least 90%, 92%, 95%, 98%, or at least 90% to SEQ ID NO: 8 The heavy chain variable region of the amino acid sequence with 99% sequence identity and the amino acid sequence shown in SEQ ID NO: 9 or at least 90%, 92%, 95%, 98% or 99% with SEQ ID NO: 9 The light chain variable region of the amino acid sequence of sequence identity.
  3. 如权利要求1所述的抗VEGFR2抗体或其抗原结合片段，其中所述抗VEGFR2抗体或其抗原结合片段是人源化抗体或嵌合抗体。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-VEGFR2 antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody.
  4. 如权利要求3所述的抗VEGFR2抗体或其抗原结合片段，其中所述抗VEGFR2抗体或其抗原结合片段包含具有如SEQ ID NO：22所示的氨基酸序列或与SEQ ID NO：22具有至少85％、90％、95％或99％序列同一性的氨基酸序列的重链可变区和具有如SEQ ID NO：23所示的氨基酸序列或与SEQ ID NO：23具有至少85％、90％、95％或99％序列同一性的轻链可变区。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to claim 3, wherein the anti-VEGFR2 antibody or antigen-binding fragment thereof has an amino acid sequence as shown in SEQ ID NO: 22 or has an amino acid sequence of at least 85 with SEQ ID NO: 22. %, 90%, 95% or 99% sequence identity of the heavy chain variable region of the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 23 or at least 85%, 90%, A light chain variable region with 95% or 99% sequence identity.
  5. 如权利要求1-4任一项所述的抗VEGFR2抗体或其抗原结合片段，其中所述抗体进一步包含轻链恒定区和重链恒定区，优选地所述轻链恒定区为氨基酸序列为SEQ ID NO:25的kappa轻链恒定区的氨基酸序列或与SEQ ID NO:25具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述重链恒定区为与氨基酸 序列为SEQ ID NO:24的IgG1重链恒定区的氨基酸序列或与SEQ ID NO:24具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody further comprises a light chain constant region and a heavy chain constant region, preferably the light chain constant region has an amino acid sequence of SEQ The amino acid sequence of the constant region of the kappa light chain of ID NO: 25 or an amino acid sequence that has at least 90%, 92%, 95%, 98% or 99% sequence identity with SEQ ID NO: 25, and/or the heavy chain The constant region is the amino acid sequence of the IgG1 heavy chain constant region with the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 24 .
  6. 如权利要求1-5中任一项所述的抗VEGFR2抗体或其抗原结合片段，其为IgG抗体，优选为IgG1抗体。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-5, which is an IgG antibody, preferably an IgG1 antibody.
  7. 如权利要求1-6中任一项所述的抗VEGFR2抗体或其抗原结合片段，其为单克隆抗体。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-6, which is a monoclonal antibody.
  8. 如权利要求1-7中任一项所述的抗VEGFR2抗体或其抗原结合片段，其与重组VEGFR2的结合亲和力K D为1-100pM，优选5-50pM，更优选10.6pM。 The anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-7, which has a binding affinity K D for recombinant VEGFR2 of 1-100 pM, preferably 5-50 pM, more preferably 10.6 pM.
  9. 如权利要求1-8任一项所述的抗VEGFR2抗体或其抗原结合片段，其中所述抗原结合片段为Fv、Fab、Fab′、Fab′-SH、F(ab′)2、Fd片段、Fd'片段、单链抗体分子或单域抗体；其中单链抗体分子优选为scFv、di-scFv、tri-scFv、双体抗体或scFab。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the antigen-binding fragment is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragments, single-chain antibody molecules or single-domain antibodies; wherein the single-chain antibody molecules are preferably scFv, di-scFv, tri-scFv, diabody or scFab.
  10. 权利要求1-9任一项所述的抗VEGFR2抗体或其抗原结合片段，其结合表位是VEGFR2的Y137/K142、R164/Y165、D257、S311/G312。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-9, whose binding epitope is Y137/K142, R164/Y165, D257, S311/G312 of VEGFR2.
  11. 一种抗体-药物缀合物，其包含如权利要求1-10任一项所述的抗VEGFR2抗体或其抗原结合片段和另外的治疗剂，优选地所述抗VEGFR2抗体或其抗原结合片段和另外的治疗剂通过接头连接。An antibody-drug conjugate comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-10 and another therapeutic agent, preferably the anti-VEGFR2 antibody or antigen-binding fragment thereof and The additional therapeutic agent is connected by a connector.
  12. 一种核酸，其编码根据权利要求1-10任一项的抗VEGFR2抗体或其抗原结合片段。A nucleic acid encoding the anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-10.
  13. 如权利要求12所述的核酸，其包含如SEQ ID NO：4所示的核苷酸序列和/或如SEQ ID NO：5所示的核苷酸序列，或者其包含如SEQ ID NO：30所示的核苷酸序列和/或如SEQ ID NO：31所示的核苷酸序列。The nucleic acid according to claim 12, which comprises a nucleotide sequence as shown in SEQ ID NO: 4 and/or a nucleotide sequence as shown in SEQ ID NO: 5, or it comprises a nucleotide sequence as shown in SEQ ID NO: 30 The nucleotide sequence shown and/or the nucleotide sequence shown in SEQ ID NO: 31.
  14. 一种表达载体，其包含如权利要求12或13所述的核酸。An expression vector comprising the nucleic acid according to claim 12 or 13.
  15. 一种宿主细胞，其包含如权利要求12或13所述的核酸或如权利要求14所述的表达载体。A host cell comprising the nucleic acid according to claim 12 or 13 or the expression vector according to claim 14.
  16. 一种用于产生如权利要求1-10任一项所述的抗VEGFR2抗体或其抗原结合片段的方法，包括在适合于抗体表达的条件下培养如权利要求15所述的宿主细胞，和从培养基中回收表达的抗体。A method for producing the anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-10, comprising culturing the host cell according to claim 15 under conditions suitable for antibody expression, and from The expressed antibody is recovered from the culture medium.
  17. 一种药物组合物，包含如权利要求1-10任一项所述的抗VEGFR2抗体或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求12-13任一项所述的核酸或如权利要求14所述的表达载体，及药学上可接受的载体。A pharmaceutical composition comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-10 or the antibody-drug conjugate according to claim 11 or any one of claims 12-13 The nucleic acid according to item or the expression vector according to claim 14, and a pharmaceutically acceptable carrier.
  18. 如权利要求1-10任一项所述的抗VEGFR2抗体或或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物，其用于治疗黑色素瘤。The anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-10 or the antibody-drug conjugate according to claim 11 or the pharmaceutical composition according to claim 17 for use in Treat melanoma.
  19. 一种用于治疗黑色素瘤的方法，包括向需要的受试者施用治疗有效量的如权利要求1-10任一项所述的抗VEGFR2抗体或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物，从而治疗所述黑色素瘤。A method for treating melanoma, comprising administering to a subject in need a therapeutically effective amount of the anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-10 or the subject according to claim 11 An antibody-drug conjugate or a pharmaceutical composition according to claim 17 to treat the melanoma.
  20. 如权利要求1-10任一项所述的抗VEGFR2抗体或其抗原结合片段或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物在制备用于治疗黑色素瘤的药物中的用途。The anti-VEGFR2 antibody or antigen-binding fragment or antigen-binding fragment thereof according to any one of claims 1-10 or the antibody-drug conjugate according to claim 11 or the pharmaceutical composition according to claim 17 Use in preparing medicine for treating melanoma.
  21. 一种药物组合，其包含如权利要求1-10任一项所述的抗VEGFR2抗体或其抗原结合片段或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物与一种或多种另外的治疗剂。A pharmaceutical combination comprising the anti-VEGFR2 antibody or antigen-binding fragment or antigen-binding fragment thereof according to any one of claims 1-10 or the antibody-drug conjugate according to claim 11 or The pharmaceutical composition of 17 and one or more additional therapeutic agents.
  22. 一种试剂盒，其包含如权利要求1-10任一项所述的抗VEGFR2抗体或其抗原结合片段或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物,优选地,还进一步包含给药的装置。A kit comprising the anti-VEGFR2 antibody or antigen-binding fragment or antigen-binding fragment thereof according to any one of claims 1-10 or the antibody-drug conjugate according to claim 11 or The pharmaceutical composition according to 17, preferably, further comprises a device for administration.
  23. 一种抗VEGFR2抗体或其抗原结合片段，其与权利要求1-10任一项所述的抗VEGFR2抗体或其抗原结合片段结合抗原上的相同的表位。An anti-VEGFR2 antibody or antigen-binding fragment thereof, which binds to the same epitope on an antigen as the anti-VEGFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-10.
  24. 一种VEGFR2分子表位，其是VEGFR2的Y137/K142、R164/Y165、D257、S311/G312。A molecular epitope of VEGFR2, which is Y137/K142, R164/Y165, D257, S311/G312 of VEGFR2.
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